Geminin is a protein involved in cell cycle progression. We aimed to evaluate the diagnostic value of geminin expression in cervical intraepithelial neoplasm (CIN).The expression of geminin, p16, and Ki67 was examined in 95 samples, including CIN1 (n = 45), CIN2/3 (n = 40), and normal cervical tissues (n = 10) by immunohistochemistry. The correlation between geminin or p16 expression and human papillomavirus (HPV) status was also evaluated.Geminin expression was negative in all normal tissues and expressed in 13.3% of CIN1 and 90.0% of CIN2/3. P16 expression was demonstrated in 24.4% of CIN1 and 87.5% of CIN2/3. The corresponding Ki67 expression was 35.6% and 95.0%. The specificity of geminin for differentiating between CIN1 and CIN2/3 was 86.7%, while for p16 and Ki67 the corresponding values were 75.6% and 64.4%. The sensitivity of geminin, p16, and Ki67 was 90.0%, 87.5%, and 95.0%, respectively. The positive predictive value (PPV) and accuracy of geminin were higher than p16 and Ki67. In addition, geminin expression showed a weak correlation with HPV status, but there was no association between p16 expression and HPV status.These results suggested that geminin had a high degree of sensitivity and specificity in determining CIN2/3. In addition to p16 and Ki67, geminin might be used as a new biomarker to distinguish between CIN1 and CIN2/3. The progression rate of CIN2/3 to invasive cervical cancer is 10% to 40%.,3 Given the malignant potential of CIN2/3, it is important to have an accurate grading of CIN and to treat these patients properly. High-risk human papillomavirus (HPV) types, including HPV-16 and HPV-18, have been identified as the major cause of cervical carcinomas and precancerous lesions. Currently, the incidence rate of cervical cancer has decreased through screening by cytological Pap smear testing and HPV testing. HPV-18, one common reason for CIN2/3, is poorly detected by cytology and colposcopy. Furthermore, HPV testing is more sensitive than cytology for predicting CIN2/3, but is of less specificity. Histological diagnosis of cervical biopsies is regarded as “gold standard.” However, the interobserver and intraobserver variabilities are high. For these reasons, it is important to have an accurate diagnosis and prediction of progression risk for clinical management of patients with CIN.Invasive cervical carcinoma is preceded by preinvasive cervical intraepithelial neoplasm (CIN). The most recent classification system of World Health Organization (WHO) applied low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL). However, CIN is previously classified into CIN1, CIN2, and CIN3 on the basis of the extent of epithelial involvement.,8 Ki67 is a marker of cell proliferation. A number of studies have shown that an increased expression of Ki67 is correlated with higher cervical CIN grade and is a highly sensitive biomarker for differentiating between CIN1 and CIN2/3, but Ki67 immunostaining is variable and less specific in many cases.,10 Currently, p16 and Ki67 have been proposed to identify persistent infections with high-risk HPV types of cervical precursor lesions. Despite the value of these 2 complementary alternative biomarkers, staining of p16 and Ki67 is not sufficient to give a definite diagnosis and an accurate differentiation in some cases.P16 , a cyclin-dependent kinase inhibitor, is a cell-cycle regulatory protein. The high-risk HPV E7 oncoproteins bind and inactivate pRb, leading to abnormal cell proliferation. P16 is an accurate marker for this event. However, p16 is a less specific marker because CIN1 and CIN2/3 sometimes show similar p16 expression, and p16 also can be found in inflammatory cervical lesions. Alterations in geminin expression are associated with cell proliferation, differentiation, and development. The expression of geminin is increased as the cell cycle progresses and downregulated when the cells exit the cell cycle. A great number of studies have reported that geminin plays a vital role in cancer pathophysiology and development. Differential geminin expression is associated with various cancers, including breast cancer, colorectal carcinomas, and small lung adenocarcinoma.–15 Suppression of geminin can inhibit cancer cell proliferation without affecting the normal cells. Using high-density microarrays, Martin et al have identified several genes involved in cell cycle regulation that are differentially expressed in premalignant and malignant cervical disease, including geminin, p16, minichromosome maintenance complex component (MCM) 3, and MCM5. Real-time polymerase chain reaction (PCR) and immunohistochemisitry were used to confirm microarray results. This study suggested that geminin might play a role in prediction of progression of cervical precursor lesions.Geminin is located on the sixth chromosome, and the protein has about 209 amino acids. Geminin is an important component of the licensing system that is involved in cell cycle progression.In this study, we aimed to evaluate and compare staining pattern for geminin, p16, and Ki67 expression in normal cervical tissues, CIN1 and CIN2/3 to determine whether geminin can serve as an additional marker for the diagnosis of CIN2/3. In addition, we investigated whether geminin could discriminate between HPV-positive and HPV-negative precursor lesions.22.1A total of 95 cervical samples were recruited from the First Affiliated Hospital of Nanjing Medical University between January 2015 and January 2016, including 10 normal cervical tissues, 45 CIN1, and 40 CIN2/3. The diagnosis of CIN was peer-reviewed according to the International Federation of Gynecology and Obstetrics criteria. All hematoxylin-eosin stained slides were reviewed by 2 independent pathologists. Exclusion criteria are patients who had a clinical record of radiotherapy or chemotherapy. The clinical characteristics of the subjects are summarized in Table 2.2HPV DNA was detected by the Hybrid Capture II System. The scrapes were tested for the presence of high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68.2.3All formalin-fixed paraffin-embedded sections were cut at 5 μm thickness and then dewaxed through xylene and dehydration with graded ethanols. The tissue sections were treated with 3% hydrogen peroxidase solution for 20 minutes to block endogenous peroxidase activity. Antigen retrieval was performed in 10 mmol/L of citrate buffer . The sections were then incubated overnight with a monoclonal mouse antibody to geminin  at 1:100 dilution, a monoclonal rabbit antibody to p16  at 1:100 dilution, and a monoclonal rabbit antibody to Ki67  at 1:500 dilution, respectively. Subsequently, tissue sections were counterstained with hematoxylin and then examined by light microscopy. For negative controls, substitution of primary antibody with TBS was run simultaneously.2.4 The cutoff value for interpretation of p16 staining is 5%. All the markers were classified into 4 groups: 0 , 1+ (positive staining in 6–25% of cells), 2+ (positive staining in 26–50% of cells), or 3+ (positive staining in more than 50% of cells). For the statistical analysis and evaluation of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy, we subdivided the staining results into 2 groups: nuclear or cytoplasmatic immunoreactivity in less than 25%  or more than 25% .Two independent authors (YX and WC) scored the expression of immunostaining slides. First, 10 normal cervical tissues were evaluated for geminin expression because there is not yet a validated cutoff value for positivity of geminin staining. We investigated that geminin expression was absent in most normal tissues. Therefore, immunostaining with Ki67 and geminin was considered positive when more than 5% of the cells showed strong positive nuclear staining. The immunoreactivity of p16 was judged as positive when there was a diffuse staining in both nuclear and cytoplasm of basal or parabasal cells.2.5P value of less than .05 was considered to be significant.Statistical analysis was performed using SPSS software package, version 21.0 . Data in this study are expressed as means ± standard deviation (SD). Chi-squared tests were used to differentiate between CIN1 and CIN2/3 and 1-way analysis of variance (ANOVA) was used to analyze the age among groups. Spearman rank correlation coefficients were analyzed to investigate the possible correlations between HPV status and geminin/p16 expression. Sensitivity, specificity, PPV, NPV, and accuracy were calculated on the basis of geminin, p16, and Ki67 staining results by the formulas of Galen and Gambino: Sensitivity=True Positive/; Specificity=True Negative/; PPV=True Positive/; NPV=True Negative/; Accuracy=True Positive+True Negative/. All statistical tests were 2-tailed and a 3P > .05). The HPV in patients with CIN1 was more frequently detected compared with the patients with CIN2/3 (P < .05).Table P < .05).The immunohistochemical staining results for geminin, p16, and Ki67 are summarized in Table P = .015). However, no correlation was found between p16 expression and HPV status .Then, we studied the performance of geminin, p16, and Ki67 in determining the clinical significant lesions and the results are summarized in Table 4The aim of this study was to assess whether geminin could be a biomarker to discriminate cervical high-grade lesions. Our results showed that geminin had a high degree of sensitivity and specificity in determining CIN2/3. The specificity of geminin (86.7%) exceeded that of the commonly used biomarkers p16 (75.6%) and Ki67 (64.4%). The sensitivity of geminin (90.0%) was higher than that of p16 (87.5%), but lower than that of Ki67 (95.0%). Therefore, it is still challenging to discriminate between CIN1 and CIN2/3.Accurate histological grading of CIN was clinically important, because CIN2 and CIN3 were regarded as precursors of invasive cervical carcinomas and therapy was indicated. Histopathology is a gold standard for diagnosis of CIN. However, the interobserver and intraobserver variabilities in interpreting cervical biopsy specimen are relatively high. Furthermore, these 2 markers were highly sensitive to detect the presence of CIN2/3, but their specificity was relatively low in our study. This is in accordance with many previous studies.,20 Our results demonstrated that the specificity, PPV, and accuracy of geminin were highest among these biomarkers. In addition, geminin expression is significantly increased in CIN2/3 compared with CIN1. This implied that the use of geminin immunohistochemical analysis might be a surrogate marker of high-grade cervical intraepithelial neoplasia.The commonly used immunohistochemical markers p16 and Ki67 were not very accurate to help for the distinction between CIN1 and CIN2/3. In the present study, HPV positivity was detected in 82.2% of CIN1 and 97.5% of CIN2/3. Our results were higher than those in a recent study. A 4-year surveillance study by Zhang et al have reported that the prevalence of HPV increased with cervical lesions severity and the HPV positivity rates were 72.4% for CIN1, 81.4% for CIN2, and 88.1% for CIN3. The discrepancy might be caused by our small sample size. Moreover, the average age of patients in our study was 47.8 ± 7.9 years, which might be another reason for the higher HPV positivity rates because age was an important factor for high-risk HPV infection clearance. In the present study, p16 expression was not associated with HPV status. The finding is in accordance with previous studies.,25 No correlation was found between p16 and HPV status in esophageal squamous cell carcinoma in Kazakh population by Wang et al, thus suggesting that p16 might be an unreliable surrogate marker for HPV status. Nevertheless, p16 expression was previously reported to exhibit a correlation with HPV status. The inconsistency might be attributed to absence of uniformity in cutoff value and variation in HPV status among patients from different geographic origins. Interestingly, a weak correlation between geminin and HPV status was observed in our study.High-risk HPV persistent infection is significantly associated with CIN2/3 and invasive cervical carcinomas.There are some limitations in our study. First, the sample size of our study is relatively small. In addition, the cutoff value for increased geminin expression used in our study needs to be validated in an independent set of cervical samples. Another limitation in this study is that we focused on the expression of geminin in CIN, but did not evaluate geminin expression in cervical carcinomas. Our results suggested that geminin might serve as a diagnostic biomarker for identifying CIN2/3; however, we are not informed about its clinical use as a prognostic variable in cervical carcinomas. Furthermore, additional mechanistic studies to investigate the role of geminin in the pathogenesis of CIN progress are needed.In conclusion, geminin is a highly sensitive and specific biomarker for CIN diagnosis. It can be used in addition to p16 and Ki67 staining in routine pathology practice when discriminating between CIN1 and CIN2/3 is difficult.5Our study showed that the immunoquantification of geminin appeared to be a new marker for the differentiation between CIN1 and CIN2/3 when there is doubt about the grading of CIN. Nonetheless, further studies are necessary before implementing geminin in clinical practice (Fig. We would like to thank all the subjects for their participation in this research, as well as the clinical coworkers form the department of Obstetrics and Gynecology.


GPs lack of time  and SHCP staff availability  were the main reported factors preventing YMSM HPV vaccination. GPs were less likely than SHCPs to believe there was sufficient evidence for vaccinating YMSM ; less likely to have skills to identify YMSM who may benefit from vaccination ; and less confident recommending YMSM vaccination . GPs appear to have different knowledge, attitudes, and skills regarding YMSM HPV vaccination when compared to SHCPs.Men who have sex with men (MSM) may be at higher risk for human papillomavirus (HPV)-associated cancers. Healthcare professionals’ recommendations can affect HPV vaccination uptake. Since 2016, MSM up to 45 years have been offered HPV vaccination at genitourinary medicine (GUM) clinics in a pilot programme, and primary care was recommended as a setting for opportunistic vaccination. Vaccination prior to potential exposure to the virus  is likely to be most efficacious, therefore a focus on young MSM (YMSM) is important. This study aimed to explore and compare the knowledge and attitudes of UK General Practitioners (GPs) and sexual healthcare professionals (SHCPs) regarding HPV vaccination for YMSM (age 16–24). A cross-sectional study using an online questionnaire examined 38 GPs and 49 SHCPs, including 59 (67.82%) females with a mean age of 40.71 years. Twenty-two participants (20 SHCPs,  Human papillomavirus (HPV) vaccination of young men who have sex with men (YMSM) age 16–24) potentially has important implications for cancer prevention worldwide. HPV is one of the most common sexually transmitted infections 6–24 pote. HPV infPrevention of HPV-related disease is a key public health issue. The United Kingdom’s (UK) current strategy is to offer publicly funded vaccination only to girls aged 12–14 , which is intended to protect males through herd immunity. This decision was made on the basis of cost effectiveness , althougVaccination is likely to be most efficacious before exposure to HPV , howeverIt is crucial to engage widely with HCPs expected to vaccinate YMSM against HPV. Patients and parents of younger children place a strong emphasis on the recommendations (or otherwise) of a HCP in decision-making regarding vaccinations ,20,21. GIn a survey of 131 sexual healthcare professionals (SHCPs), 95% of clinicians supported a targeted HPV vaccination programme in MSM within GUM services but expressed concern that alone this strategy was too late and too limited for most MSM . This stAn exploratory cross-sectional survey of GPs and SHCPs was conducted as part of a mixed-methods study. SCHPs included GUM consultants, doctors-in-training, and nurses working in sexual health clinics. Between September 2016 and January 2017, convenience sampling was used to recruit participants through an email invitation distributed by the Royal College of General Practitioners (RCGP), British HIV Association (BHIVA), British Association for Sexual Health and HIV (BASHH), Society of Sexual Health Advisors (SSHV), Society for the Study of Sexually Transmitted Diseases in Ireland (SSSTDI), and the University of Bristol Centre for Academic Primary Care (CAPC). The email invitation included a link to the online survey and a participant information sheet which explained that participation implied consent. At the end of the anonymous survey, GPs were invited to provide contact details if they wished to take part in a follow-up interview (findings not yet published). Similar to Nadarzynski et al. , particiThe questionnaire aimed to capture knowledge and attitudes towards HPV vaccination for YMSM, as well as any barriers or facilitators. YMSM were chosen as the focus for this study as greater understanding of factors affecting HPV vaccination in this age group could improve the efficacy of HPV vaccination programmes for MSM by targeting younger men before they engage in sexual activity. Questionnaire content was informed by a study steering group comprising two lesbian, gay, bisexual, transgender and queer (LGBTQ) group stakeholders and three MSM sexual health researchers from England and Northern Ireland, and piloted with HCPs prior to wider distribution. The questionnaire was adapted from a HCP HPV attitude scale developed by Nadarzynski et al. , and a Ht-tests were utilised for comparison of demographics. Due to null responses for some categories, sexual orientation was converted to a binary variable  for the analysis. Ordinal knowledge variables were converted to binary responses (“high/medium” vs. “low/none”). Attitudinal responses were converted into positive (“yes”) or negative (“unsure” or “no”) binary variables. Simple and multiple logistic regression techniques were utilised to compare the responses of GPs and SHCPs to the knowledge and attitude questions. Adjusted analysis controlled for the effects of participant age, gender, sexual orientation, and years of experience. Hosmer–Lemeshow goodness of fit testing was performed to assess accuracy of multiple logistic regression models. All analysis was conducted using Stata version 14 .Descriptive statistics summarised demographic, attitude, and knowledge data. Fisher’s exact testing and unpaired This study received ethical approval from the Queens University (Belfast) School of Nursing and Midwifery Research Ethics Committee (ref. 39.GPrue.05.16.M8.V2). In total, 87 participants completed the survey. Demographic data was incomplete for three SHCPs, but overall individual question response rates were high (range: 94.25–100%). Thirty-eight GPs and 49 SHCPs  completed the questionnaire. Participants included 59 females (67.82%), with a mean age of 40.71 years, and a median 14 years of experience (IQR (Interquartile Range) 8, 24). There were no significant differences between GPs and SHCPs. Further demographics are shown in p < 0.001), more likely to be aware of the recent JCVI recommendations , and more likely to report they knew enough to have an informed discussion with MSM about HPV vaccination . Thirty GPs (78.95%) stated they have a “low level of knowledge” or “no knowledge” of HPV vaccination for YMSM, compared to 6 (12.24%) SHCPs , but there were no significant differences in knowledge ratings regarding overall HPV knowledge or HPV in females (see SHCPS were more likely than GPs to have vaccinated a YMSM patient against HPV (20/49 (47.83%) vs. 2/38 (5.6%), ales see . p = 0.36).GPs attitudes towards HPV vaccination in YMSM differed from SHCPs see . GPs werp = 0.57). GPs were much less likely to believe that a young person would disclose their sexual orientation to them , less confident that they had the skills to identify YMSM who may benefit from HPV vaccination , and they reported lower levels of confidence in recommending HPV vaccination for YMSM .When asked about whether they ask patients about sexual orientation “if it is relevant to the consultation”, there was no difference between the responses of GPs  and SHCPs  has proved insightful, given the JCVI recommendations that both settings could be used to deliver the vaccine. The lower levels of confidence and knowledge among GPs may help to explain the low uptake of HPV vaccination for MSM in the current pilot programme to date .There are a number of limitations that be considered in the interpretation of this study’s findings. The cross-sectional design, convenience sampling approach, and exploratory nature of the study—using pre-determined survey statements—limits the ability to draw sound inferences about the reasons behind participant responses. The sample size is small, and while a response rate cannot be accurately calculated it is presumably quite poor considering the RCGP has over 50,000 members and BASHH has over 1000 members (some of whom are not based in the UK). There were no incentives offered for participation; a practice which is known to raise study participation rates in similar studies. Interviews with GPs who participated in this survey will provide more in-depth insight into their views and opinions regarding HPV vaccination for YMSM. GPs can potentially play a crucial role in the prevention of HPV-related diseases in YMSM. In order to implement the JCVI recommendation regarding HPV vaccination for MSM most effectively, YMSM should be identified early and offered the HPV vaccine with clear information. However, barriers to such implementation in primary care appear to still remain. If the findings of this exploratory work were confirmed in future research, interventions could be developed to raise awareness and educate GPs about the benefits of HPV vaccination for MSM, and to improve the skills of GPs in sensitively eliciting a patient’s sexual orientation to benefit the consultation and the patient–doctor relationship. There are also other potential settings for delivering HPV vaccination to YMSM to improve access, such as pharmacies and schools, which have not yet been explored.


Detection and quantification of human Papillomavirus (HPV) genome in oral carcinoma play an important role in diagnosis, as well as implications for progression of disease.E6 gene of HPV genotypes was amplified by specific primers. The sensitivity of PCR assay was analyzed and determined using HPV-DNA-containing plasmids. Real-time PCR was utilized to determine the prevalence and HPV viral load in patients with oral cavity squamous cell carcinoma.We evaluated tissues from 50 esopharyngeal cancers collected from different regions of Iran for HPV E6 using the two type-specific primers sets. Eighteen (36%) specimens were positive for HPV. Among the 18 positive specimens, 10 showed HPV-18 (55.55%), and 8 specimens were positive for HPV-11 (44.44%). Of the 18 infected specimens, 6 (33.32%) and 12 (66.65%) were identified as high-titer and low-titer viral load, respectively.The PCR-based assay, developed in the current study, could be used for HPV detection, quantification, and genotyping in epidemiological and clinical studies. Human papillomavirus (HPV), is small, dsDNA virus and is related to a variety of clinical conditions, ranging from benign to malignant tumors and cancers such as oral cavity squamous cell carcinoma (OCSCC). Two disHPV-16, -18, -6, -11, and -33 have been indicated to be the most important and prevalent genotypes associated with the oral cavity carcinoma. HPV-16 and HPV-18 have been confirmed to be associated with head and neck carcinoma. HPV E6 E6 gene that allow the identification of high-risk as well as low-risk genotypes. The sensitivity of this PCR assay was determined and obtained using HPV DNA-containing plasmids. Furthermore, we report a real-time analysis in order to assess the prevalence rate and the viral load of HPV in patients with OCSCC.Currently, the prognostic value of HPV infection in OCSCC has been understated, and numerous studies have reported conflicting results. However, HPV detection and typing in OCSCC specimens is one of the most important developments in oncology. There iParticipants in this study were 50 patients with histological diagnosis of high-grade intraepithelial lesions and 10 healthy subjects admitted at the Imam Khomeini Hospital (Tehran) during the years 2012 to 2014. All participants completed a self-administered questionnaire including information about demographic features, cigarette smoking, alcohol consumption, sexual practice, and the numbers of sexual partners and signed a written informed consent. Oral cavity specimens, such as lip, tongue, floor of mouth, buccal, gingival, and hard palate were obtained from the patients after surgery, transported to the virology laboratory and stored in -70ºC. This study was approved by the Department of Virology, School of Medicine, Tarbiat Modares University, Tehran, Iran.2, 0.5% (v/v) Tween-20, and 0.5% (v/v) Nonidet P-40. Samples were incubated at 95ºC for 30 min, mixed for 2 min and digested with 50 μl proteinase K (20 μg/ul). After an overnight incubation at 56ºC, specimens were heated at 95ºC for 10 min to inactivate the proteinase K. The precipitated nucleic acid was pelleted by centrifugation at 10,000 ×g for 15 min, washed twice with 70% ethanol and air-dried. The specimens were resuspended in 30 μl sterile distilled water[Extraction of total DNA was performed using a manual procedure. The manual method included organic extraction (phenol-chloroform) of the specimens. Briefly, cellular pellets were resuspended in 500 μl lysis solution containing 100 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.5 mM MgClled water. Then 10The E6 genomic sequences were obtained from the GenBank, aligned by MEGA version 4 and finally identified as relatively well-conserved regions from nucleotides 157 to 174 and 338 to 351 according to the HPV-16/-18 sequence. The appropriate E6 region for HPV-11/-6-related genotypes was located from nucleotides 234 to 257 and 365 to 385. Designing of primer was performed using the Oligo version 7 and Allele ID version 6 software.The 190-bp HPV-16/-18 E6 amplicons (157-351) were PCR amplified, using Taq polymerase and standard PCR conditions, from positive subjects; positive clinical specimen for HeLa cell genomic DNA as positive control for HPV-18. Both amplicons were cloned into pTZR57 vector  according to the manufacturer’s instructions to obtain the relevant plasmids. The 150-bp HPV-6/-11 E6 amplicons were PCR amplified from positive clinical specimen, and cloning was carried out similar to HPV-16/-18 E6 amplicons. Purified plasmids were sequenced to verify the accuracy of the sequences and also were used to be tested for their sensitivity and specificity in the conventional PCR and derive standard curves related to the real-time PCR assay.1 to 108 copies/reaction. The specificity was tested by determining the ability of primers to discriminate plasmids with different HPV types. There was no specific signal for the independent real-time PCR assays (data not shown).To determine the sensitivities of the PCR assays, the prepared plasmids were quantified by UV spectroscopy, and the viral copy number was calculated. The 10fold serial dilutions of all HPV DNA-containing plasmids were made. For sensitivity, PCR was performed on a 10fold dilution of plasmids ranging from 102 , 0.2 mM each dNTP , 5 pmol each primer, and 1 U Taq polymerase . Amplification was carried out for 35 cycles  after an initial denaturation step of 94°C for 5 min, on a Techne Thermal Cycler . The cycles were followed by a 5-min extension at 72°C, and the PCR product was visualized on a 1.5% agarose gel by ethidium bromide staining and UV photography . Regions of HPV-16/-18 and HPV-6/-11 genomes were detected by PCR using the primers 5’-F: GATTTATTTGTGGTGT ATAGAGAC-3’ and 5’-R: TTCTGCTGGATTCAACG G-3’ and the primers 5’-F: ACACTTTGATTATGCTG GATA TGC-3’ and 5’-R: GCTTTATGAACCGTGCC TTGG-3’, respectively to amplify two segments of approximately 190 bp related to HPV-16/-18 and 150 bp related to HPV-6/-11. PCR mixture consisted of 10 pmol of each primer, 50 ng DNA specimen, 1.5 mM MgCl2 in final concentration, 0.2 mM each dNTP in final concentration, and 1 U Taq polymerase . Amplifications were done for 35 cycles the same as β-globin gene. Finally, to confirm the genotyping of E6-specefic PCR for each sample, the sequence analysis was obtained from BLAST (http://www.ncbi. nlm.nih.gov/blast).DNA extraction was assessed by PCR amplification of a 260-bp fragment of β-globin gene using the forward 5′-GAAGAGCCAAGGACAGGTAC-3′ and reverse 5′-CAACTTCATCCACGTTCACC-3′ primers, 50 μg DNA specimen, 1.5 mM MgCl2, 0.2 mM each dNTP, 1 Unit Taq polymerase, and SYBR Green dye . Thermal cycle conditions started with first denaturation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 30 s. For standard curves, real-time PCR was performed on a 10fold dilution series of the plasmids as described above.Real-time PCR reactions targeting the E6 region of HPV-16, -18, -6, -11 were performed using SYBR Green Master Mix . The master mix was prepared for all specimens, as well as for positive and negative control samples. Real-time master mix consisted of 1.5 mM MgClP value <0.05 was considered statistically significant.SPSS 18 for windows  was used for statistical analysis. Categorical variables, including HPV infection and OSCC grade were compared by the Chi-squared or fisher’s exact test. All The HPV-6, -11, -16, and -18 E6 gene fragments were amplified by PCR, and the amplicons were ligated into pTZR57 cloning vector. The recombinant plasmids were then identified and confirmed by PCR using E6-specific primers .1 plasmid copies and ended to 108 plasmid copies. This assay showed significant sensitivity about 100 viral copies for all four HPV genotypes . A linear relationship between the virus copy number and Ct values was constructed for each of the HPV genotypes based on serial dilution associated to each plasmid. PCR assay on 50 clinical specimens showed that 18 (36%) were positive for HPV. Of these HPV-positive specimens, 10 (55.56%) and 8 (44.44%) were positive for HPV-18 and HPV-11, respectively. In 1982, Syrjanen reported his observation on correlation between HPV infectionand development of esophageal carcinoma. This obRegarding this fact that HPV is considered as an etiologic agent of oral squamous tumors, screening the patient with an increased risk of developing oral squamous tumors seems to be necessary. There aet al.[et al.[et al. [Here, we have described for the first time in Iran a PCR-based method for the accurate and sensitive detection of high-risk genotypes (HPV-16/-18) and low-risk genotypes (HPV-6/-11) using the ur designed E6-specific primers. PCR-based detection of different types of HPV using the general or consensus primers necessitates confirming the underlying genotypes with time-consuming and costly techniques, such as sequencing, line immunoassay, restriction fragment length polymorphism, Southern-blot hybridization, or enzyme immunoassays. In addition, this method could potentially predict the progression of malignancy caused by high-risk HPV genotypes. In two different studies by Sotlar et al. and Kenal.[et al., PCR ass.[et al. , we founet al.[The result of the current investigation indicated that 36% (18 of 50) of clinical specimens tested were positive for HPV. Of these 18 investigated samples, 55.5% (10 of 18) were found to be positive for HPV-18, and the 45.5% (8 of 18) of the specimens were positive for HPV-11. In a recent study conducted by Tabatabai et al., the freet al. detectedet al.[et al.[According to the growing reports on the incidence of OSCC worldwide, the accurate determination of HPV levels in OSCC patients is serious. Real-time PCR has been shown to be a useful tool in quantifying HPV viral load,23. In tet al.. In othePCR-based assay utilizing the E6 region, as primer-binding site, represents a highly sensitive and specific, rapid, and valid method for identification of HPV, especially the high-risk oncogenic HPV. The spectrum of this method could be extended by the addition of multiplex set of primers for other HPV genotypes. Furthermore, by using the real-time quantitative reverse transcription PCR for amplification of E6 oncogene transcripts, it may be possible to detect a further risk factor for the establishment or progression of a malignant lesion. This assay could also be employed as a potentially sensitive method in epidemiological and clinical follow-up studies, especially when exact HPV typing is required. Our results revealed that risk factors other than HPV infection play a possible role in OCSCCs among the Iranian population. Large-scale studies are needed to determine that HPV, as a risk factor, is independent of environmental factors such as alcohol and tobacco consumption, and sexual practices can be effective in creating OSCC.


Blue aleurone 1 (Ba1) transferred from Thinopyrum ponticum. In the genes involved in anthocyanin biosynthesis, only a basic helix-loop-helix (bHLH) transcription factor, ThMYC4E, had a higher transcript level in blue aleurone phenotype, and was homologous to the genes on chromosome 4 of Triticum aestivum. ThMYC4E carried the characteristic domains  of a bHLH transcription factor, and clustered with genes regulating anthocyanin biosynthesis upon phylogenetic analysis. The over-expression of ThMYC4E regulated anthocyanin biosynthesis with the coexpression of the MYB transcription factor ZmC1 from maize. ThMYC4E existed in the genomes of the addition, substitution and near isogenic lines with the blue aleurone trait derived from Th. ponticum, and could not be detected in any germplasm of T. urartu, T. monococcum, T. turgidum, Aegilops tauschii or T. aestivum, with white aleurone. These results suggested that ThMYC4E was candidate Ba1 gene controlling the blue aleurone trait in T. aestivum genotypes carrying Th. ponticum introgression. The ThMYC4E isolation aids in better understanding the genetic mechanisms of the blue aleurone trait and in its more effective use during wheat breeding.Blue aleurone is a useful and interesting trait in common wheat that was derived from related species. Here, transcriptomes of blue and white aleurone were compared for isolating  Blue grain wheat cultivars have a blue aleurone layer that contains a group of anthocyanin pigments that differ from those in purple, red or white wheat grains –3. BecauAgropyron species and diploid wheat . Th. ThF3'5'on wheat . So ThMYS and Ra , 36, whiinterest .ThMYC4E was distinguished from the bHLH proteins from T. uratu, Ae. tauschii and T. aestivum. ‘Sebesta Blue 1’, ‘Sebesta Blue 2’ ‘Blue 1’, ‘Blue 2’ and ‘Blue Norco’ have disomic additions of chromosome 4E [ThMYC4E existed in these cultivars, but did not exist in T. uratu, T. monococcum, T. turgidum, Ae. tauschiior, and T. aestivum. Compared with common wheat, the whole or segment of chromosome 4E should be the only unique chromosome in ‘Blue 1’, ‘Blue 2’, ‘Blue Norco’, ‘Sebesta Blue 1’, ‘Sebesta Blue 2’, and ‘Sebesta Blue 3’, which implies that ThMYC4E originally resided on chromosome 4E.In the phylogenetic tree composed of bHLH proteins regulating anthocyanin biosynthesis, osome 4E , 7, 44, osome 4E , 7. ThMYThMYC4E was only a bHLH transcription factor with high expression in blue aleurone Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 FigThe tree was constructed using MEGA6, neighboring-joining phylogeny testing, and 1,000 boot strap replicates. The accession number of these proteins (ortranslated products) areas follows in the GenBank database: Rice\Ra: AAC49219; Rice\R-Sx2: XP_006653664; Triticum urartu\R-S: KD049651.1; Aegilops tauschii\RS: KD512407.1; Barley\R accession AK361387.1; Triticum urartu\RS: KD032825.1; Aegilops tauschii\R-S: KD566857.1; Maize\R-S like: XP_008669036; Maize\B1: KC771884.1; Maize\CP1: NP_001105706; Sorghum\b1-1: AY542311.1; Maize/RS: NP_001106073; Maize\Hopi: CAB92300; Maize\LC: NP_001105339; Maize\SN: NP_001105339; Maize\r1-B3: NP_001105339; Arabidopsis\GL3: NP_680372; Arabidopsis\EGL3: NP_176552; petunia\AF13: AAC39455; Arabidopsis\MYC1: NP_191957; Tobacco\AN1a: AEE99257; Tobacco\AN1-like: NM_001302566.1; Tobacco\AN1b: HQ589209.1; Petunia\AN1: AF260918.1; Medicago\TT8: AF260918.1; Arabidopsis\TT8: CAC14865; Maize\IN1: AAB03841; ThMYC4E: KX914905.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Despite the increasing incidence of laryngeal squamous cell carcinoma (LSCC) in Algeria, scarce information is available on the importance of the preventable etiological factors which may drive the disease. Remarkably, a significant number of cases occur in nonsmoker and nondrinker patients; hence, suggesting that alternative risk factors, like Human papillomavirus (HPV), might be etiologically involved. To gain more insight on the risk factors associated with the disease in the country, we evaluated the etiological fraction of HPV in comparison to tobacco and alcohol intake in LSCC patients.To evaluate the etiopathologic fraction (EF) for HPV compared to history of tobacco and alcohol in LSCC, HPV DNA presence in 46 invasive and 3 non-invasive formalin-fixed paraffin-embedded laryngeal tumors was screened using the SPF10-DEIA-LiPA25 Assay. Demographic data and information related to exposure to the risk factors were gathered through interviewer-assisted questionnaires.We observed that 40.8% of all LSCC cases were associated with smoking, 40.8% had combined tobacco and alcohol exposure history, and 14.3% did not show prior exposure to either risk factor. 1 out of 3 in-situ carcinoma cases was positive for HPV-6. HPV prevalence was null in the invasive tumors. HPV DNA was detected in 2.38% for all studied cases. 10.2% of LSCC patients did not associate with any of the studied risk factors.Here we show that HPV etiological fraction in LSCC Algerian patients is low and smoking and alcohol remain the principal etiopathologic risk for LSCC burden in Algeria. It accounted for 1.6% of all new cancer cases and 1.1% of deaths in 2012 worldwide . The conTobacco and alcohol are the main risk factors for LSCC. Both act as powerful forces driving epigenetic reprogramming and genetic instability to induce oncogenesis , 7, 8. TINK4a) [Interestingly, approximately 5% of all LSCC showed to happen in nonsmoker and nondrinker patients, thus suggesting that alternative carcinogenic factors may induce LC . In the INK4a) , 17, 18.INK4a) . To the INK4a) .To gain more insight on the prevalence of HPV infection in LSCC Algerian patients, a strict standardized protocol was applied to assess the etiological fraction of HPV compared to tobacco and alcohol in Algeria.Tumors specimens were prospectively obtained from 58 patients, admitted between 2012 and 2016 at the Otolaryngology department of Algiers University Hospital Mustapha Pacha, upon informed consent. Cases were randomly recruited during two days per week; biopsies were performed on patients presenting a radiological report in favor of a laryngeal tumor, and fragments were used for the present study. The specimens were immediately fixed in formol (10%). Following fixation, the biopsies were rinsed twice in phosphate-buffered saline (PBS) and then manually dehydrated in ascending series of ethanol solutions . The samples were then cleared in xylene and embedded in paraffin.Demographic  and risk factors data  were gathered for the purpose of this study through an interviewer-assisted questionnaire led by the treating physician. This study was approved by the ethics committee of the Algerian National Agency for Research and Development in Health (ATRSS).The histopathological evaluation was performed for all collected specimens by expert pathologists based on the conventional classification of the world health organization (WHO). The type of infiltration and degree of differentiation of the tumors was evaluated, and the presence of HPV cytopathic effect (koilocytosis) was verified. Specimens with epithelial tumor in hematoxylin and eosin (HE)-stained sections were kept for analysis.A sandwich sectioning method was used to prepare paraffin sections for HPV screening protocol as previously described . In brie25) that detects 25 high and low-risk HPV types . Results were interpreted following manufacturer’s instructions. DNA quality of the samples was assessed by amplification of human tubulin gene with a specific set of primers. DEIA negative and tubulin positive samples were considered negative for HPV, while DEIA negative and tubulin negative samples were excluded from the HPV analysis.Paraffin sections of histologically confirmed cases were used to extract DNA. 250 μl of freshly prepared proteinase K solution (pH 8.0) was added directly to the paraffin sections and incubated at 70 °C until full digestion of the tissues. After proteinase K heat inactivation, HPV DNA detection was performed using SPF10-DEIA-LiPA25 version 1 system . In brief, a 65-bp fragment of the L1 open reading frame of HPV was amplified employing the SPF10 consensus primers. Presence of HPV DNA was determined by DNA enzyme immunoassay (DEIA) using 10 μl of the amplified product that was hybridized to a mixture of conservative probes recognizing at least 54 HPV genotypes. Optical densities were read at 450 nm, and samples were categorized as HPV DNA negative, positive or borderline. Positive amplimers were genotyped using reverse hybridization line  were located in the glottis and two (4.1%) were located in the epiglottis. Tumors invading glottis and epiglottis or glottis and subraglottic areas were identified in 13 (26.5%) and two (4.1%) cases respectively. Three patients (6.1%) had tumors invading the three laryngeal regions. The majority of cases (93.9%) were diagnosed at invasive stage and only three tumors (6.1%) were in-situ carcinoma histological grade. Conventional type squamous cell carcinoma was the predominant histological type (87.8%).To screen for presence of HPV DNA, our samples were tested following the procedure outlined in Fig. To compare the etiopathologic fraction (EF) due to HPV with those due to the classical risk factors of LSCC, information on oral sex practice, tobacco, alcohol intake and HPV presence were confronted for each patient Fig. . PatientTo evaluate the burden of the risk factors in the studied patients, tobacco units were defined as number of cigarettes/day; alcohol exposure was defined according to the frequency of drinks per week, and risk factors exposure was stratified based on the gender Fig. . Among mAs HPV related HNC has been described to be likely diagnosed in young, nonsmoker/nondrinker patients practicing oral sex , we nextp = 0.005)  Fig. . We concp = 0.17)  Fig. . SurprisSimilarly to the rest of the world, Algeria has witnessed during the last 30 years a dramatic increase in LSCC incidence rate . This epHere, we estimated the EF for HPV in LSCC at 2.38%. Our observation is in agreement with the most recent evaluations which showed that the EF for HPV in LC is generally low in comparison to other HNC sites , 17. IntConsidering our observation, that the single HPV-LC positive case was found in a women, the recent demonstrations that oral HPV would be frequently detected in patients with HPV cervical lesions , and theIn this study, we also observed that, while among the invasive LSCC cases HPV prevalence was null, 1 out of 3 in-situ carcinoma cases were positive for HPV6. These findings are in agreement with reports from Castellsague et al. (2016), which described HPV6 as figuring among the most frequently detected HPV genotypes associating with LC . It is nOur observation that LSCC occurs in absence of HPV, enforces the idea that tobacco and alcohol may be central to laryngeal carcinogenesis. Indeed, from a total of 49 patients, 85.7% were active smokers, 40.8% were alcohol drinkers and 40.8% had a mixed profile. Considering the heavy frequency of smoking (> 20 cigarettes/day) observed in our patients, the exposure to tobacco seems to constitute the principal cause of the disease. This idea is further supported by the reports indicating a wider access to tobacco in the society over alcohol, as indicated by the recent estimates indicating that 25.3% of men, 0.3% of women and 18% of boys would daily use tobacco  and WHO By contrast to the literature, we found that 10.2% of the tumors occurred in patients showing no prior exposure to any of the studied risk factors; this observation exceeded by two folds the generally observed rates . Hence, In summary, our data suggest that HPV infection would have little impact on the development of LSCC, while tobacco and alcohol, would represent its major preventable risk factors. Considering the growing accessibility to tobacco and alcohol for the youngest, it is probable that the disease burden will remain high for the next decades in the country. Although our findings need to be confronted to larger case series, they constitute, to the best of our knowledge, the first rational arguments for the national health authorities to consider policies to reduce tobacco and alcohol consumption to decrease LSCC prevalence in Algeria and warrant the evaluation of the inherited risk to develop LSCC in the general population.


Patients down-staged according to the HPV-dependant TNM are at risk for undertreatment and data in clinical trials may be skewed due to false patient inclusion. We performed a meta-analysis to classify clinical outcomes of the distinct subgroups with combined p16 and HPV detection. 25 out of 1677 publications fulfilled the inclusion criteria. The proportion of the subgroups was 35.6% for HPV+/p16+, 50.4% for HPV−/p16−, 6.7% for HPV−/p16+ and 7.3% for HPV+/P16−. The HPV+/p16+ subgroup had a significantly improved 5-year overall-survival (OS) and disease-free-survival in comparison to others both for HNSCC and oropharyngeal cancers. The 5-year OS of the HPV−/p16+ HNSCC was intermediate while HPV+/p16− and HPV−/p16− had the shortest survival outcomes. The clearly distinct survival of HPV−/p16+ cancers may characterize a new relevant HPV-independent subtype yet to be biologically characterized. The possibility also exists that in some HPV+/p16+ cancers HPV is an innocent bystander and p16 is independently positive. Therefore, in perspective, HPV-testing should distinguish between bystander HPV and truly HPV-driven cancers to avoid potential undertreatment in HPV+ but non-HPV-driven HNSCC.Consistent discrepancies in the p16/HPV-positivity have been observed in head and neck squamous cell carcinoma (HNSCC). It is therefore questionable, if all HPV From an epidemiological perspective, exogenous carcinogen-exposure -induced HNSCC is declining, while human papillomavirus (HPV) infection-driven HNSCC is increasing in younger individuals in recent years2. Due to a lack of screening-test the incidence of HPV+ HNSCC is increasing, while cervical cancer is decreasing in the USA3 and Europe for example in Germany4 and France5. If HNSCC is HPV+ this may be, because HPV drives carcinogenesis and can then be considered as causal, or HPV is detected as an innocent bystander of concurrent infection6. This bystander infection can be imagined as an infection located next to the tumor and co-sampled with the tumor material or as an integrated virus copy that has been silenced e.g. by promoter methylation or gene deletion with no active gene transcription. Thus, only if the expression of HPV-related oncogenes is detected, the tumor is proven to be HPV-driven8 indicated by the existence of HPV+/p16− cases. Thus, unless HPV-oncogene expression has been determined the tumor should be referred to as HPV-associated.Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide+ tumors have been identified in many regions of head and neck9, especially in oropharyngeal squamous cell carcinomas (OPSCC)11. These tumors differ from HPV-unrelated cancers at the molecular level15 and likely, as a consequence, have a much more favorable prognosis, despite the generally more advanced stage at presentation16. Given by the nature of HPV-driven tumors, the recently released 8th Edition of the American Joint Committee on Cancer (AJCC) created a separate staging algorithm for high-risk-HPV-associated cancer of the oropharynx distinguishing it from HPV− OPSCC17. HPV+ OPSCC, that used to be advanced stage are now categorized into lower stage. This gives a much more accurate and reasonable prediction of survival for patients with HPV+ OPSCC.HPV− tumors20. Because of the differences in biological features and prognosis, individually optimized therapy for patients with HPV-driven tumors would minimize treatment-related toxicity and improve outcomes. Consequently, de-escalated treatment protocols are under investigation.Preclinical studies also demonstrated that biological features depending on tumor HPV status would influence the effectiveness of treatment. HPV-driven tumors respond better to chemotherapy and X-ray or proton therapy than HPVink4a (cyclin-dependent kinase 2a) immunohistochemical staining are both well-established methods in identifying HPV+ tumors and often regarded delivering equal information on HPV positivity. This is due to the correlation of high-risk HPV E7 expression and in consequence an upregulation of p16. Since, p16 immunohistochemical staining is inexpensive, convenient in use, and the interpretation of results is established it is widely used for detection of HPV-related HNSCC. Therefore, in the modified 8th AJCC/UICC, p16 was recommended as HPV surrogate marker with the cutoff point for diffuse (≥75%) overexpression in a histological section and at least moderate (+2/3) staining intensity17. Although both p16 overexpression and HPV-DNA-positivity have shown their independent prognostic value, as we summarized before21, there are p16+/HPV− and p16−/HPV+ subgroups in which surprisingly different prognosis relative to p16- and HPV-status was observed. Survival of patients with HNSCC was better if associated with HPV+/p16+ or HPV−/p16+. Therefore, in addition to the HPV-related prognostic feature, the biological relevance of p16 independent of HPV infection is currently of interest and under investigation, possibly describing another subgroup of HNSCC with a role of p16 in HPV-independent HNSCC.Most importantly, for a correct classification a reliable distinction between the different entities is crucial. HPV-DNA testing and p16+ and HPV− tumors were also discussed.In this meta-analysis, we included all current clinical studies and evaluated the clinical relevance of HPVDNA-positivity and p16 overexpression in HNSCC. Current observations in elucidating the biological role of p16 in HPVhttp://www.ncbi.nlm.nih.gov/pubmed), OVID (www.ovid.com), EMBASE (www.embase.com), and Wanfang (www.wanfangdata.com.cn). This search included publication dates up to April 20, 2017, adding an additional two years to the previously performed literature search21. We searched for the terms “HPV, p16, head neck”. We also included references quoted in original or review articles that may not have been found during the initial literature search. We screened the articles and included all studies of HNSCC patients which investigated survival rates by the p16 and HPV status of the tumor. Our search strategy was performed in accordance with PRISMA criteria and registered in the PROSPERO register (CRD42017062330). We excluded studies that met the following criteria: missing patient survival information, evaluation of only one marker (HPV or p16), non-HNSCC primary cancer , cell culture or animal models, and reviews or case reports. We also excluded studies with duplicate patient data from the same or similar populations (based on the authors’ names and institutions) in a second round selection process. Where this occurred, we selected the study which was either more recent or had larger patient numbers. We also excluded studies with insufficient survival data. Finally, we included studies with the following criteria: (1) the numerical portion of the subgroups HPV+/p16+ versus HPV−/p16− versus HPV+/p16− versus HPV−/p16+ in HNSCC patients; (2) the numerical survival data of these subgroups (Hazard ratio (HR); overall survival (OS); disease free survival (DFS)) or Kaplan-Meier curves of the subgroups of OS or DFS.Four database searches were performed for publications that statistically analysed subgroup survival after detection of both HPV and p16 markers in PubMed  for data processing in studies where the OS or DFS was displayed as Kaplan-Meier plot.Two authors (A.C. and A.E.A.) extracted the relevant data from the selected publications according to the aforementioned inclusion criteria21, calculating summary RR estimates and 95% confidence intervals (CI) using maximum-likelihood methods for linear mixed models. We assessed study heterogeneity using a chi-squared based Q test. An absence of heterogeneity between the studies was indicated by a p-value greater than 0.05. Existing heterogeneity was examined using the I2 index in the meta-analysis, which was represented as a percentage value between 0 and 100. We initially applied a fixed-effects model (Mantel-Haenszel method and chi-squared test) to the data. Where there was significant heterogeneity, we used the random-effects model (DerSimonian-Liard method). We examined the RR of the 5-year OS and DFS of all subgroups correlating with the HPV+/p16+ and HPV−/p16− groups, depending on the data we extracted from each publication. In cases where the HR was described in the studies, we performed the same analysis. We compared all of the studies using a forest plot.The OS and DFS of all subgroups was evaluated using relative risk (RR)22.Publication bias was examined using a funnel plot. We used the R Version 3.1.0 (R Core Team 2014) computing environment for all statistical analysesThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.47 . The main characteristics of the eligible studies were summarized in Tables in situ hybridization (ISH) without earlier PCR. The identified subgroups discriminated by HPV and p16-status are represented in Table 47. In all studies combined, the subgroup of HPV+/p16+ was 35.6%, of HPV−/p16− 50.4%, of HPV−/p16+ 6.7% and of HPV+/p16− 7.3%. In the studies that only investigated OPSCC, the subgroup of HPV+/p16+ was 44.9%, of HPV−/p16− 44.4%, of HPV−/p16+ 5.4% and of HPV+/p16− 5.3%. The studies investigating correlations between HPV-status and clinicopathological characteristics like tumor size, lymph node involvement, smoking etc. summarized in Table The initial search yielded 1677 citations . Eleven studies that enrolled 1617 patients have data for subgroup HPV+/p16+ and HPV−/p16+ analysis, and 11 studies that enrolled 1545 patients for subgroup HPV+/p16+ and HPV+/p16−. As shown in Fig. +/p16+ has an improved OS compared with both the HPV−/p16+  and the HPV+/p16− .Pooled outcome (5-year OS) results of 19 studies for all four distinct HPV/p16 subgroups can be seen in Fig. −/p16− and HPV−/p16+ and 10 studies (1527 patients) of subgroup HPV−/p16− and HPV+/p16−. The forest plot in Fig. −/p16− does not significantly differ from the HPV+/p16− . However, the 5-year OS of the HPV−/p16− subgroup was inferior compared to the HPV−/p16+  displayed data for the 5-year OS of subgroup HPV+/p16− and HPV−/p16+  was excluded, the 5-year OS of the subgroup HPV+/p16− was inferior compared with the HPV−/p16+ . The Ramshankar et al.42 study reported a small correlation between HPV16-DNA and p16 expression in oral tongue SCC patients. Those patients with p16 overexpression showed an increased risk of death and disease recurrence regardless of their HPV16-DNA status.Additionally, 8 studies (428 patients) reported the 5-year OS of subgroup HPV16+ Fig. . There w+/p16+ showed a better survival than the HPV−/p16− , the HPV−/p16+  and the HPV+/p16− . Figure −/p16− and the HPV−/p16+, and 3 studies of the HPV−/p16− and the HPV+/p16− subgroup. The forest plot in Fig. −/p16+ compared with HPV−/p16− . The subgroup HPV−/p16− did not differ significantly to the HPV+/p16− . The 5-year DFS of the subgroup HPV+/p16+ was also significantly improved compared to the HPV−/p16+ and the HPV+/p16−. The 5-year DFS of the subgroup HPV−/p16− did not differ significantly from the HPV−/p16+ and the HPV+/p16−, even when the Ramshankar et al.42 study was excluded . Additionally, 4 studies enrolling 342 patients had the data of HPV+/p16− and HPV−/p16+ patients for a 5-year DFS meta-analysis. Both subgroups did not differ to each other significantly, even when the Ramshankar et al.42 study was excluded. The DFS-HR was investigated in 6 studies33. The subgroup HPV+/p16+ was associated with a significantly improved 5-year DFS .Eleven studies investigated 5-year DFS. We summarize the individual meta-analyses of the different subgroups in relation to HPV and p16 status individually in Table +/p16+ and HPV-/p16- based on where the study was performed by continent. The results from sub-meta-analyses in the region of the North America46, Europe47, and Asia44 were comparable with the complete meta-analyses including all regions (p > 0.05)  Fig. . The fix+/p16+ and HPV−/p16− into two groups classified by HPV detection methods: one group using PCR47 and one group using ISH without PCR44. These two meta-analyses showed comparable results to the complete data meta-analyses  inherent to some detection methods (e.g. ISH) or testing of only individual HPV types ), we investigated the HPV−/p16+ subgroup further, by excluding the following studies40. However, meta-analysis of the 5-year OS of the subgroup HPV+/p16+ and HPV-/p16+ 47, as well as the HPV−/p16− and the HPV−/p16+ 47 showed comparable results to the meta-analyses with all data included 47, the HPV−/p16+ 47 and the HPV+/p16− 47. The 5-year OS of the HPV−/p16− subgroup was inferior compared to the HPV−/p16+ 47 while the survival of the HPV+/p16− was not significantly different 47. The 5-year OS of the subgroup HPV+/p16− and HPV−/p16+ was not statistically significant , even when the Ramshankar et al. study42 was excluded 47. Therefore, the sub meta-analyses confirmed the whole meta-analyses results with the complete international studies data set  was consistently found. And, moreover, these subtypes based on HPV/p16 status have shown different clinical outcomes21. In this meta-analysis, we confirm and expand to recent investigations to increase the knowledge about (a) the clinical relevance of HPV+ HNSCC and OPSCC; (b) the incidence and clinical course of subtypes of HPV+ tumors.The incidence of high-risk-HPV− cases in the HPV+ compartment and HPV− cases in the p16+ compartment. The number of studies included for evaluation of the discrepant cases of HPV+/p16− and HPV−/p16+ was increased by eight with addition of 428 new patients in this meta-analysis. The relative incidences of HPV+/p16− and HPV−/p16+ HNSCC were 7.3% and 6.7%, respectively. The survival data showed that HNSCC patients with HPV+ status defined as HPV+/p16+ have a better 5-year OS and DFS than subgroups with HPV−/p16−, HPV+/p16− and HPV−/p16+. These significant observations have been made in all head and neck regions and in OPSCC in particular and were consistent with previous studies. The sensitivity analysis confirmed the consistency of the 8 new additional studies. Thus, the survival benefit of the HPV+/p16+ subgroup is obvious. HPV−/p16+ HNSCC have a better 5-year OS than the HPV+/p16− subtype after excluding the study from Ramshankar et al.42 who found in early staged oral tongue squamous cell carcinoma patients that p16 overexpression was associated with lower survival and increased risk for disease recurrence irrespective of the HPV16 DNA status. Compared to the HPV−/p16− subgroup, 5-year OS of HPV−/p16+ HNSCC is better while HPV+/p16− is not. HPV+ in p16− HNSCC may be an innocent bystander with no functional involvement. Therefore, a careful investigation is required why HPV is negative to exclude false negative results of HPV tests or to prove truly HPV-independent development. Better survival in p16+ subgroups raises the question for its cause if independent of HPV. Consequently, in clinical trials these subtypes should be investigated separately to clarify if cancers displaying the HPV−/p16+ phenotype also qualify to be considered for de-escalation protocols.There is a discordant group of p1649. In HPV-driven tumors, high-risk HPV E7 protein triggers a cellular defense response mediated by p16 and inactivating the retinoblastoma (Rb) pathway. Therefore, p16 overexpression is an excellent biomarker for high-risk HPV-associated malignancies including cervical cancer50 and HNSCC21. In a number of premalignant lesions and non-HPV driven tumors21, however, a p16 overexpression is also present. The underlying mechanisms of p16 overexpression in these non-HPV driven tumors is currently undetermined. These tumors often harbor mutations such as RAS and BRAF51. But a study in p16+/HPV− head and neck and anogenital SCCs has shown that overexpression of p16 in these tumors is not an attribute to KRAS mutations52. It has been also suggested that deregulation of Rb or Rb loss results in increased p16 expression in tumor cells which is associated with uncontrolled cell proliferation in malignant tumors54. A recent study identified in HPV− high-grade neuroendocrine carcinomas of the head and neck an overexpression of p1655. Most of these tumors had Rb loss and a low or absent cyclin D1 expression. Therefore, we hypothesize that mechanisms other than HPV infection may affect the p16-Rb-cyclin D1 pathway and induce cell cycle activation in HPV− HNSCC. However, future studies are required to clarify the pathogenic mechanisms between these subgroups.p16 is a member of the INK4 class of cell-cycle inhibitors (INK4a) and functions as tumor suppressor. It binds to cyclin-dependent-kinases (CDK) 4 and CDK6 and prevents their association with cyclin D1, and consequently, the phosphorylation and inactivation of the retinoblastoma protein (Rb)+ tumors and p53 mutation-type staining in HPV− tumors. However, in a cervical adenocarcinoma, diffuse p16 immunoreactivity is not necessarily indicative of a high-risk HPV-associated tumor56. p16INK4A enhances the transcriptional and the apoptotic functions of p53 through DNA-dependent interaction57. Therefore, we hypothesize that in the HPV−/p16+ subgroup, which are E6 negative, the p53 is presumably mutated and this subgroup represents the HPV independent cases.As p53 is a key event in transformation and not directly associated with p16, p53 wild-type has been shown in HPVet al.32 discussed that HPV testing was performed regardless of DNA quality which may have revealed a high false negative rate in DNA-based HPV detection methods (PCR and ISH). DNA degradation does not have any effect on p16 IHC testing results32. To determine the HPV status, Zafereo et al. presented an algorithm of p16 immunohistochemistry and HPV ISH and PCR58. Ou et al. demonstrated an algorithm consisting of two PCR assays and p16 immunohistochemistry59. Prigge et al. demonstrated in a meta-analysis the high sensitivity but only moderate specificity of p16INK4a and HPV DNA PCR when used as single tests to detect a transforming HPV infection in OPSCC. However, by combining the two tests, specificity was significantly optimized without altering the sensitivity6.The prevalence of HPV-associated HNSCC potentially depends on the sensitivity and specificity of the detection method. Therefore, the detection technique for HPV may be another cause for discordant results in HPV/p16 testing. Therefore, we analysed the data separately according to the HPV-detection method used. HPV detection by PCR and by ISH only (without PCR) were used as HPV detection methods in 20 and 5 studies, respectively. Sensitivity analysis showed that the results of the 5-year OS were comparable using both HPV detection methods. Evans 64. Immunohistochemical evaluation of E6/E7 oncoprotein expression is another method for HPV detection which is independent from RNA or DNA degradation65. False negative results in HPV PCR testing may be caused by gene losses when L1 targets were used (15 of 20 studies testing for HPV DNA). These gene losses can cause the PCR to be negative, even though HPV is present . For E6/E7 targets this is not the case. However, studies using E6/E7 targets also detected patients belonging to the HPV−/p16+ subgroup46. Therefore, the HPV−/p16+ subgroup should be tested using the uniform high-sensitivity method for E6/E7 expression.During HPV infection, the HPV E6/E7 oncogenes are expressed at low levels. Sensitive techniques such as qPCR detect HPV E6/E7 transcripts despite very limited expression64. To identify a truly driven HPV-infection of OPSCC, the RNAscope HPV-test showed comparable results with p16-based algorithms combined with HPV PCR or HPV ISH. The RNAscope HPV-test performed better than p16 alone66. The quality of the results using mRNA detection are still controversial67. As a first step to analyze causal oncogenic HPV involvement, the detection of viral DNA is more practicable as viral RNA is sensitive to degradation68.Detection of E6 and E7 mRNA expression is highly associated with p16 expression71. Other HPV types than genotype 16 may explain the identified subgroups if HPV tests have a restricted genotype spectrum. Most studies investigated multiple HPV types, however the number of investigated HPV types varied. Some studies investigated only p1637. After excluding studies which tested only individual HPV types or used HPV detection methods with low sensitivity, we confirmed the distinct 5- year OS of the HPV−/p16+ subgroup to be inferior compared with the HPV+/p16+ subgroup and superior compared with the HPV−/p16− subgroup. Thus, the HPV−/p16+ subgroup should be tested for all possibly involved HPV genotypes. In addition, the HPV−/p16+ subgroup may be caused by HPV independent mechanisms.In about 90% of HPV-associated OPSCC, the high-risk-HPV-type 16 is found+ OPSCCs have similar survival benefits in Brazil (GENCAPO study), the US (CHANCE study), and Europe (ARCAGE study)72. The present sensitivity analysis showed comparable results for the 5-year OS in Europe, the US, and Asia. Therefore, a geographic differentiation of the study origin is not necessary. As anti-smoking campaigns and their success differ in these countries overall there is a difference in the portion of the HPV positive HNSCC. However, HPV+ patients with tobacco consumption have to be distinguished from HPV+ non-smokers75 because the true etiology may be drug-associated and HPV an innocent bystander infection.HPV72. Chung et al.40 found that p16+ non-OPSCC have better outcomes compared to the corresponding patients with p16− non-OPSCC. Salazar et al.37 found no survival benefit for non-OPSCC in p16+ patients. However, when both p16 and HPV DNA were considered, concordantly positive non-OPSCC had significantly better survival. There were not sufficient data to perform a meta-analysis for non-OPSCC in the present study, therefore, a definitive conclusion cannot be drawn at this time.The prognostic utility of HPV among non-oropharyngeal-derived HNSCC is limited. The effect of HPV16/p16 was significantly different in non-OPSCC compared with OPSCC+ cancers and trials evaluating de-escalation protocols, HPV detection that includes detection of activity by measuring mRNA and protein of HPV oncogenes is an important step to correctly interpret data: HPV oncogene expression is prognosis relevant while HPV DNA as a bystander is not. One example for such a misinterpretation that is under current circumstances possible is p16+HNSCC with HPV bystander infection that results in a HPV+/p16+ phenotype which would possibly be undertreated in a de-escalation protocol. Therefore, as an ideal detection method for HPV-associated/driven cancers we propose the following algorithm for detection: perform IHC to determine the p16-status and if the staining is positive, determine positivity for HPV-oncogene mRNA or protein.After the recent introduction of a specific TNM system for HPV+ needs to be avoided. It remains important to elucidate the risk for progression and therapy failure especially for the HPV−/p16+ subgroup and potentially HPV false negative patients to prevent erroneous classification of patients for downscaling treatment by de-escalation-therapy. Additionally, re-examining patient’s specimens in a cluster by testing HPV-oncogene mRNA or protein can identify additional epidemiologic links between HPV-driven and non-HPV-driven tumors.In conclusion, the information obtained from our meta analysis has revealed a potential new biologic subtype of HNSCC. From a research and clinical perspective, recruiting HPV-driven patients would be critical for the success of clinical trials towards redirecting the treatment of those patients. Further undertreatment in cases of bystander HPV and HPV-independent p16


Vascular smooth muscle cells (VSMCs) are highly phenotypically plastic, and loss of the contractile phenotype in VSMCs has been recognized at the early onset of the pathology of a variety of vascular diseases. However, the endogenous regulatory mechanism to maintain contractile phenotype in VSMCs remains elusive. Moreover, little has been known about the role of the mitochondrial bioenergetics in terms of VSMC homeostasis. Herein, we asked if glycoprotein COMP (Cartilage oligomeric matrix protein) is involved in mitochondrial bioenergetics and therefore regulates VSMCs homeostasis. By using fluorescence assay, subcellular western blot and liquid chromatography tandem mass spectrometry analysis, we found that extracellular matrix protein COMP unexpectedly localized within mitochondria. Further mitochondrial transplantation revealed that both mitochondrial and non-mitochondrial COMP maintained VSMC identity. Moreover, microarray analysis revealed that COMP deficiency impaired mitochondrial oxidative phosphorylation in VSMCs. Further study confirmed that COMP deficiency caused mitochondrial oxidative phosphorylation dysfunction accompanied by morphological abnormality. Moreover, the interactome of mitochondrial COMP revealed that COMP interacted with prohibitin 2, and COMP–prohibitin 2 interaction maintained mitochondrial homeostasis. Additionally, disruption of COMP–prohibitin 2 interaction caused VSMC dedifferentiation in vitro and enhanced the neointima formation post rat carotid artery injury in vivo. In conclusion, COMP–prohibitin 2 interaction in mitochondria plays an important role in maintaining the contractile phenotype of VSMCs by regulating mitochondrial oxidative phosphorylation. Maintaining the homeostasis of mitochondrial respiration through COMP–prohibitin 2 interaction may shed light on prevention of vascular disease. In mature blood vessels, VSMCs exhibit a “contractile” or “differentiated” phenotype characterized by the expression of contractile markers, including smooth muscle myosin heavy chain (SM-MHA), SM α-actin, SM-22 and calponin. However, under certain circumstances, VSMCs can undergo phenotype transition toward a non-contractile or dedifferentiated phenotype characterized by reduced expression of contractile markers and increased capacity for cell proliferation, migration, or secretion of various extracellular matrix (ECM) proteins and cytokines. Accumulating evidence has suggested that phenotypic transition of VSMCs occurs at the early onset of the pathology of several vascular diseases, including atherosclerosis, post-injury restenosis, vascular calcification and aortic aneurysms5. To date, compelling studies have identified the contribution of growth factors, mitogenic cytokines, reactive oxygen species, hypoxia, stretch and injury to cell dedifferentiation9. Recent studies have also revealed the importance of CArG-SRF-myocardin-dependent transcriptional mechanisms and epigenetic controls in the regulation of VSMC differentiation10. Nevertheless, how quiescent VSMCs maintain the differentiated state upon environmental cues is much less understood.Vascular smooth muscle cells (VSMCs) are of high plasticity and can undergo reversible changes in phenotype in response to alterations in local environmental cues11. Until recently, little has been known about the role of the bioenergetics of VSMC mitochondria in terms of vascular function and disease. The bioenergetics of human VSMCs via mitochondrial metabolism mainly relies on oxygen-consuming mitochondrial oxidative phosphorylation (OXPHOS) due to high efficiency of ATP production, whereas glycolysis is also involved13. It has become increasingly evident that under certain disease conditions, there is damage to the mitochondria that limits their ability to generate ATP via oxidative phosphorylation, leading to mitochondrial dysfunction. Subsequently, mitochondrial dysfunction causes the overproduction of reactive oxygen species (ROS), mitochondrial DNA damage, aberrant mitochondrial dynamics and disturbed calcium homeostasis, ultimately contributing to cardiovascular diseases such as atherosclerosis17. A few studies have shown that during aortic VSMC dedifferentiation induced by serum or PDGF-BB, mitochondria undergo metabolic reprogramming and decreased ATP production18. Moreover, complex I dysfunction underlies the oxidative phosphorylation-to-glycolysis switch in pulmonary hypertensive smooth muscle cells19. However, the definitive role of mitochondrial respiration in the VSMC phenotypic transition and the underlying mechanism have been little studied.Mitochondria are the energy powerhouses for ATP production and are particularly abundant in muscle cells, including human VSMCs21. We recently also demonstrated that COMP negatively regulates atherosclerosis and lesional calcification formation via direct interaction with integrin β322. In the current study, we identified an unexpected intracellular localization of COMP and a role of the COMP–prohibitin 2 interaction in the maintenance of mitochondrial homeostasis and the regulation of VSMC phenotype switching. The prohibitins are highly conserved proteins in eukaryotic cells that are present in multiple cellular compartments including mitochondrion, cytoplasm and nucleus of a variety of cells24. In the mitochondrial inner membrane, prohibitin 1 and prohibitin 2 form a ring-like structure, thus maintains mitochondrial genome stability, mitochondrial dynamics and promotes mitochondrial respiratory chain complex assembling28. So far, little is known about the role of prohibitin 2 in VSMC homeostasis.Cartilage oligomeric matrix protein (COMP), a 524-kDa pentameric noncollagenous glycoprotein, is an extracellular matrix protein found in both the musculoskeletal and cardiovascular systems. Our recent studies have demonstrated that COMP plays key roles in the maintenance of VSMC homeostasis. COMP maintains the contractile phenotype of VSMCs via integrin α7β1 and prevents osteochondrogenic transdifferentiation of VSMCs by directly binding to BMP-2, which inhibits post-injury neointima formation and vascular calcification respectivelyAntibodies against COMP, TGN46, ERp5, SM22, calponin, ATP synthase subunit β, β-tubulin, α-actin and β-actin used for the western blot analysis, were purchased from Abcam . Antibodies against GAPDH, His were obtained from Cell Signaling Technology . Antibodies against prohibitin 1, prohibitin 2 and eIF-5 were purchased from Santa Cruz Biotechnology . IRDye-conjugated secondary antibodies for western blotting were purchased from Rockland, Inc. . JC-1, Mito-Tracker and antibody against COMP used for confocal fluorescence microscopy were purchased from Thermo Fisher Scientific . Mito stress kit and cell energy phenotype kit were purchased from Agilent Seahorse XF Technology . PGC-1α plasmid was kindly provided by Prof. Changtao Jiang from Peking university. Other reagents were obtained from Sigma-Aldrich  unless specified.−/− mice in the C57BL/6J background were kindly provided by Professor Äke Oldberg 29. Eight-week-old male and female COMP−/− mice and their wild-type (WT) littermates were used for VSMC isolation. All animal studies followed the guidelines of the Animal Care and Use Committee of Peking University.COMP30. All steps were performed at 0–4 °C. Briefly, cells (5 × 107) were collected and washed twice with cold PBS. The cells were subsequently washed with 10 to 20 ml of TD buffer . Following centrifugation, the pellet was resuspended in 10 ml of MgRSB buffer  and incubated for 10 min to swell. Swollen cells were collected in a glass homogenizer with 40 strokes, when ~70% of the cells were disrupted. To stabilize the mitochondria, 2.5× mannitol-sucrose buffer was added to a final concentration . Following the removal of the nuclei , the mitochondria were pelleted from this supernatant at 15,000 r.p.m. for 20 min. Following resuspension in 5 ml of mannitol-sucrose buffer, the pellet was laid over a discontinuous sucrose gradient consisting of 15 ml of 1.0 M sucrose, 5 mM EDTA, and 10 mM Tris, pH 7.5, on top of 15 ml of 1.5 M sucrose, 5 mM EDTA, and 10 mM Tris, pH 7.5, and then was centrifuged at 4 °C for 30 min at 22,000 rpm. The components at the 1.0–1.5 M sucrose interface were collected as mitochondria.Mitochondria were isolated by sucrose density gradient centrifugation according to a well-established protocol31. Cells and mouse tissue extracts that contained equal amounts of total protein were resolved by 10 or 12% SDS-PAGE and were subsequently transferred onto nitrocellulose or PVDF membranes. The membranes were blocked with 5% milk in TBST, followed by incubation with primary antibody at 4 °C overnight. Following 1 h of incubation with IRDye-conjugated secondary antibodies , the membranes were analyzed using an Odyssey infrared imaging system  to detect the immunofluorescence signal.Western blotting was performed as previously described32. All steps were performed at 0–4 °C. Briefly, mitochondria were suspended with 40 µl of T10E20 buffer  and were subsequently incubated with digitonin . Three volumes of 250 mM sucrose in T10E20 buffer were added. Following centrifugation at 10,000 × g for 15 min, the inner mitochondrial membrane and matrix of the mitochondria were precipitated, whereas the outer membrane and intermembrane components were in the supernatant.Approximately 1 mg of mitochondria was sufficient for subfractionation as describedThe mitochondrial and residual cytoplasmic fractions were isolated from HEK293A cells transfected with the full-length COMP plasmid as previously described, and the lysates were incubated with anti-COMP antibody. The precipitated proteins were denatured with 2× SDS sample buffer at 95 °C for 5 min and were subsequently loaded onto 8% gradient gels. Following coomassie brilliant blue staining, the protein bands present exclusively (110 kDa) were excised respectively. The N-terminus of each protein in the mitochondrial and non-mitochondrial cytoplasmic bands was labeled with a dimethyl moiety before mass spectrometric analysis, and each N-terminus of the trypsin post-digestive peptide fragment was sequenced for analysis.34. To remove endogenous mitochondria, VSMCs at 80% confluence in culture were either maintained in DMEM as control or treated in DMEM supplemented with 110 µg/ml sodium pyruvate, 50 µg/ml uridine, and different concentrations of ethidium bromide , respectively. After 2 weeks, mtDNA copy number was detected to verify removal efficiency. Then the VSMCs were incubated with 2.35 × 108/ml GFP-labeled exogenous mitochondria prepared from scrambled siRNA or COMP siRNA-transfected VSMCs at 37 °C for 24 h. The transplantation of exogenous mitochondria was evaluated and quantified by live cell imaging using mitochondria-targeted GFP as tracer. The oxygen consumption rate measurement was performed to detect the function of transplanted mitochondria.Mitochondria were transplanted as previously described−/− mice, and the mRNA was extracted from 5 × 106 cells isolated from 6 mice per sample using Trizol reagent . The RNA quantity and quality were measured using a NanoDrop ND-1000 spectrophotometer, and the RNA integrity was assessed using standard denaturing agarose gel electrophoresis. The microarray experiments were performed using Kang Chen Bio-technology Corp  according to the standard protocol in three independent repeats. A Mouse DNA Array  was used to compare the gene expression between WT and COMP−/− VSMCs.Primary aortic VSMCs were isolated from WT and COMPp value of t test between the two groups were corrected by false discovery rate (FDR). Then a volcano plot was introduced to display log-fold-change against −log10 from the t test. The cutoff of p value is 0.01. Differentially expressed genes were identified through fold change filtering . Pathway Analysis (KEGG) and GO analysis were used to analyze the functions of the differentially expressed genes.Briefly, the total RNA from each sample was used for labeling and array hybridization via the following steps: (1) reverse transcription using Invitrogen SuperScript ds-cDNA synthesis kit; (2) ds-cDNA labeling using the NimbleGen one-color DNA labeling kit; (3) array hybridization using the NimbleGen Hybridization System, followed by washing with the NimbleGen wash buffer kit; and (4) array scanning using the Axon GenePix 4000B microarray scanner (Molecular Devices Corporation). Scanned images (TIFFs) were subsequently imported into NimbleScan software (Version 2.5) for grid alignment and expression data analysis. The expression data were normalized using quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software. All gene level files were imported into Agilent GeneSpring GX software (Version 11.5.1) for further analysis. Genes that were expressed at significantly different levels between the groups were identified through volcano plot filtering. Briefly, the 35. Rat VSMCs were seeded in XF24 culture plates at a density of 20,000 cells per well overnight and were cultured with DMEM that contained 10% FBS. The direct measurement was recorded as the baseline OCR, followed by the addition of oligomycin (2 μM) to measure the ATP-linked OCR, and the oxidative phosphorylation uncoupler FCCP (2 μM) to indicate the maximal respiration. Finally, rotenone (1 μM) and antimycin A (1 μM) were injected to determine non-mitochondrial respiration. The operations followed the manufacturer’s protocol. The OCR was normalized by the amount in 100 μg of cellular protein in each well.The OCRs were measured as previously described using an XF24 Extracellular Flux Analyzer (Seahorse Biosciences)An adenovirus for full-length mouse COMP  was constructed and amplified according to the manufacturer’s protocol (BD Biosciences). An adenovirus that carried GFP (Ad-GFP) was used as a negative control. Cells cultured at ~80% confluence were infected with recombinant adenovirus (50 multiplicity of infection) for 48 h.The cDNA fragment that encoded full-length rat prohibitin 2 (NCBI Reference sequence: NM_001013035.1) was cloned into the BamHI/XbaI sites of the pcDNA3.1 plasmid. The full-length COMP was subcloned into an C-terminal RFP vector for expression in primary mouse vascular smooth muscle cells. The full-length COMP coding sequence and fragments that encoded the 4 functional domains of mouse COMP  repeat domain , type III repeat domain , and C-terminus ) were subcloned into an N-terminal FLAG vector for expression in COS-7 cells. The full-length prohibitin 2 coding sequence and fragments that encoded the 3 functional domains of mouse prohibitin 2  domain , and C-terminus ) were subcloned into an N-terminal 6 × His vector for expression in HEK293A cells. In vitro plasmid transfection was performed using jetPEI . The transfection procedures followed the manufacturers’ instructions and all the primers used for subcloning are shown in Supplemental Table Male Sprague–Dawley rats (210–230 g) were used for the carotid artery injury model. All studies followed the guidelines of the Animal Care and Use Committee of Peking University. Briefly, rats were anesthetized by intraperitoneal injection of chloralhydrate (300 mg/kg). A 1.5-mm diameter balloon catheter  was introduced through the left external carotid artery and distended then pulled back with constant rotation. This procedure was repeated for 5 times. Contralateral carotid arteries served as sham controls.10 pfu of adenovirus dissolved in 30% pluronic gel solution were perivascularly delivered to the carotid arteries post balloon injury as described previously21.The prohibitin 2 dominant-negative fragment  was subcloned into an adenovirus vector pAdTrack-CMV. Subsequently, the recombinant adenovirus plasmids pDC316-mCMV-prohibitin 2-DN were transfected into HEK293A cells to yield the final expression clone Ad-prohibitin 2-DN. Ad-prohibitin 2-DN was amplified by infecting HEK293A cells and purified by PD-10 Sephadex  precipitation. An adenovirus vector carrying green fluorescence protein (Ad-GFP) was applied as negative control. For in vivo studies, 1 × 10t test for two-group comparisons and ANOVA for more than two-group comparisons if evaluation of similar variances was passed. Nonparametric tests were used where data were not normally distributed. In all cases, statistical significance was concluded where the two-tailed probability was less than 0.05. Briefly, paired two-tailed Student’s t test was applied for the comparisons of the protein expression and mitochondrial membrane potential between the VSMCs transfected with scrambled siRNA and COMP siRNA, whereas unpaired two-tailed Student’s t-test was applied for the comparisons of the percentages of ATP production and amount of mtDNA between the VSMCs transfected with scrambled siRNA and COMP siRNA, as well as the comparisons of the mRNA levels between the WT and COMP−/− VSMCs, the comparation of neointima area, neointima/media ratio, circumference of EEL, the media area between Ad-GFP and Ad-prohibitin 2-DN infected rat carotid arteries. The comparisons of the protein expression among more than 2 groups by western blot were analyzed using one-way ANOVA followed by the Student–Newman–Keuls test for post-hoc comparison. The comparisons of the OCRs between the VSMCs treated with scrambled siRNA or COMP siRNA and control or COMP-deficient mitochondria as well as vector or prohibitin 2-DN plasmid were analyzed by unpaired two-tailed Student’s t test. Specific statistical analysis used in each experiment was described in detail in figure legends.All the results of the experimental studies are expressed as the mean ± SD. Statistical analysis was performed using GraphPad prism 6.0 software . Whether data are normally distributed was first evaluated. Then Brown-Forsythe test was performed for checking similar variances among normally distributed data followed by Student’s 20. Whether this is true in COMP−/− VSMCs was not explored. In the current study, we isolated primary VSMCs from COMP−/− and wild-type mice. COMP−/− VSMCs exhibited the decreased levels of contractile proteins, including α-actin, calponin and SM22 as well as morphological alteration to polygonal-shaped cells, which was rescued by the ectopic expression of COMP with red fluorescence protein labeled COMP (COMP-RFP) plasmid  software, the N-terminus (residues 12–23) of COMP is predicted to be responsible for mitochondrial targeting (data not shown). To investigate the definite sequence of mitochondrial COMP and cytoplasmic COMP, we subcloned the full-length of COMP and transfected it into HEK293A cells. Mitochondrial and mitochondria-free cytoplasmic fractions were isolated followed by immunoprecipitation. After Coomassie brilliant blue staining, the protein band of COMP (between 100 and 130 kDa) in each fraction was excised, and the N-terminus was labeled with a dimethyl moiety, followed by subjection to LC-MS/MS analysis . Similar results were obtained , reduced mitochondrial DNA (mtDNA) number, reduced autophagic flux and mitochondrial morphological changes. We firstly measured the mitochondrial membrane potential (△Ψm) using JC-1 dye staining. COMP silencing consistently decreased the intracellular red fluorescence signaling but enhanced green fluorescence signaling in the VSMCs, suggesting that △Ψm was significantly attenuated in the COMP-silenced VSMCs Fig. . Additio38. Decreased fusion or increased fission results in mitochondrial fragmentation, an increase in the number of abnormal mitochondria and VSMC proliferation39. COMP-silenced VSMCs exhibited substantial mitochondrial fragmentation, as indicated by Mito-Tracker staining  and fusion , which maintains mitochondrial homeostasising Fig. .Together, the data showed that COMP deficiency impaired mitochondrial oxidative phosphorylation and caused mitochondrial dysfunction.12. We first examined the cell energy phenotype and mitochondrial respiration rate of VSMCs under different differentiation states upon PDGF-BB or TGF-β stimulation. PDGF-BB-induced VSMC dedifferentiation paralleled with decreased respiration, whereas the TGF-β-driven contractile phenotype of VSMCs exhibited enhanced mitochondrial respiration  in rats  domain to block the binding between COMP and prohibitin 2  damage and progressive respiratory chain dysfunction50. However, the molecular link of mitochondrial dysfunction to vascular diseases is not fully understood. We previously identified COMP as an important modulator in maintaining vascular homeostasis. COMP−/− mice are more susceptible to post-injury restenosis, atherosclerosis, vascular calcification and thrombosis formation52. COMP-degrading proteinase ADAMTS-7 has been demonstrated to be involved in the pathogenesis of atherosclerosis by both human genome-wide association studies and in vivo mouse models53. Herein, we revealed an unexpected intracellular distribution of COMP and its undisputable role in the maintenance of mitochondrial homeostasis in VSMCs. COMP deficiency led to impaired mitochondrial respiration and ATP production and systemic mitochondrial dysfunction, which may also be involved in the aberrant vascular phenotype in COMP−/− mice.Mitochondria not only are the powerhouses for ATP production but also play critical roles in ROS generation, inflammation, metabolism and apoptosis54. In addition, Prohibitin 2 deficiency inhibits the activities of complex I, II and IV in mitochondrial respiratory chain25. Although the mRNA levels of mitochondrial respiration complex subunits  were significantly dowregulated by COMP deficiency, whether COMP regulates mitochondrial respiration complex activity needs to be further studied. Prohibitin 2 is also associated with many critical mitochondrial proteins, including components of the mitochondrial respiratory chain, mitochondrial structure and cristae formation, mitochondrial transporters, membrane translocases, mitochondrial-mediated translation, and mitochondrial apoptosis56. A recent study also revealed prohibitin 2 as an inner membrane mitophagy receptor57. In the current study, we found that COMP and prohibitin 2 are functionally interdependent in the regulation of mitochondrial respiration via direct interaction. Whether the COMP–prohibitin 2 interaction affects other mitochondrial structures and functions needs to be further explored. In addition, prohibitin 2 is ubiquitously expressed in many other tissues, such as the heart58. The mitochondrial COMP–prohibitin 2 interaction may not be limited to VSMCs. We did observe COMP expressed in the mitochondria in cardiomyocytes (data not shown), and COMP deficiency caused spontaneous dilated cardiomyopathy59. Whether the COMP–prohibitin 2 interaction in the heart plays essential roles in mitochondrial function and heart failure requires further investigation.Of interest, our proteomics analysis identified a mitochondrial binding partner of COMP, prohibitin 2. COMP deficiency shares similar identities with prohibitin 2 deficiency in mitochondrial dysfunction, as previous studies revealed that lack of prohibitin 2 impaired oxidative phosphorylation, reduced mtDNA copy number and attenuated mitochondrial membrane potential11. Aging cells or PDGF-BB-treated dedifferentiated VSMCs have lower oxidative phosphorylation and reduced reserve capacity18. Moreover, a metabolic shift from oxidative phosphorylation to glycolysis and substrate selectivity between glucose and fat oxidation were observed in synthetic VSMCs60. Recent research reported lactate could promote synthetic phenotype in VSMCs61.This process was similar to the Warburg effect present in cancer cells that is known to favor glycolysis even when well oxygenated. Our study not only provides further direct evidence that the manipulation of mitochondrial respiration led to VSMC phenotypic transition but also reveals maintaining mitochondrial respiration may inhibit VSMC phenotype switching and alleviate vascular diseases. However, how the alteration of mitochondrial metabolism contributes to the VSMC phenotypic transition needs further investigation.Another interesting finding of our study is that mitochondrial bioenergetics regulated by COMP–prohibitin 2 directs VSMC phenotypic transition. Until recently, little has been known regarding mitochondrial respiration in terms of VSMC function. Interestingly, direct measurement of the cellular bioenergetics of human coronary artery smooth muscle revealed that VSMCs appear to be very similar to cardiac and skeletal muscle cells in terms of oxidative phosphorylation capacity, although VSMCs have a lower mitochondrial content64. To the best of our knowledge, this is the first study to demonstrate that COMP intracellularly resides in an organelle and plays a role in mitochondrial function under physiological conditions. Previously, mutations in the human COMP gene have been linked to the development of two inherited chondrodysplasia and osteoarthritic phenotype, pseudoachondroplasia and multiple epithelial dysplasia, because of aberrant COMP retention in the ER and subsequent ER stress62. Of interest, TSP-4, which is also a thrombospondin family member, has been reported as an intracellular adaptive ER stress response effector via binding with the ER luminal domain of activating transcription factor 6a (ATF6a)65. More intriguingly, MMP-12, an extracellular matrix metalloproteinase, is transported into virus-infected cells and further translocated into the nucleus, where it acts as a transcription factor66. The notion that the same protein with different subcellular localizations plays various roles in cell behavior highlights the complexities of the ECM superfamily proteins.A surprising finding in this study is the localization of the ECM protein COMP in the mitochondria. COMP belongs to the thrombospondins (TSP) family and is also referred to as TSP-5. Accumulating evidence has indicated that COMP, as an extracellular protein, exerts its functions in both the musculoskeletal and cardiovascular systems by binding to other ECM proteins , mitogens , membrane proteins  or thrombin20. Moreover, we also identified COMP degradation by metalloproteinase ADAMTS-7 during vascular injury or calcification67. How mitochondrial COMP level was regulated needs further exploration.Our previous study showed the COMP is unregulated by serum starving or TGF-β and repressed by PDGF-BB or serum supplement in VSMCs during phenotype switchingThus, COMP deficiency aggravates VSMC phenotypic switching, at least in part, by disrupting mitochondrial homeostasis maintained by the interaction between COMP and prohibitin 2. Increasing endogenous COMP or improving mitochondrial respiration may help maintain the VSMC contractile phenotype to inhibit several cardiovascular diseases.Supplemental materialSupplementary figure legends