AR) gene.Only approximately 85% of patients with a clinical diagnosis complete androgen insensitivity syndrome and less than 30% with partial androgen insensitivity syndrome can be explained by inactivating mutations in the androgen receptor  and male controls.APOD) in cultured genital fibroblasts (GFs) (APOD assay) and next-generation sequencing of the complete coding and noncoding AR locus.Quantification of DHT-dependent transcriptional induction of the AR target gene apolipoprotein D , and previously undiagnosed DSD including patients with a clinical suspicion of AIS (n = 46).GFs from 169 individuals were studied encompassing control males (n = 68), molecular defined DSD other than androgen insensitivity syndrome , There were no interventions.APOD expression in cultured GF and AR mutation status in 169 individuals was measured.DHT-dependent APOD-induction; 100% sensitivity, 93.3% specificity, P < .0001). Of 46 DSD individuals with no AR mutation, 17 (37%) fell below the cutoff, indicating disrupted androgen signaling.The APOD assay clearly separated control individuals  from genetically proven AIS (cutoff < 2.3-fold AR mutation-positive AIS can be reliably identified by the APOD assay. Its combination with next-generation sequencing of the AR locus uncovered an AR mutation-negative, new class of androgen resistance, which we propose to name AIS type II. Our data support the existence of cellular components outside the AR affecting androgen signaling during sexual differentiation with high clinical relevance. The use of Apolipoprotein D as a biomarker for androgen sensitivity identifies a new type of androgen insensitivity syndrome that is not associated with a mutation in the androgen receptor gene. AR gene. AIS is often suspected to be a common cause of DSD in a 46,XY individual and may be associated with complete feminization of the external genitalia due to a complete lack of AR transcriptional activity (complete AIS [CAIS])  –3. Andro–APOD) has been reported to exhibit the most significant induction upon DHT treatment. APOD is a direct transcriptional target of the AR  in more than 85% of cases, the clinical diagnosis of PAIS is more difficult, and, in addition, less than 30% of cases that are clinically suspected of PAIS are associated with a mutation in the AR  (APOD assay) enabled us to discover a new AR mutation-negative class of androgen resistance, which we propose to name AIS type II.To understand the possible coexistence of androgen resistance without any genetic evidence of a defect in the The study was performed in agreement with the vote of the Ethical Committee of the Medical Faculty of the Christian-Albrechts-University . GFs received from collaborating partners were included in this study according to the recommendations of the local ethical committees. All GFs included in this study were double encrypted and numbered from 1 to 169.The GFs herein analyzed belonged to four major clinical groups.The first group (group 1) was established in collaboration with local urologists and pediatric surgeons and includes scrotum-derived control GFs from fertile adult patients with normal virilization of the external genitalia, who underwent vasectomy (n = 30). We included scrotal biopsies of patients under the age of 18 years who underwent orchidopexy due to maldescended testes (n = 13) with normal external genitalia, ie, no hypospadias. In addition, we used control foreskin fibroblasts from patients who underwent circumcision due to cultural reasons or phimosis (n = 25). Genomic DNA of all male control GF cultures was sequenced using our custom haloplex next-generation sequencing (NGS) panel including up- and downstream sequences, untranslated regions, and the introns A.NR5A1) (n = 2), the 17α-hydroxylase gene (CYP17A1) (n = 2), the 17β-hydroxysteroid-dehydrogenase type III gene (HSD17B3) (n = 4), and the 5α-reductase type II gene (SRD5A2) (n = 8) in conjunction with ambiguous or female external genitalia. Biopsies were taken from either labioscrotal or foreskin/labia minora tissue. We added GFs from female  individuals with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CYP21A2) (n = 2). Genomic DNA derived from the GF cultures of the second group was sequenced via the custom haloplex NGS AR panel.The second group consists of GFs from previously characterized 46,XY DSD individuals with a defined molecular diagnosis other than AIS (group 2). In particular, these individuals carried mutations in the steroidogenic factor 1 gene . All GFs that revealed an AR-CDS mutation via the custom haloplex NGS AR panel were validated by Sanger sequencing.The third group contains labioscrotal and foreskin/labia minora-derived GFs with a genetic proof of AIS, in whom mutations in the Supplemental Tables 1 and 2). This group may also contain individuals with a yet-undiagnosed form of DSD other than AIS. All GFs of group 4 were sequenced through our custom haloplex NGS AR panel. If an AR-CDS mutation was detected by NGS, Sanger sequencing was used for confirmation and the GFs were subsequently reallocated to group 3.The fourth group was compiled from a collection of labioscrotal and foreskin/labia minora-derived GF samples from 46,XY DSD individuals without an established molecular diagnosis (group 4). It includes individuals with apparently unaffected androgen biosynthesis based on available hormone data supporting a clinical suspicion of androgen resistance (n = 46). When available, data on the external genital appearance, basal and stimulated T levels (HCG test), and measurements on AR ligand binding  were collected , there were also no AR-CDS or intron-exon boundary mutations. In all classical AIS individuals  previously identified by Sanger sequencing, mutations in the AR-CDS could be validated by our custom NGS AR panel, underlining the validity of the NGS approach. All those individuals in group 4 with previously undiagnosed forms of DSD in whom we identified a mutation in the AR-CDS by NGS (n = 9) were reallocated to group 3. In the remaining GF samples of group 4, including DSD samples in which AIS was suspected (n = 46), neither mutations in the AR-CDS nor in the intron-exon boundaries could be detected. A schematic representation of all four groups is shown in AR-CDS mutations within group 3 is schematically shown in Supplemental Table 4. Eight AR-CDS mutations are not currently listed in the AR mutation database . Outside the coding region, numerous nonannotated single-nucleotide polymorphisms (SNPs) were found in all four groups. A distribution of those SNPs is shown in In the group of male controls (group 1), no mutations were detected in the coding sequence (CDS) and the intron-exon boundaries of the database  and, to APOD (APOD assay). Male control scrotum-derived GFs from group 1  showed a mean DHT-mediated APOD induction of 3.5-fold (SD 0.85), defining the normal range of transcriptional function of the AR in this group  (P < .001). CAIS-derived GFs showed on average no induction (0.96), whereas PAIS-derived GFs demonstrated an average induction of 1.62 .We now functionally characterized all 169 sequenced GF by measuring the DHT-triggered ability of the AR to induce transcription of its target gene is group A. Scrotuse cells A. Only osistance . In cont of 1.62 A. This cAR-CDS mutation positive AIS) of 2.36-fold APOD induction. Hence, a DHT-mediated APOD induction less than 2.36 distinguishes genetically proven AIS from other molecular-defined DSDs with a sensitivity of 100% and a specificity of 93.33% .From the clinical perspective, it is of much greater relevance to distinguish AIS from other forms of DSD rather than from clinically unsuspicious male controls. We calculated a cutoff between labioscrotal-derived GFs from group 2 (molecular defined DSD other than AIS) and group 3 (AR-CDS mutation (group 3) . The average DHT-mediated APOD induction in CAIS was 0.97 (no APOD induction) and 1.92 in PAIS   and GFs from AIS individuals harboring an group 3) C. A cuto in PAIS B. Althougroup 1) C, no cutomal sex , A and BAPOD induction, we found a significant difference between PAIS and CAIS in both labioscrotal and labia minora/foreskin-derived tissues (P < .01). However, there was some overlap due to a few GF cultures  never showed any APOD induction and always belonged to the CAIS group (apart from the p.Leu174stop mosaic), whereas missense mutations had a variable APOD induction and were present in both PAIS- and CAIS-derived GFs .We also compared Supplemental Figure 8D).In conclusion, the APOD assay does distinguish CAIS individuals from PAIS individuals, albeit with slightly lower sensitivity (88.2%) and specificity (90%)  using the APOD assay and applied the above calculated cutoffs. Looking at labioscrotal-derived GFs, 24% (n = 8) of fibroblast cultures from group 4 fell below the cutoff of 2.29 and therefore have to be defined as functionally androgen resistant . One of the GF cultures was from an individual with the suspected clinical diagnosis of CAIS and showed strongly reduced APOD induction. In contrast, the remaining 76% (n = 25) GF cell lines showed an APOD induction above the cutoff and had an AR activity comparable with that of control groups 1 and 2 . The remaining 31% of foreskin/labia minora GF cultures (n = 4) behaved like male foreskin controls (group 1) in terms of APOD induction, and hence, androgen insensitivity can be ruled out. In summary, 17 of the 46 GFs from group 4 have functionally proven androgen resistance based on androgen-induced APOD transcription despite the absence of an AR-CDS mutation.We then analyzed the large group of  1 and 2 A. TherefAR locus but outside the AR-CDS in individuals of group 4 could potentially have influenced AR activity. Of the 17 AR-CDS-mutation-negative, androgen-resistant GFs, nine had one or more not annotated SNPs outside the AR-CDS, whereas eight had only previously annotated and clinically unsuspicious SNPs in the region covered by our NGS approach. The distribution of the nonannotated SNPs along the analyzed AR locus is shown in APOD induction could be due to a reduced AR protein expression or stability in these GFs. We checked the AR protein levels in all the 17 GF cultures and compared them with their appropriate control groups   . A lower the CDS . In concAR-CDS-negative new class of AIS, which we suggest to call AIS type II. This is not only a significant addition to the current classification of 46,XY DSDs but also a starting point for a better understanding of AR signaling, including the identification of new AR cofactors in future clinical and molecular DSD studies.Functional assays for AR activity in GFs have been described before . LackingAPOD in induction shown in this paper and in previously published DHT binding and dissociation studies  deficiency (group2) showed reduced AR activity in the APOD assay. A possible explanation could be that this individual has a defect both in DHT synthesis and in androgen action. An additional 5αRD deficiency may also be responsible for the CAIS phenotype of an individual carrying a p.Tyr781Asp mutation in the ligand binding domain of the AR because biochemical data indicate reduced 5αRD activity in the GFs of this patient (identification in C31 ). Both t studies  indicateAPOD induction with 10 nM DHT despite a clinical CAIS. Interestingly, no APOD induction was measured when using 1 nM DHT, suggesting that the CAIS phenotype might have originated from low local genital DHT concentrations during embryogenesis. This observation is supported by the literature associating this mutation with different AIS phenotypes, ranging from CAIS through PAIS to MAIS (APOD induction could be detected, well in line with the high percentage of the mutation in this PAIS cell culture. Hence, whereas the APOD assay correctly identified AIS in this situation, somatic mosaicism may influence the detected level of functional impairment, which may be in contrast to the clinical phenotype. We have previously described this phenomenon of discrepancy between molecular studies and the clinical phenotype in mosaic AIS, using other functional approaches (Also, the APOD assay did not distinguish between PAIS and CAIS in all cases. This overlap may be affected by specific functional and molecular conditions in some individual GF cultures. In GFs from a CAIS individual carrying a p.Val867Met mutation in the AR ligand binding domain, we observed residual  to MAIS . Another to MAIS , 19. In  to MAIS  present proaches . UltimatAR locus outside the AR-CDS, we could detect as-yet nonannotated SNPs within potentially regulatory regions. Some of these SNPs are potential candidates for influencing AR activity because they are paralleled by reduced AR protein expression in the corresponding GF cultures, which could explain the lower AR activity in the AIS type II individuals. Interestingly, we previously detected a mutation in the 5′untranslated region of the AR in an individual having CAIS and experimentally showed that this mutation is sufficient to strongly reduce AR protein levels and AR activity (AR-CDS. Another promising group of factors outside the AR gene region that might contribute to AIS type II are AR cofactors, which are needed for proper AR activity (AR gene was cloned in 1988 (By analyzing sequencing data of the activity . This unactivity . Numerouactivity , but a c in 1988 , 27, onl in 1988 , but thiAR-CDS-negative AIS type II cohort in comparison with the other three cohorts of this study is one of the next important experimental steps planned. Furthermore, we cannot exclude that mild functional AIS type II may play a role as secondary modifier contributing to a DSD phenotype, even in certain molecular-defined DSDs and in unknown DSDs. This is supported by previous reports documenting the existence of more than one compromised molecular factor in the same DSD individual (–AR-CDS-negative in AIS type II cohort, we found documented minor syndromic signs in 4 of 46 cases, hence 9%. In addition, prenatal conditions leading to low birth weight may have programming effects on androgen responsiveness of genital cells as a correlation of a low birth weight and a PAIS-like phenotype in individuals without an AR gene mutation has been described before (We do not yet know whether the AIS type II cohort identified in this study has a monogenic origin or whether multiple aberrant genes may contribute to this entity. Exome sequencing of the dividual –31. Accoividual –, associad before .APOD induction may be associated with a reduced future AR sensitivity during puberty and may influence clinical response to androgen treatment. Prospective data are needed to correlate APOD expression with clinical outcome parameters in affected individuals. Given the high significance of the data provided in this manuscript, the scientific community in DSD research should revisit the clinical indication of a diagnostic genital skin biopsy in specific unclear DSD cases.Currently our data are based on retrospective analyses of fibroblasts obtained from our DSD biobank, but they can nevertheless be of potential value for the clinical endocrinologist. Apart from being an explanation for the phenotypic development of a DSD individual, reduced


Detection of occlusal caries with visual examination using ICDAS correlates strongly with histology under stereomicroscopy (SM), but dentin aspects under SM are ambiguous regarding mineral content. Thus, our aim was to test two null hypotheses: SM and microradiography result in similar correlations between ICDAS and histology; SM and microradiography result in similar positive (PPV) and negative predictive values (NPV) of ICDAS cut-off 1–2 (scores 0–2 as sound) with histological threshold D3 . Occlusal surfaces of extracted permanent teeth (n = 115) were scored using ICDAS. Undemineralized ground sections were histologically scored using both SM without contrast solution and microradiography after immersion in Thoulet’s solution 1.47 for 24 h (MRC). Correlation between ICDAS and histology differed from SM (0.782) to MRC (0.511) (p = 0.0002), with a large effect size “q” of 0.49 (95% CI: 0.638/0.338). For ICDAS cut-off 1–2 and D3, PPV from MRC (0.56) was higher than that from SM (0.28) , and NPV from MRC (0.72) was lower than that from SM  . In conclusion, SM overestimated the correlation between ICDAS and lesion depth, and underestimated the number of occlusal surfaces with ICDAS cut-off 1–2 and deep dentin demineralization. Visual caries detection of the occlusal tooth surface with visual scoring systems that include early noncavitated caries lesions, like the International Caries Detection and Assessment System (ICDAS), has been satisfactorily validated with the histological depth of carious demineralization measured with the aid of stereomicroscopy (SM) –3. From Microradiography is considered as a gold-standard technique for detecting variations in mineral content in hard dental tissues ,11. AsidIn this context, differences in lesion depth measured by SM and microradiography and the implication in the validation of ICDAS should be investigated. In order to identify dentin demineralization with improved contrast, microradiography with iodide solution as a contrast solution (MRC) was used, following its use to identify larger pores in rocks . The aimThis study was approved by the ethical committee on research in humans of the Lauro Wanderley Hospital of the Federal University of Paraiba in João Pessoa, Brazil (register number 39014214.0.0000.5188).The sample consisted of extracted human premolars and permanent third molars donated by volunteers (living in an area without water fluoridation) that provided signed free informed written consent. Teeth presenting crown fracture, calculus deposits, endodontic preparation, and dental restorations were excluded. All teeth were cleaned with pumice and rotating brush, washed with distilled water and kept in 0.02% sodium azyde solution.mean = (0.58+0.68+0.54+0.43+0.54)/5 = 0.554)] reported by others [18 [3+(1240–3)/100q2 = 94). With an estimated loss of 22%, the final sample size was 115 teeth.Sample size calculation for testing the difference between correlation coefficients requires predicted values of each correlation coefficient. The predicted correlation coefficient between ICDAS and lesion depth from SM was the mean of the five values [ry others  as the oy others :q=z1−z2The second null hypotheses tested involved the difference between two proportions, and a large effect size was predicted. Using an effect size h of 0.8  . two-taiOcclusal surfaces were positioned at 25 cm from the eyes of each of two examiners, illuminated with room illumination, and examined wet and then after drying with compressed air for 5 s independently by the two examiners, who were previously trained with the e-learning program provided by the ICDAS Foundation and with 60 sample extracted teeth. The examiners applied independently the ICDAS scoring system  with no In order to select the site with the deepest lesion depth for longitudinal histological sectioning , each tooth was examined with real-time digital microradiography . The occlusal surface was positioned perpendicular to the X rays path, and the tooth was rotated to 360° so that a video file was produced. Each video was analyzed, the site of the deepest lesion depth was identified and its distance from the buccal surface measured. This procedure guided the histological sectioning of each occlusal surface.A 1 mm-thick longitudinal undemineralized section oriented mesio-distally to the tooth crown, cut with a diamond disc (0.15 mm thick) mounted in a low speed motor (with water irrigation), was prepared for each occlusal surface including the site where the ICDAS score was determined, as oriented by previous microradiographic examination. Each section received a consensus score of lesion depth according the following histological scoring system : 0 = no Each section was scored histologically with regard to lesion depth using both SM  and digital microradiography after immersion in contrast solution (MRC) for 24 h. A digital photograph of the histological aspect in each technique was obtained. The intra and inter-examiners reliability of the same two examiners were tested for each technique by analyzing 30 sections twice between an interval of 2 weeks. The final SM and MRC lesion depth scores were determined by consensus between examiners.The contrast solution consisted of a Thoulet’s solution (aqueous solution of potassium iodide and mercuric iodide) with refractive index 1.47 and pH 6.7, as determined in an Abbe refractometer and in a pHmeter. This solution was used because it has been previously shown to infiltrate natural enamel caries after 24 h of immersion at room temperature . The higThe intra and inter-examiner reliabilities on ICDAS, SM, and MRC were determined using Kappa statistics. The Spearman correlations between ICDAS and lesion depth (from SM and MRC) were calculated along with corresponding p values, 95% confidence intervals, and statistical power using the equations available elsewhere .The effect size q (difference between correlations) was calculated with Eqs We also tested the null hypothesis that the positive predictive value  and the negative predictive value  using ICDAS cut-off 1–2  with histological threshold D3 from both SM and MRC are similar. The statistical significance was tested with a Z test , and the effect size h (for the difference between proportions) and the statistical power were calculated using the methods described elsewhere .Intra and inter-examiner reliabilities were the following: for ICDAS, the intra-examiner Kappa scores were 0.97 and 0.97, and the inter-examiner Kappa score was 0.85; for SM, the intra-examiner Kappa scores were 0.82 and 0.89, and the inter-examiner Kappa score was 0.95; and for MRC, the intra-examiner Kappa scores were 0.94 and 0.91, and the inter-examiner Kappa score was 0.93.There was no sample loss in this study. The distribution of ICDAS score in relation to the histological scores from both SM and MRC are shown in The correlation between ICDAS and lesion depth had the correlation coefficients of 0.78289 and 0.51138, for SM and MRC, respectively . The difThe PPVs for ICDAS cut-off 1–2 and histological threshold D3  were the following: 0.278 using SM, and 0.555 using MRC. The corresponding NPVs were: 1.00 for SM and 0.72 for MRC. Both PPV and NPV differed statistically (p < 0.0001), with an effect size h of 0.81 for PPV and 1.58 for NPV, both with statistical power > 99.9%.The correlation of 0.783 between ICDAS and lesion depth determined with SM is consistent with previous reports in the literature using similar scoring system with eight codes (0.87)  and fiveIn histology, carious dentin is the sum of caries-infected and caries-affected dentin, with the former including demineralized infected dentin and the later including both demineralized non-infected and sclerotic  dentin . Caries The morphology of the dentin areas infiltrated with the contrast solution provide evidence that the infiltration of the contrast solution was not at random; it followed the demineralized dentinal tubules from the enamel-dentin junction to the roof of the dental pulp chamber, and the infiltrated dentinal tubules were related to the enamel surface of the site of choice on the occlusal surface for visual examination. The use of the Thoulet’s solution contributed to the histological examination with MRC by improving the identification of demineralized dentin Figs  and 2.MRC = 0.555) with a large effect size (h = 0.81), and NPV decreased (NPVMRC = 0.72), also with a large effect size (h = 1.58). The PPV for ICDAS 1–2 cut-off and D3 threshold is the proportion of carious occlusal surfaces with deep dentin demineralization, while the NPV for ICDAS 1–2 cut-off and D3 threshold is the proportion of sound occlusal surfaces with deep dentin demineralization. Our results show that the proportions of both sound and carious occlusal surfaces (detected by ICDAS cut-off 1–2) with deep dentin demineralization increased significantly when lesion depth was measured with MRC. This is consistent with the recent report of occlusal caries lesion with ICDAS score 2 showing that, when lesion depth was measured histologically, SM showed only the aspect of “sound” dentin, but deep carious dentin demineralization was observed under microradiography even without contrast solution [Both the PPV and the NPV of ICDAS cut-off 1–2 with histological threshold D3 differed significantly from SM to MRC. The low PPV of 0.278 from SM is close to the PPV of 0.34 from SM calculated with data available in the literature [solution .Visual scoring systems for dental caries are important for investigating the prognosis of tooth surfaces according to their different visual aspects, and thus contribute to treatment decisions. In a 4-yrs follow up study of occlusal surfaces with ICDAS score 0–4 in high-risk children, the surfaces that progressed to cavitated carious lesion at the shortest time interval included some with ICDAS scores 0 and 1 at the initial examination, while most had ICDAS score 4 at the initial examination . The occIn conclusion, the two null hypotheses were rejected. The correlation between ICDAS and lesion depth measured with SM was lower (with a large effect size q of 0.49) than the correlation between ICDAS and lesion depth measured with MRC. The proportions of both sound and carious occlusal surfaces diagnosed with ICDAS 1–2 cut-off presenting deep dentin demineralization detected with MRC increased significantly  compared to the same proportions when SM was used to detect dentin demineralization. This has important repercussions for the prognosis of occlusal surfaces screened by ICDAS.S1 FileThis is an Excel file containing tabulated data on sample code and ICDAS, MRC, and SM scores.(XLSX)Click here for additional data file.


In patients with disease relapse, AR positivity was significantly correlated with better prognosis . Conclusions: AR expression may be useful as a subclassification marker for prognosis in TNBC.Background: Triple-negative breast cancer (TNBC) has a poor prognosis because of frequent recurrence. Androgen receptor (AR) is involved in the pathogenesis of breast cancer, but its role is not clearly defined. The aim of this study was to explore the expression of AR and its relationship with clinicopathologic features in TNBC. Methods: This study investigated 1036 cases of sporadic invasive breast carcinoma. Immunohistochemical assays were performed to determine the expression of AR in 190 TNBC samples. The relationships between AR expression and clinicopathologic data and prognosis were analyzed. Results: In 190 TNBC cases, the prognosis of AR-positive patients was significantly better ( Breast cancer is a highly diverse disease that can be classified into subtypes comprising different clinical or cellular characteristics. It is commonly subclassified into five subtypes including luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-enriched, basal-like, and normal-like, according to the mRNA expression profile; these breast cancer types are frequently referred to as the “intrinsic subtype” ,2,3,4. THowever, recent research indicates that TNBC can be further classified according to its genetic profile. Androgen receptor (AR)-positive TNBC is one of these subtypes . AR-posiIn this study, we classified 190 cases of breast cancer with the triple-negative phenotype from 1036 breast carcinomas. We addressed the significance of clinicopathologic features and AR expression in order to identify additional prognostic markers that can help identify tumors with more aggressive behavior.This study investigated a consecutive series of 1036 patients with primary infiltrating breast cancer who underwent operations at the Osaka City University Hospital from 2000 to 2006. Additionally, 190 patients with TNBC treated at the Osaka City General Hospital were included. All of the patients who had undergone conservative breast surgery received postoperative radiotherapy to the residual breast. TNBC patients received adjuvant chemotherapy by either an anthracycline-based regimen (doxorubicin or epirubicin) or a 5-fluorouracil (5-FU)-based regimen, depending on the stage or risk of recurrence, in accordance with the National Comprehensive Cancer Network guidelines or the guidelines for breast cancer in Japan. The median follow-up time was 6.6 years . Relapse-free survival (RFS) was defined as the interval between the date of surgical removal of the primary tumor and the date at which relapse was confirmed or the date of the last follow-up (for censored patients). Cancer-specific survival (CSS) was the time, in years, from the date of the primary surgery to the time of breast cancer-related death. Tumors were confirmed histopathologically and staged according to the TNM classification. This research conformed to the provisions of the Declaration of Helsinki in 1995. All patients were informed of the investigational nature of this study and provided their written, informed consent. The study protocol was approved by the Ethics Committee of Osaka City University (#926).2 in Target Retrieval Solution . The specimens were then incubated with 3% hydrogen peroxide in methanol for 15 min to block endogenous peroxidase activity, and then incubated in 10% normal goat or rabbit serum to block nonspecific reactions.Immunohistochemical studies were performed as previously described . The tumPrimary monoclonal antibodies directed against ER , PR , HER2 , Ki67 , and AR  were used. The tissue sections were incubated with antibody for 70 min at room temperature or overnight at 4 °C , and were then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse Ig polymer as a secondary antibody (HISTOFINE (PO)™ kit; Nichirei, Tokyo, Japan). The slides were subsequently treated with streptavidin–peroxidase reagent and incubated in phosphate-buffered saline-diaminobenzidine and 1% hydrogen peroxide (v/v), followed by counterstaining with Mayer’s hematoxylin. Positive and negative controls for each marker were used according to the supplier’s data sheet.(ASCO)/College of American Pathologists (CAP) guidelines for HER2 immunohistochemistry classification for positive breast cancer if the HER2/CEP17 ratio was higher than 2.0 [Immunohistochemical scoring was performed by two pathologists who specialized in mammary gland pathology using the blind method to confirm the objectivity and reproducibility of the diagnosis. In line with previous studies, the cut-off value for ER and PR positivity was set at ≥1%, and the same cut-off was adopted for AR positivity. HER2 expression was graded according to the accepted grading system as 0, 1+, 2+, or 3+. The following criteria were used for scoring: 0, no reactivity or membranous reactivity in <10% of cells; 1+, faint/barely perceptible membranous reactivity in ≥10% of cells or reactivity in only part of the cell membrane; 2+, weak to moderate complete or basolateral membranous reactivity in ≥10% of tumor cells; 3+, strong complete or basolateral membranous reactivity in ≥10% of tumor cells. HER2 was considered positive if the grade of immunostaining was 3+, or 2+ with gene amplification via fluorescent in situ hybridization (FISH). In the FISH analysis, each copy of the HER2 gene and a reference gene  was counted. The interpretation followed the criteria of the American Society of Clinical Oncology than 2.0 . A Ki67-than 2.0 . Immunohthan 2.0 ,29,30. Athan 2.0 ,29,30.® version 19.0 statistical software package . Categorical data are reported with numbers and percentage, and continuous data as median and range. The association between TNBC and other clinicopathologic variables and the significance of different prognostic markers were analyzed using the chi-squared test (or Fisher’s exact test when necessary). Association with survival was analyzed by the Kaplan-Meier plot and log-rank test. The Cox proportional hazards model was used to compute univariate and multivariate hazard ratios (HRs) for the study parameters with 95% confidence intervals (95% CIs). In all of the tests, a p-value of less than 0.05 was considered statistically significant. Cutoff values for different biomarkers included in this study were chosen before statistical analysis.Statistical analysis was performed using the SPSSp < 0.001, log-rank)   were diagnosed with TNBC, and 846 (81.7%) with non-TNBC. Adjuvant chemotherapy was provided to 138/190 (72.6%) TNBC patients; 60 patients received an anthracycline-based regimen and 78 received a 5-FU-based regimen. Patients with TNBC had a significantly higher relapse rate compared to those with non-TNBC (og-rank) A. Furtheog-rank) B.p = 0.348, log-rank)  . In addition, AR expression was found to be an independent factor indicating favorable prognosis in patients with TNBC  (Fifty-six of 190 (29.5%) TNBC tumors expressed AR. No correlation was found between clinicopathologic characteristics and AR expression . Additioog-rank) A. Howeveog-rank) B. A stat = 0.36) .p = 0.034, log-rank)  had AR-positive TNBC. When CSS after the relapse was investigated, patients with AR-positive TNBC had a significantly better prognosis than those with AR-negative TNBC (og-rank) . Howeverog-rank) .In recent studies, it has been determined that TNBC may further be classified into seven subtypes according to its gene expression profile ,11, and As has been suggested in previous reports, we observed a significant difference in disease-free survival between patients with AR-positive and -negative TNBC ,32,34. PAR expression also had a significant effect on CSS. Patients with AR-positive TNBCs survived longer after recurrence than those with AR-negative TNBCs. This clearly suggests a difference in malignant potential between AR-positive and -negative TNBC. However, we could not identify any specific factor responsible for this increase in survival. This was a retrospective study, and thus we could not alter the treatment strategies as the result of AR expression for any patient. Therefore, the difference in survival might be caused by differences in sensitivity to conventional treatments or by the innate nature of the AR-positive TNBC phenotype. Further investigation is required to identify the precise characteristics of AR-positive TNBCs. We have previously reported that TNBCs that are positive for AR expression have a significantly lower rate of pathological complete response (pCR) in neoadjuvant chemotherapy (NAC) and are chemotherapy-resistant .A study examining treatment with AR antagonists in AR-expressing breast cancers is currently underway for clinical application . There hIn this study, we carried out protein expression analysis on TNBCs using immunohistochemical staining and investigated the clinical significance of AR expression. Although we observed no significant difference in the RFS rate between AR-positive and -negative TNBC cases, many late relapse cases (4 or more years to recurrence) showed luminal type relapse. Thus, we believe that AR-positive TNBC has biological properties different from those of the basal-like TNBCs that display a high degree of malignancy, and that are more similar to the hormone receptor-positive luminal subtypes. We believe that among the TNBC subtypes, the biological malignancy of AR-positive TNBC is lower than other subtypes.We conclude that AR expression may be useful as a subclassification marker for good prognosis in TNBC, and that AR-positive TNBCs may be responsive to anti-androgen endocrine therapy.


Anion exchanger 1 catalyzes the transmembrane antiport of chloride and bicarbonate ions through a mechanism that has remained unclear. By modeling its inward-facing state and comparing it with the known outward-facing form, Ficici et al. hypothesize that this transporter features an elevator-like mechanism. The membrane transporter anion exchanger 1 (AE1), or band 3, is a key component in the processes of carbon-dioxide transport in the blood and urinary acidification in the renal collecting duct. In both erythrocytes and the basolateral membrane of the collecting-duct α-intercalated cells, the role of AE1 is to catalyze a one-for-one exchange of chloride for bicarbonate. After decades of biochemical and functional studies, the structure of the transmembrane region of AE1, which catalyzes the anion-exchange reaction, has finally been determined. Each protomer of the AE1 dimer comprises two repeats with inverted transmembrane topologies, but the structures of these repeats differ. This asymmetry causes the putative substrate-binding site to be exposed only to the extracellular space, consistent with the expectation that anion exchange occurs via an alternating-access mechanism. Here, we hypothesize that the unknown, inward-facing conformation results from inversion of this asymmetry, and we propose a model of this state constructed using repeat-swap homology modeling. By comparing this inward-facing model with the outward-facing experimental structure, we predict that the mechanism of AE1 involves an elevator-like motion of the substrate-binding domain relative to the nearly stationary dimerization domain and to the membrane plane. This hypothesis is in qualitative agreement with a wide range of biochemical and functional data, which we review in detail, and suggests new avenues of experimentation. The AE1e kidney , where ie kidney . AE1 is mporters . In addinditions .2DIDS; AE1 is an ∼95-kD glycoprotein consisting of two major domains with distinct functions . The N-t− or HCO3−) is likely to be precisely between the ends of TM3 and TM10  is not immediately apparent from the existing crystal structure. To gain insights into this mechanism, several studies have compared OF AE1 with structurally related transporters. Specifically, two recent studies have described low-resolution structures of SLC4-type boron transporters, namely a 4.1-Å-resolution crystal structure of occluded-state Bor1 from thaliana , and a 6croscopy . Confusicroscopy  and rockcroscopy . Divergecroscopy . It is lAn alternate strategy to predict the IF conformation of AE1, and thereby the type of transport mechanism used by this protein, is to infer its features from those revealed by a systematic analysis of the known structure. AE1, like most secondary-active transporters , containIt follows from this analysis that a hypothetical model in which repeat A is assumed to adopt the conformation of repeat B, and vice versa, ought to be an IF state. Indeed, this “repeat-swapping” modeling strategy has reliably predicted the global conformational changes of transporters with widely varying structural folds, revealing both rocking-like motions  and elev4YZF, chain A; An IF model of the membrane domain of AE1 was produced using the repeat-swapping modeling technique described previously . That isA model of the AE1 dimer was then generated by superimposing the dimerization domain in the protomer model onto each of the equivalent domains in the x-ray structure. This IF structure of dimeric AE1 was then energy-minimized with CHARMM version 41b1 , 2009 usVideo 1 shows the predicted conformational change obtained by morphing the OF structure into the IF model. Data S1, included as a TXT file, shows the IF model in Protein Data Bank format.2DIDS-bound crystal structure of AE1  stems from the asymmetry of the two inverted-topology seven-helix repeats  TM12 in the dimerization domain.2DIDS binds in this pathway  that connect the two domains . The axi2DIDS-bound structure of AE1 is less outward-open than the actual unbound state. The monoclonal antibody fragment used to scaffold the AE1 crystal might also restrict the degree to which the anion-binding site is exposed to the extracellular space because this Fab fragment specifically recognizes the transport domain and docks onto that side of the protein. A repeat-swapped IF model derived from a more outward-open structure would reveal an even larger vertical displacement of the transport domain, providing further support to the proposed elevator-like mechanism.In summary, the repeat-swapped model of AE1 predicts an elevator-type transport mechanism rather than a mechanism involving rocking of helix bundles around a stationary substrate-binding site. Instead, in this elevator-like mechanism, it is the dimerization domain that remains stationary, whereas the transport domain, which encloses the substrate, moves vertically relative to the membrane plane and rotates around the membrane perpendicular. Although these motions are apparent when the OF crystal structure is compared with our IF model, it is important to note that our prediction might underestimate the actual range of these motions. It is quite possible that the HFrom comparison of the crystal structure of OF AE1 and the IF model we constructed, it can be clearly inferred that this antiporter features an elevator-like mechanism. It is important to note that the strategy used to develop the IF model does not presuppose any type of mechanism; indeed, based on the same procedure, it has been concluded that other transporters feature a rocking-type mechanism . Althoug3 at sites in the TM5–TM6 loop (K551 on one subunit and K562 on the other) without inhibiting transport . Chemical modification of E681, however, also strongly affects the intracellular pH dependence of SO42− translocation, indicating that E681 traverses the permeability barrier during transport , cleavage of the TM7–TM8 loop accelerates transport  results from the deletion of nine amino acid residues (A400–A408) near the N terminus of the AE1 membrane domain . SAO-AE12DIDS), leading to the observed loss of transport function. Thus, the SAO mutation appears to be consistent with an elevator-like mechanism.In the OF crystal structure, residues 400–408 comprise the C terminus of cytoplasmic helix H1 and the first six residues of TM1 . If the max for anion exchange to be two- to threefold higher than that of the wild-type transporter  in human red blood cells. Further analysis of the F792A mutation at the interface between the dimerization and transport domains is also likely to be revealing.Experimentally, it may be possible to test the predicted elevator-like mechanism using cross-linking experiments with selected cysteine substitutions, following an approach similar to that used to study other elevator-like transporters , or usin+-HCO3− cotransporters in the SLC4 family (Finally, it is of interest to ask whether the elevator model also applies to the Na4 family , 2013. T


Overexpression of let-7a inhibited β1-AR expression in neonatal rat ventricular cells (NRVCs), which was abolished by anti-let-7a antisense inhibitor. The lentivirus vector containing precursor let-7a (len-pre-let-7a) further down-regulated the reduced β1-AR level by CIHF and the effect was reversed by len-AMO-let-7a. Len-negative control did not produce any significant influence on β1-AR expression. Importantly, there exists a negative feedback loop associated with β1-AR regulation through β1-AR/cAMP/PKA/GATA4/let-7a/β1-AR signaling pathway in CIHF. As demonstrated, GATA4 was activated by β1-AR up-regulation through cAMP-PKA signaling pathway in early phase of ischemia, then GATA4 positively regulated let-7a expression which in turn suppressed β1-AR expression.CIHF model was established by occlusion of coronary artery for 4 weeks. β1-AR expression and forms a negative feedback loop with β1-AR signaling pathway in ischemic heart failure. This study provides a new insight into the differential expression of β1-AR in early and later phase of myocardial ischemia.Let-7a regulates β However, the molecular mechanism of down-regulated expression of β1-AR as an adaptive effect for cardioprotection in CIHF remains unclear.Chronic ischemic heart failure (CIHF) remains the leading cause of morbidity and mortality worldwide . CIHF is1-AR, β2-AR and β3-AR [1-AR is the main subtype for cardiac function regulation, and it acts through activating the G-protein/adenylate cyclase/protein kinase A (PKA) signaling pathway [1-AR changes dynamically at different stages of myocardial ischemia [1-AR-cAMP/PKA signaling pathway. In this way, it is critically involved in the regulation of cardiac development [The β-adrenergic receptor (β-AR) and its mediated sympathetic activities play a primary role in governing cardiac rhythm/heart rate, cardiac excitation/conduction, and cardiac contraction through regulating the associated cellular signal transductions . β-ARs cnd β3-AR . The β1- pathway . Interesischemia , being dischemia . GATA4 ielopment , cardiacelopment , and genelopment  caused b1-Ars [MicroRNAs (miRNAs) are small single-stranded non-coding RNAs, which mostly suppress protein translation by binding to the 3’UTR of mRNAs . Numerou1-Ars .1-ARs and the associated arrhythmogenesis and dysfunction of the heart. Let-7 replacement rescues cardiac function mainly through its anti-arrhythmic efficacy [Let-7 belongs to a miRNA family containing 13 members sharing the same seed sequence thereby the same set of target genes , and is efficacy . Intrigu1-ARs in heart failure.The present work was conducted to elucidate the mechanism by which let-7a is up-regulated and the influence it produces to the heart in a model of CIHF. Our findings allowed us to establish a negative feedback circuit as a mechanism for abnormal up-regulation of let-7a and to verify the up-regulation of let-7a as a mechanism for the down-regulation of β1-ARs was decreased by 34.0 ± 6.3% in failing hearts compared with those in non-failing hearts  and FS from 21.1 ± 6.3% to 36.4 ± 7.7% (P < 0.05) compared to the len-let-7a group   and fractional shortening (FS) in CIHF rats. Oppositely, len-AMO-let-7a enhanced EF from 41.5 ± 10.6% to 66.6 ± 8.7%  Figure . ReducedGATA4 is a cardiac specific transcription factor that plays a key role in the regulation of cardiac physiology and development. Our initial computational analysis using the JASPAR database predicted that there are putative binding regions for GATA4 in the upstream regulatory domain of the let-7a gene.1-AR was pronouncedly up-regulated at both protein and mRNA levels  for GATA4 was transfected into NRVCs. As expected, the levels of total GATA4 and the phosphorylated form of GATA4 remained unaffected by the decoy ODN Figure . Howevers Figure  and 4D. A Figure . Let-7a A Figure , and thed Figure  and 4H. 1-AR agonist, 10 μM) for 72 h. We observed that the levels of the total protein (t-GATA4) and phosphorylated form of GATA4 (p-GATA4) were increased compared with the control groups  was used to treat the NRVCs for 72 h. The results showed the levels of t-GATA4) and p-GATA4 were upregulated by FSK, which were unaffected by the co-administration of GATA4 decoy ODN Figure . FSK up-t Figure  and 6D. t Figure . Let-7a t Figure . The dowt Figure  and 6H. 1-ARs are down-regulated in the setting of chronic ischemic heart failure (CIHF) [1-AR down-regulation in CIHF and role of let-7a in regulating the β1-AR/cAMP/PKA/GATA-4 signaling pathway. Our results revealed the following aspects. First, the level of let-7 family members, especially let-7a, was significantly up-regulated in CIHF, along with down-regulation of β1-ARs. Second, let-7a directly targeted ADRB1 mRNA 3’UTR to repress β1-AR expression. Third, GATA4 positively regulated the transcription of let-7a. It is conceivable based on our findings that let-7 participates in the β1-AR/cAMP/PKA/GATA-4 signaling pathway as an upstream component and its participation forms a negative feedback loop. We therefore proposed the modified signaling pathway: β1-AR↑→cAMP↑→PKA↑→GATA-4↑→let-7↑→β1-AR↓. This can be translated into a more physiological term: in the early stage, or the acute phase, of ischemia, the enhanced sympathetic tone induces β1-AR activation and the subsequent signal transduction, leading to activation of GATA4 thereby the expression of let-7a. The latter then serves to tune down the signal transduction by down-regulating expression of β1-ARs, as occurring in CIHF. A schematic diagram illustrating the feedback circuit is presented in Figure It has been long recognized that βe (CIHF) . The mai1-AR accounts for 70% in heart and played a major role in the development of CIHF [1-AR shows up-regulation in acute phase of ischemic heart failure and down-regulation in late phase of heart failure, both changes as adaptive effects for cardiac function and cardioprotection. In the early stage of ischemic heart failure, the up-regulation of β1-AR is due to ischemia caused by sympathetic nerve excitability, adrenergic hyperactivity and endogenous catecholamines substance secretion. The activation of β1-AR could accelerate sinus rhythm, strengthen the heart of the positive effect of muscle strength, improve cardiac output and enhance the perfusion of peripheral tissue, to compensate for the lack of oxygen caused by ischemia. However, the positive inotropic effect of persistent hyperexcitability of β1-AR leads to increased consumption of myocardial oxygen and relative shortage of energy supply to myocytes, which will induce myocardial organic hypertrophy, myocardial apoptosis, damage to the heart etc [1-AR with long-term stimulation by circulating catecholamine could lead to the down-regulation of β1-AR expression [1-AR will reduce myocardial sensitivity to catecholamines, and attenuate positive effects of myocardium. It reduces myocardial energy consumption for the energy hungry state in heart failure, and results in saving the viability of the cells and resisting the excessive sympathetic nervous excitement caused by myocardial injury. It is helpful to slow its further deterioration and progression of heart failure. The internalization and desensitization of receptors were the mechanisms for the decrease of β1-AR in the cell membrane. Long-term excessive stimulation of β1-AR could induce the phosphorylation of the receptor, which initiates the process of receptor internalization [It was well known that β-AR played a leading role in mediating cardiac function, and β of CIHF –25. β1-Aeart etc –28. In tpression . The dowlization , 31.1-AR stayed activated and the sensitivity of β1-AR to catecholamines decreased. The biological function of β1-AR was reduced or lost, which is manifested by desensitization of the receptor to catecholamines. The number of non intrinsic receptors might be associated with the decrease of mRNA and the degradation of receptor molecules by cAMP dependent β1-AR [1-AR belongs to G protein-coupled-receptors that its desensitization is mainly divided into G proteins decoupled work and mRNA transcription or translation inhibition [1-AR down-regulation in CIHF remains unclear.Receptors by endocytosis, from the cell membrane into the cell, can bind with the β-arrestins, which are degradated by the lysosomal, the other part can be returned to the cell membrane . This innt β1-AR . The stant β1-AR . β1-AR bhibition , 36. How1-AR/cAMP/PKA signaling pathway in CIHF [Heart failure involves multiple pathophysiological processes such as interstitial fibrosis, cardiomyocyte apoptosis, and inflammatory response etc. In the present study, we found that let-7a, b, c, d, f and I were significantly upregulated and let-7e downregulated in rat failure hearts. This discrepancy of let-7 family expression may be attributed to the family members’ transcript from different gene clusters which contribute to different processes in heart failure. Nuclear GATA4 is a critical transcription factor for cardiac development and a variety of cardiac pathophysiological processes, and can be activated by phosphorylation through the cAMP-PKA signaling pathway . Phospho in CIHF .et al. demonstrated that miR-223 targets and inhibits E2F1 which binds to the miR-223 promoter in AML blast cells and inhibits miR-223 transcription, generating negative feedback loop between these two molecules [Negative feedback regulation is one of the crucial physiological mechanisms and has been demonstrated to mediate a wide range of biological processes . miRNAs olecules .1-AR expression by acting on the 3’UTR of ADRB1 mRNA. It may be therefore conceived that there is a negative feedback loop which regulates β1-AR expression in CIHF. This negative feedback pathway is described as that β1-AR is excited and up-regulated in early phase of myocardial ischemia, which activates transcription factor GATA4 via cAMP/PKA signaling pathway, leading to up-regulation of let-7a which inhibits β1-AR expression in CIHF. Thus, it forms a critical feedback loop signaling pathway of β1-AR/cAMP/PKA/GATA-4/let-7/β1-AR, and the decreasing of β1-AR in CIHF is induced by β1-AR activation in early phase of CIHF, which is a kind of self-regulation of β1-AR in the course of ischemia induced heart failure. The limitations of the study may be that (1) there is a lack of transgenic overexpression or knockout model to further confirm the function of let-7a;(2) the role of let-7a in human heart failure has not been explored.We employed the decoy ODN and siRNA assays to inhibit GATA4, and used DOB and FSK to stimulate cAMP-PKA signaling pathway to activate GATA4. Our results revealed that GATA4 positively regulated let-7a expression that in turn reduced β1-AR negative feedback loop which had been opened out as our work for the first time. It has a significant influence on the research of cardiac pathophysiology. It could reduce the damage of myocardium caused by β1-AR activation, and protect cardiac function. It was helpful to further cognize the pathological process of CIHF with the dynamic changes of β1-AR expression by means of clarifying the negative feedback loop. Artificial mediation on this feedback circuit by interfering let-7 expression may be a potential strategy and new idea for the regulation of β1-AR expression in the prevention and treatment of heart failure.This signaling pathway of βHealthy adult male Sprague-Dawley (SD) rats  used in the present study were cared under the standard animal room conditions  with daily food and water for 1 week prior to experiments. All experimental procedures were in accordance with and approved by the institutional Animal Care and Use Committees of Harbin Medical University.5 units/day, im) for 7 days. After surgery, the rats were given food and water ad libitum.All rats were anesthetized with intraperitoneal injection (i.p.) of ketamine (60 mg/kg) and xylazine (6 mg/kg). Then, tracheal cannula was performed with a polyethylene tube and ventilated with the TOPO small animal Ventilato , and then the chest was opened through the third intercostal space and propped ribs by a rib spreader. The pericardium was opened carefully to expose the heart. The left anterior descending coronary artery (LAD) was ligated using a 5/0 silk thread to create infarction of the LV free wall for 4 week to establish a rat model of chronic heart failure. After operation, rats were given penicillin  were isolated from 1 to 3-day-old SD rats. The neonatal rats were sterilized with 75% ethanol before decapitated. The hearts were obtained and rapidly removed into DMEM . The isolated hearts were cut into l~3 mm pieces. The tissues were dissociated in 0.25% trypsin at 37°C and cell suspension was collected. After centrifugation , the isolated cells were resuspended in DMEM containing 10% fetal bovine serum , 100 units/ml penicillin and 100 μg/ml streptomycin. Then the cells were cultured in DMEM at 37°C in humidified air with 5% COLet-7a mimics (sequence: 5’-UGAGGUAGUAGG UUGUAUAGUU-3’) and its antisense inhibitor AMO-let-7a (sequence: 5’-AACUAUACAACCUC CUACCUCA-3’) were synthesized by RiboBio . Additionally, a scrambled sequence was used as negative control (NC) (sequence: 5’-UUCUCCGAACGUGUCACGU-3’) that was designed according to the Blast search of the human/rat genomic. The X-treme GENE siRNA Transfection Reagent (Roche) was used to perform for transfection. After transfection with let-7a mimics (100 nM) or AMO-let-7a (200 nM) for 36 h, the NRVCs were used for qRT-PCR and Western blot analysis.1-AR was obtained from Santa Cruz . The antibodies against GATA-4 total and GATA-4 phosphate S262 were obtained from Abcam . The antibody against GAPDH was obtained from Jinshan . The fluorescence conjugated secondary antibodies .Total protein was extracted with RIPA Lysis buffer  mixed with 1% protease inhibitor cocktail  and admixing with 5X loading buffer (Beyitime) at 100°C for 5 min. The protein concentration was determined by BCA kit  according to the instruction. For Western blot analysis, denatured proteins(120 μg from NRVCs and 80 μg from tissues) were separated in 10% SDS-polyacrylamide gels and blotted to nitrocellulose membranes. Prior to incubating with primary antibody, the membranes were blocked with 5% skimmed milk in PBS for 2 h in room temperature. The membranes were incubated with diluted antibody in PBS at 4°C overnight. The membranes were washed with PBS-T (PBS containing 0.5% Tween 20), followed by re-probing with the fluorescence conjugated secondary antibody at room temperature for 1 h. Membranes were washed again with PBS-T prior to detection on Odyssey infrared scanning system . The Western blot bands were quantified using Odyssey 3.0 software and normalized to loading control. The antibody resources and dilution used are as follows: The antibody against βTotal RNA was extracted from cultured cells and tissues using a Trizol standard protocol . The integrity, quantity, and purity of RNA were examined using Nano-Drop 3000 Spectrophotometer . For each sample, 500 ng of total RNA was converted to cDNA using High Capacity cDNA Reverse Transcripition Kit . The relative expression levels of mRNAs and miRNAs were quantified by the mirVana qRT-PCR miRNA Detection Kit in conjunction with real-time RT-PCR with SYBR Green I (Applied Biosystems). The threshold cycle (Ct) was determined and relative mRNA and miRNA levels were calculated based on the Ct values and normalized to GAPDH or U6 level for each sample. The sequences of primers used in our qRT-PCR experiments are shown in Table The sequence (5’-TGTGTCTGATAAATCAGAGATAACCCCACC-3’) of decoy oligonucleotides fragment for GATA-4 (GATA ODN), a scrambled oligonucleotides fragment (5’-TAAATTGGCCAAGTGTAGCTCCG TTTGTGA-3’) as a negative control (control ODN), GAGA-4 siRNA  and its negative control  were synthesized by Invitrogen . The ODNs (100 nM) and siRNAs (100 nM) were transfected into NRVCs with the X-treme GENE siRNA Transfection Reagent (Roche) for 48h.1-AR, which is highly conserved among mammals. A segment containing the let-7 miRNA binding sites flanked by the Hand III and Sac I restriction sites and a scramble sequence as a negative control (NC) were synthesized by Invitrogen. The sequences were inserted separately into the pMD18T-simple vector (Invitrogen), and then transferred into the pMIR-REPORTTM Luciferase miRNA Expression Reporter Vector . pRLRenilla Luciferase Reporter vector  was used as an internal control. The plasmids and miRNAs were co-transfected into HEK293 cells using X-treme GENE siRNA transfection reagent . The dual luciferase reporter assay kit (Promega) and the GloMax biological fluorescent tester were used to detect luciferase activity 36 h after transfection. The results were expressed as fold changes by normalizing the scaled data. The sequences of human and rat ADRB13’UTR containing the binding site for let-7a inserted into the vectors were as follows: The human β1-AR 3’UTR : 5’-CGAGCTCTTAAGCTCTTCTTGGAACAAGCCCCACCTTGCTTTCCTTGTGTAGGGCAAACCCGCTGTCCCCCGCGCGCCTGGGTGGTCAGGCTGAGGGATTTCTACCTCACCTGTGCATTTGCACAGCAGATAGAAAGACTTGTTTATATTAAGCTTGGG-3’. The rat β1-AR 3’UTR :5’-CGAGCTCCTCGGTGGTCCTGCTGTGGGTCCTCTACCTCACTCTGTGCATATTGCACAGCAAGATAGAAAGACTTGTTTATATTAAACAGCTTATTTATGTATCAATATTAGTTGGAAGGACCAGGCGCTGAAGCTTGGG-3’.Targetscan predicts the presence of a putative binding site for let-7 in the 3’UTR of ADRB1 mRNA, the gene encoding β8TU) including pre-let-7a, pre-AMO-let-7a and pre-NC were injected into the cavity of the left ventricle of rat heart with ascending aortic artery clamped. After surgery, the rats were given food and water ad libitum and received penicillin  for 7 days.Lentivirus vectors expressing mature let-7a, anti-miRNA-oligo of let-7a (AMO-let-7a) or NC sequence were constructed . The heart of rats was exposed by performing the thoractomy in the third left intercostal space. Virus-containing solution  4 weeks after LAD ligation and infection with virus-containing solution. Rats were positioned on a Rat Pad  with an integrated heater. Body temperature of rats was maintained at 37°C. The M-mode tracings were recorded in both parasternal long and short axis views. Ventricular parameters including diastolic anterior wall thicknesses, diastolic posterior wall thicknesses and left ventricular systolic internal diameters were measured. Fractional shortening (FS) and ejection fraction (EF) were calculated automatically.P < 0.05 was considered statistically significant.All data are presented as mean ± SD. Statistical analysis was performed using Student's non-paired t test or One-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test. A value of


The use of walking aids is prevalent among older people and people with mobility impairment. Rollators are designed to support outdoor mobility and require the user to negotiate curbs and slopes in the urban environment. Despite the prevalence of rollators, analysis of their use outside of controlled environments has received relatively little attention. This Letter reports on an initial study to characterise rollator movement. An inertial measurement unit (IMU) was used to measure the motion of the rollator and analytical approaches were developed to extract features characterising the rollator movement, properties of the surface and push events. The analytics were tested in two situations: first, a healthy participant used a rollator in a laboratory using a motion capture system to obtain ground truth. Second, the IMU was used to measure the movement of a rollator being used by a user with multiple sclerosis on a flat surface, cross-slope, up and down slopes and up and down a step. The results showed that surface inclination and distance travelled measured by the IMU have close approximation to the results from ground truth; therefore, demonstrating the potential for IMU-derived metrics to characterise rollator movement and user's pushing style in the outdoor environment. A EuropRollators are the most and second most common walking aids in Sweden  and Canaet al. [et al. [A small number of studies have reported on user views on rollators. Brandt et al.  carried  [et al.  found th [et al.  and Japa [et al.  were fouet al. [et al. [et al. [From a biomechanics perspective, despite their prevalence amongst the older population, the literature on characterisation of rollator–user interaction is very limited. Kegelmeyer et al.  studied  [et al.  studied  [et al.  investiget al. [In one of the most recent papers, Tung et al.  studied Despite the recent advances in low-cost computing and sensing, there is no data on the patterns of use of rollator devices outside of controlled environments, whereas in other areas of mobility aids research such as wheelchairs the usage and activity levels can be measured by accelerometers and inertial measurement units (IMUs) –15. ThisIn the light of this, this Letter reports on a feasibility study to characterise rollator use in the laboratory using real-world surfaces. Two experiments are presented, first an experiment with a healthy user, and second an experiment with a user who has MS. The first experiment demonstrates how a single IMU mounted on the rollator frame together with sensors on the user's feet can be used to characterise basic features of rollator use. These features are number of push events, distance travelled, average distance and duration of each push, and the push events in relation to the foot movements. The second experiment applies this technique to one rollator user with MS in a simulated urban environment (SUE) and demonstrates the potential to obtain information on the environment including surface slope and curb crossing events from a rollator-mounted sensor, in addition to the basic gait features.2The aim of the feasibility study was to establish the capability of the IMU to capture the interaction between the rollator, the user and the walking environment. To evaluate the capability of the IMU, the experiment was two-fold, containing (i) testing of protocols and software algorithm using a gold standard motion capture system and (ii) testing of the protocols and algorithm in an SUE.2.1A healthy participant was recruited for understanding baseline performance. Subsequently, a participant with 3 years of MS participated in tasks in the SUE. Ethical approval was obtained from the University College London Research Ethics Committee (4721/002).2.2Y-axis for anterior–posterior movements, the X-axis for mediolateral movements and Z-axis for vertical movements. The IMU is oriented such that a negative value in the Y-axis corresponds to forward movement. The rollator was banged onto the force plates by the participant before the start of each trial to get a peak force in both VICON and Xsens to synchronise the two datasets.The gold standard test comprised a 6 m straight-line walking assessment with a rollator. The healthy participant had IMUs of Xsens MTw2 Awinda  attached to the pelvis and both feet, operating at a sampling frequency of 100 Hz. To obtain ground truth, the three-dimensional coordinate data of the pelvis and both feet were captured using an eight-camera VICON Motion Capture System at a sampling frequency of 100 Hz. On the rollator, there were an IMU horizontally attached to the frame and a cluster of markers to each of the left, right and front side of the frame as shown in Fig. 2.3The tests in the simulated environment used the same IMU placement as the gold standard testing, but did not use the motion capture system. The tests consisted of a participant moving along four straight lanes including an 8.4 m flat path, an 8.4 m 4% cross-slope (2.29° elevation across the distance of travel), a 4.8 m 6% slope (3.44° elevation in the distance of travel) and a step of 80 mm, which were set up at the Pedestrian Accessibility Movement Environment Laboratory at University College London as shown in Fig. 3The results of the gold standard testing from the motion capture system served as the ground truth to examine the analysis of the IMU data for distance travelled, whilst the known characteristics of the surface of the SUE served as ground truth for surface detection. Gait phase data was obtained from the foot-worn IMU, which was measured alongside the push events of the rollator. The analysis was utilised to measure the characteristics of rollator usage in the laboratory and SUE.3.1X, Y and Z axes are vectors with length n of the formThe raw data in the 3.2Y-axis is used to calculate the longitudinal tilt of the rollator on the flat surface, slope and step; the acceleration of the X-axis is used to calculate the cross-sectional tilt of the rollator on the cross-slope.The acceleration of the The low-pass data are used for surface detection. From these data, the orientation of the rollator with respect to the gravitational pull of the earth is estimated, which provides the angle of the horizontal plane of the rollator to the earth. From this angle the direction of the surface slope, if any, can be determined. Orientation is calculated as3.3Y-axis (corresponding to the anterior–posterior orientation of the rollator) has been used. This axis is oriented approximately parallel to the ground in the direction of movement and thus captures the majority of the motion of interest.Distance travelled is obtained principally from a double integration of the accelerometer signal in the direction of travel. For this work, only the a and b are the indices of Y between which an integral is required. Equation (The band-pass filtered data were used to calculate distance travelled. After filtering, the signal was cumulatively, numerically integrated to obtain velocity over time,  case as\document−3. The set of zero-points and their associated timestamps are interpolated to get a baseline signal with the same timestamps as the velocity signal using MATLAB's pchip interpolation, which is based on work by Fritsch and Carlson [et al. [To achieve this, a baseline signal is created by interpolating between velocity points where the gradient is below 0.5 × 10 Carlson  and Kaha [et al. . Pchip iOnce a baseline signal is created, it is added to the velocity signal to correct the offset. The adjusted velocity signal is then cumulatively integrated a second time, using , to get 44.1Results from the ground truth test with the healthy participant showed that calculated distance travelled is a very close approximation to ground truth for both tests. Fig. The basic features of rollator use of the healthy participant including the number of push events, average distance and distance of each push, and mean velocity of rollator movement are shown in Table 4.2The basic features of rollator use of the MS participant measured by the analysis of IMU data developed in the gold standard testing and applied to the SUE data are presented in Table Similar to the results in the gold standard testing, the characteristics of the rollator movement of the MS participant along the flat surface are comparatively steady, as shown in Fig. X-axis, as shown in Figs. Y-axis (Figs. The mediolateral inclination of the rollator movement along the 6% cross-slope is identified by the degrees elevation across the distance of travel, around −2° to −3° on the is Figs.  and 11.Y-axis.During the step-up and step-down, the regular movement of the rollator is shown to have been interfered with the step. Figs. Y-axis.Figs. 5The results of the tests in the laboratory and SUE show that it is feasible to use an IMU to characterise the rollator movement and measure the interaction between the rollator, the user and the urban environment. The results also show that by using an IMU alone, the travel pattern can be reconstructed offline, which can provide researchers and physiotherapists with insight into a user's performance while walking and using a rollator.Past studies have demonstrated the difference in the movement behaviour between laboratory assessments and real environment and call for a better understanding of the interaction , 12. ThiThe property of the surface and distance travelled can be detected by the IMU by the degree of the inclination of the rollator and integration of the acceleration of the rollator movement. Along with the push events in relation to the foot movement and average distance and duration of each push, the user's balance mechanism and coping strategy used to deal with the uneven surface in the urban environment can be further understood.Investigating the characterisation of rollator use has helped shed some light on the understanding of the quality, difficulty and risk of the use of rollators in the urban environment. Furthermore, studies need to investigate how the understanding of this interaction between the rollator, the user and the urban environment can help physiotherapists provide training, rehabilitation and assessments for rollator users of different physical, cognitive and sensory capabilities.We do, however, acknowledge several limitations of this Letter. As a pilot study exploring the interaction between the rollator, the user and the environment, only one participant was measured in each of the laboratory and SUE. This Letter does not intend to demonstrate the generalisability of findings, but explore the potential and validation of using low cost, portable IMUs to characterise rollator use outside the laboratory setting. This Letter provides initial evidence to conduct future research with larger sample sizes, more types of surfaces and longer walking distances. Furthermore, work will focus on creating a generalised set of algorithms to extract rollator characterisation data from IMUs and the applications of this approach to different user groups.6The work presented in this Letter provides a first examination of the interaction between the rollator, the user and the environment using potable IMUs to characterise the rollator movements. A healthy participant performed walking tests using a rollator on a flat surface in the laboratory to examine the IMU measures with the gold standard ground truth from a motion capture system. Subsequently, a participant with MS performed walking tests using a rollator on a flat surface, cross-slope, up and down-slope and up and down a step in an SUE with an IMU alone attached. The use of IMUs to measure the pushing style, property of surface and travel distance has been examined by the motion capture system and can be utilised to detect these movement characteristics of a rollator user with MS on different surfaces. The results of this Letter show the potential to provide insight into the quality of the use of rollators, fall risks associated to rollators and quality of the provision of rehabilitation for rollator users.


Clear cell renal cell carcinoma (ccRCC) is a gender-biased tumor. Here we report that there is also a gender difference between pulmonary metastasis and lymph node metastasis showing that the androgen receptor (AR)-positive ccRCC may prefer to metastasize to lung rather than to lymph nodes. A higher AR expression increases ccRCC hematogenous metastasis yet decreases ccRCC lymphatic metastases. Mechanism dissection indicates that AR enhances miR-185-5p expression via binding to the androgen response elements located on the promoter of miR-185-5p, which suppresses VEGF-C expression via binding to its 3′ UTR. In contrast, AR-enhanced miR-185-5p also promotes HIF2α/VEGF-A expression via binding to the promoter region of HIF2α. Together, these results provide a unique mechanism by which AR can either increase or decrease ccRCC metastasis at different sites and may help us to develop combined therapies using anti-AR and anti-VEGF-C compounds to better suppress ccRCC progression. The incidence of renal cell carcinoma is higher in males than in females due to the different androgen receptor signaling but the molecular mechanisms behind this gender bias are unclear. Here the authors show how androgen receptor expression influences the metastatic route through the regulation of miR-185 and VEGF isoforms. Approximately 30% of RCC patients had metastatic lesions detected at initial diagnosis2, however, the detailed mechanisms behind the development of these metastases remain unclear. The most common histologic subtype, clear cell RCC (ccRCC), derives from the epithelial cells of the proximal renal tubule and may be resistant to many chemotherapies and radiotherapies. Early studies indicated that the majority of ccRCC were associated with a loss of the von Hippel–Lindau (VHL) gene with induction of hypoxia-inducible factors (HIF) and vascular endothelium growth factors (VEGF)4, and anti-VEGF targeted therapy remains the first-line treatment for metastatic ccRCC.Renal cell carcinoma (RCC) is the most lethal of all urological malignancies6 that involves the alterations of the androgen/androgen receptor (AR) signals9. Early studies indicated that the AR acted as a stimulator to promote the ccRCC progression7. However, some clinical surveys also suggested that AR expression was associated with less malignancy8 and AR-negative tumors might be associated with more metastatic ccRCC9 with 10 of 16 cases of lymph node metastatic RCC vs. one case of lung metastatic ccRCC9. This contrasting difference for the AR roles in ccRCC progression suggests that the AR may play distinct roles in a stage- or tissue-specific manner. The detailed mechanisms, however, remain unclear.The ccRCC is a gender-biased neoplasm10. Interestingly, from a survey of about 119 ccRCC specimens, we found increased AR expression in the pulmonary metastases (PM), the most common hematogenous metastatic site, yet decreased AR expression was linked with metastases to the lymph nodes. These differential manifestations of the two ccRCC metastatic sites suggested that the role of AR in hematogenous vs. lymphatic ccRCC metastasis may vary and may function through different mechanisms to modulate different target genes.Previous studies indicated that hematogenous metastasis and lymph node metastasis of RCC were characterized with varying microvessel density (MVD) and angiogenesis-specific factorsHere we investigated the underlying mechanisms behind these clinical findings and found AR might differentially modulate VEGF-A vs. VEGF-C expression via regulation of the microRNA (miRNA), miR-185-5p, to either promote or suppress RCC metastasis to different sites.Using the epidemiological survey of 3989 RCC cases from Chinese PLA General Hospital, we found the gender difference with male to female incidence ratio near 2.7:1 Fig. . This clWe then focused on the AR and examined the AR mRNA expression in 119 randomly extracted ccRCC patients . The results in Fig. These results suggest AR may play key roles for the gender difference in the differential metastatic destinations, and VEGF-A and VEGF-C may play a role consistent with their respective function associated with differential metastatic locations.9 that are associated with RCC progression. We also used AR expression (positive or negative) as a dependent variable, and we found that the gender, Fuhrman grade, and pulmonary metastatic status were correlated with AR expression showing a higher AR-staining-positive ratio in PM than in NM and LM specimens. Importantly, we found that the risk of AR positivity in PM was 6.7-fold higher than that found in LM and NM specimens (Table n = 42) was also higher than that in females . In addition, among female patients, AR-positive RCC was more likely to metastasize to lung than AR-negative RCC (2/7 vs. 0/12). Therefore, it was likely that AR level rather than gender (or hormone) determines RCC metastasis.We next detected the AR protein expression by IHC in 89 randomly extracted cases of AR-positive or AR-negative ccRCC specimens, using prostate tissues as positive controls  staining, indicative of more angiogenesis  cell lines SN12-PM6, Caki-1, and ACHN and VHL-mutant (VHL-mut) cell lines A498, SW839, OSRC-2, 769-P, and 786-O cells17. The prostate cancer cell line C4-2 expressing AR was used as positive control  harbor the VHL gene mutationrol Fig. . Among trol Fig. . Therefo19, while the latter is directly linked with lymphangiogenesis22. These cells were cultured in phenol red-free media with charcoal-stripped serum for 24 h and then treated with the androgen dihydrotestosterone (DHT), vehicle, or the anti-androgen enzalutamide (Enz). The western blot results showed that HIF2α and VEGF-A were increased by DHT, but not by vehicle or Enz in SW839 cells , invasion, and tube formation  and public literature to identify the potential miRNAs candidates  that could enhance HIF2α/VEGF-A signal yet suppress VEGF-C and VEGF-D signals .To dissect the mechanism(s) how AR can differentially modulate the HIF2α→VEGF-A signal vs. VEGF-C signal in the VHL-mut ccRCC cells, we focused on modulation of the miRNAs. The miRNAs have been reported to be able to suppress gene expression via targeting the 3′ untranslated region (UTR) of mRNA. In contrast, they can also enhance gene expressions via targeting their 5′ promoter regions29 or promote HIF2α and VEGF-A expression via binding to the 5′ promoter region30.Among these miRNA candidates that can respond to AR modulation in A498 cells Fig. , we foun30 and RegRNA 2.031, respectively. Importantly, a clinical survey of 22 VHL-mut ccRCC tumors also found that the miR-185-5p was positively correlated with the AR expression  of HIF2α as well as CMV-driven luciferase followed by the wild-type and mutant 3′ UTR of VEGF-C Fig.  and measTogether, results from Fig. We next asked how AR could modulate the expression of miR-185-5p. We applied the miRStart to determine the transcriptional start site of pre-miR-185 and then examined the potential AR binding element (AREs) up to 5000 bases upstream from the transcriptional start site, and results revealed that this promoter region contains 10 potential AR binding elements that may allow AR to bind Fig. . We applTogether, results from Fig. 32. We first transduced the SN12-PM6 cells with luciferase and knocked down VHL simultaneously with lentiviral vectors pLV-luci-U6-VHL to generate the stable luciferase expressing VHL-mut cell line . We also performed the ex vivo bioluminescent imaging immediately after mice were killed to monitor the lung, liver, and retroperitoneal lymph node metastases . Anatomic studies were carried out to examine the macroscopic appearance of primary tumors and the metastases . In addition, histological stainings were performed to confirm the tumor type .Sixty male nude mice were randomly divided into four groups for injections with SN16-PM6 control, overexpressed AR (OE-AR), knocked down miR-185-5p (Sh-miR-185), or overexpressed AR together with knocked down miR-185-5p (OE-AR + Sh-miR-185) cell xenografts. The tumors were grown for 12 weeks, with bioluminescent imaging monitoring of both primary and metastatic tumors at 4, 8, 10, and 12 weeks  overexpression of AR (middle panels) increased HIF2α/VEGF-A expression while suppressing VEGF-C expression. However, knocking down miR-185-5p could reverse the impact of AR . A new and better therapy with clear mechanistic foundations to suppress RCC metastasis is therefore needed to extend patients survival. Our finding that AR can differentially regulate VEGF-A and VEGF-C through the miR-185-5p expression may explain the clinical phenomenon why patients with higher AR-positive ccRCC may have pulmonary metastasis vs. those patients with lower AR-positive ccRCC may have preferential invasion into the lymph nodes. Importantly, these findings suggest that anti-AR therapy may be biased in favor of ccRCC patients with a propensity for lung metastasis and combining anti-AR and anti-VEGF-C therapy should be better to treat this metastatic ccRCC.The ccRCC resists chemotherapy and radiotherapy with a limited therapeutic duration of the anti-angiogenesis targeted therapy , the broad spectrum tyrosine kinase inhibitors, can extend RCC patients survival, yet have a therapeutic durability of only 6–15 monthsThe epidemiological survey of 3989 cases of RCC patients (from January 2006 to December 2014) was performed on the clinical database of Chinese PLA General Hospital. A total of 119 specimens of ccRCC for real-time PCR or 89 cases of ccRCC for immunohistochemistry staining were randomly extracted from the patient population stratified by metastatic status. All patients provided signed informed consent for the use of their tissues for scientific research. The current study was approved by the Institutional Review Board. All ccRCC cases were confirmed by a senior pathologist  and staged based on the 2011 Union for International Cancer Control TNM classification of malignant tumors.2 environment at 37 °C. The vascular endothelium HUVEC-C cell line was acquired from ATCC and maintained in media containing 10 ng/ml VEGF165 . HDLMVEC cells were purchased from Cell Applications, Inc.  and maintained with endothelial cell growth medium in attachment factor solution pre-coated flasks. Cells were authenticated by STR typing and tested to have no mycoplasma contamination before experiments.The human normal cell line 293T and RCC cell lines, ACHN, Caki-1, A498, OSRC-2, 769-P, and 786-O were originally purchased from American Type Culture Collection . The SW839 cell line was purchased from Cell Resource Center for Biomedical Research, Tohoku University. The SN12-PM6 cell line was kindly provided by Dr. X.P. Zhang from Union Hospital in Wuhan, China. All RCC cells were cultured in DMEM supplemented with 10% FBS in the humidified 5% CO41. The lentiviruses were produced in 293T cells with the psAX2 packaging plasmid and pMD2G envelope plasmid together with the transfer plasmid. After 48 h transfection, virus supernatants were collected for immediate use and/or frozen at −80 °C for later use.To generate AR overexpressing or AR knocked down stable cell populations, SN12-PM6, A498, and SW839 cells were infected with lentiviral vectors, pWPI-AR/pWPI-Vec or pLKO1-sh-AR/pLKO1-scr or pLV-sh-AR/pLV-scr. To overexpress miR-185-5p, the cells were infected with lentiviral vector pLVTHM-miR-185-5p. To knock down miR-185-5p, the cells were infected with lentiviral vector pLVTHM-sh-miRNA-185 that overexpressed the complementary sequence of miR-185-5p as “miRNA scavengers”The transient transfection was performed using the Lipofectamine 3000 (Invitrogen) reverse transfection protocol according to the manufacturer’s instructions. The miR-185-5p mimic, miR-185-5p inhibitor, negative controls, and unspecific All Stars negative control RNA, were from Qiagen and used at the final concentrations of 30 nM.For RNA extraction, total RNAs were isolated using Trizol reagent  and 1 µg of total RNA was subjected to reverse transcription using Superscript III transcriptase (Invitrogen).The miRNAs were also reversed transcribed from total RNA. Briefly, 1 µg of total RNA was processed for poly A addition by adding two units of polymerase with 1 mM ATP in 1× RT buffer at 37 °C for 20 min in 10 μl volume, and then adding 50 pmol anchor primer to 11 μl, and incubating at 65 °C for 5 min, then 4 °C for 2 min. For the last step of complimentary DNA synthesis, we added 2 µl 5× RT buffer, 2 µl 10 mM dNTP, 1 µl reverse transcriptase to total 20 µl, and incubated at 42 °C for 1 h. Quantitative real-time PCR (qRT-PCR) was conducted using a Bio-Rad CFX96 system with SYBR green to determine the mRNA expression level of a gene of interest or with Tagman probe to determine the miRNA expressions. Expression levels were normalized to the expression of TATA box binding protein TBP RNA, or 5s RNA and/or U6. The primers used for the genes of interest were listed in Supplementary Table Cells were lysed in RIPA buffer and proteins (30 µg) were separated on 8–10% SDS/PAGE gel and then transferred onto PVDF membranes . After blocking membranes with non-fat milk solution, they were incubated with appropriate dilutions of specific primary antibodies. For human ccRCC samples and cells, the primary antibodies of the rabbit anti-AR , the mouse anti-HIF2α , rabbit anti-VEGF-A , rabbit anti-VEGF-C , and rabbit anti-VEGF-D  were used for blotting at dilutions of 1:2000, 1:500, 1:1000, 1:500, and 1:1000, respectively. They were then incubated with HRP-conjugated secondary antibodies and visualized using the ECL system . The antibodies used were listed in Supplementary Table 5 cells/well, and the bottom chambers contained mixed CM with 10% FBS, and then incubated for 24 h at 37 °C. Following incubation, the invaded cells attached to the lower surface of the membrane were fixed by 4% paraformaldehyde and stained with 1% crystal violet, while the upper surface and non-attached cells were washed and removed, then cell numbers were counted in five randomly chosen microscopic fields (×400) per membrane.Endothelial cells (HUVEC and HDLMVEC) were co-cultured with RCC cells with indicated treatments (with/without overexpression/knockdown of AR) for 72 h, and then the endothelial cells were harvested and subjected to the migration and invasion capability of endothelial cells (HUVEC and HDLMVEC) as determined by the wound-healing and transwell assays, respectively. For these experiments, the endothelial cells were maintained in mixed CM, media from the co-culture system with fresh media at the ratio of 1:1. Wound-healing assays of HUVEC and HDLMVEC cells co-cultured with A498 and SW839 with altered AR expression were conducted at indicated time points. The cells migrated into the wounds were counted after 24 h. For the invasion assay, before seeding the cells, 100 µl of Matrigel  was dissolved in 1.5 ml serum-free DMEM and applied to the upper chambers of transwells with 8 µm-pore-size polycarbonate membrane filters  and then were incubated at 37 °C for 5 h. Endothelial cells were then harvested and seeded with serum-free DMEM into the upper chambers of the transwells at 1 × 104 cells were harvested and seeded to 48-well plates pre-coated with the BME Reduced Growth Factor. After 6–12 h, the fluorescence microscope (485 nm excitation/520 nm emission) was used to visualize tube formation of the endothelial cells.This experiment was performed using the In Vitro Angiogenesis Assay Kit Tube Formation  according to the manufacturer’s instructions. Briefly, after co-culturing with RCC cells, endothelial cells were stained by treating with 2 μM Calcein AM for 30 min, then 5 × 10The 3′ UTR of VEGF-C was constructed into PWPI-LUC- vector  and promoter regions of miR-185-5p and HIF-2α were constructed into pGL3-basic vector (Promega). Cells were plated in 24-well plates and the complimentary DNA were transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. The pRL-TK was used as an internal control. Luciferase activity was measured by Dual-Luciferase Assay reagent (Promega) according to the manufacturer’s manual.Cell lysates were precleared sequentially with normal rabbit IgG  and protein A-agarose. Anti-AR antibody (2.0 µg) was added to the cell lysates and incubated at 4 °C overnight. For the negative control, IgG was used in the reaction. Specific primer sets designed to amplify a target sequence within the human miR-185-5p promoter were listed in the Supplementary Table 6, mixed with Matrigel, 1:1) with various transduction listed above were then injected orthotopically into capsule of left kidney of nude mice (n = 15). Two mice died of anesthesia in the control group and in the Sh-miR-185 group. A bioluminecent imaging system  was used to monitor the primary and metastatic lesions following abdominal injection of 150 mg/kg Luciferin at four different time points  weeks. The animals were killed at the end of 12 weeks, metastases were detected by in/ex vivo bioluminecent imaging, gross examination, and histological staining with haematoxylin and eosin (H & E).Four-to-six-week-old male BALB/c nude mice were housed and fed under specific pathogen-free conditions. The animal models in this study were approved by the Animal Ethical Committee of the Chinese PLA General Hospital. We first generated stable SN12-PM6 cells with lentiviral vectors of pLV-Luci-U6-VHL and luciferase. This stable cell line was further divided and infected with pLV-puro, pLV-AR, pLV-sh-miRNA-185, or pLV-AR-sh-miR-185. About 60 male nude mice were randomly divided into four groups without blinding for injection of cells with control, overexpression of AR (OE-AR), knockdown of miR-185-5p (Sh-miR-185), and overexpression of AR with simultaneous knockdown of miR-185-5p (OE-AR + Sh-miR-185). The luciferase stable expressing SN12-PM6-sh-VHL cells , the rabbit anti-HIF2α (Abcam), the rabbit anti-CD34 (Abcam), the mouse anti-D2-40 (Abcam), rabbit anti-VEGF-A (Abcam), and rabbit anti-VEGF-C (Abcam) were used for staining at dilutions of 1:100, 1:1000, 1:100, 1:40, 1:100, and 1:50, respectively. For mouse samples, we used rabbit polyclonal antibody to HIF2α (Abcam) and D2-40 (GeneTex) at dilutions of 1:1000 and 1:40, respectively. The primary antibody was recognized by the biotinylated secondary antibody (Vector), and visualized by VECTASTAIN ABC peroxidase system and peroxidase substrate DAB kit (Vector).t-test or ANOVA with SPSS 17.0  after estimating the variance between groups. Linear correlation analyses were performed to determine the correlation between the gene expression levels. The logistic regression model was performed to select the risk factors associated with AR expression. The sample size of human tissues or animals was estimated by preliminary data. p < 0.05 was considered statistically significant.Data were expressed as mean ± SEM from at least three independent experiments. Statistical analyses involved All relevant data are available within the article and Supplementary Files, or available from the authors upon request.Supplementary Information