The intracerebral hemorrhagic stroke (ICH) is associated with high mortality and severe disability in survivors, which causing about 42% of the disability-adjusted life years lost and 50% of all stroke patients dead within 1 year. Although early functional training is recommended to facility rehabilitation after the stroke, the benefit and safety are still controversial.This systematic review aims to investigate whether early physical rehabilitation could have a beneficial effect for the patients with ICH compared with standard rehabilitation care.I2) and Chi-Squared will be applied for heterogeneity detection between clinical trials. A value of P < .05 will be considered statistically significant.Pubmed, Embase, and Cochrane library will be searched to include randomized control trials which investigate the rehabilitation effective of the early mobilization for patients with ICH compared with routine nursing or standard care. Rev-Man version 5.3 will be used to perform all calculations related to the meta-analysis. Dichotomous data will be calculated in terms of a fixed or random effect model and expressed by the relative risk (RR) with 95% confidence interval (CI). The Cochrane collaborations tool in the following aspects was used to assess the risk of bias (ROB) in included studies. The inconsistency index (This study will explore the role of early physical rehabilitation and provide insight for clinicals to improve rehabilitation results of ICH.INPLASY2020110068. The hemorrhagic stroke is caused by leaking or bursting of the blood vessel, which account for about 20% of all strokes. The intracerebral hemorrhagic stroke (ICH) is associated with high mortality and severe disability in survivors,,4 which causing about 42% of the disability-adjusted life years lost and 50% of all stroke patients dead within 1 year.Stroke is one of the main factors leading to major disability and death in most countries in recent years, which reported that more than 795,000 people are affected in America.–9 However, the optimal initial rehabilitation time of functional activity is not clear. Several studies indicated that early physical rehabilitations might have a benefit for the rehabilitation among the patients with ICH.–12 One study by Hsiao-Ching Yen etc. suggested that the early out-of-bed mobilization whin 24 to 72 hours might improve the functional independence compared with the standard rehabilitation (in-bed training). Ning Liu etc. conducted a multicenter, randomized controlled study compared early physical rehabilitation starting within 48 hours with the standard care group commencing rehabilitation after 7 days. They showed that early physical rehabilitation within 48 hours had a beneficial effect for the patients with ICH. Moreover, previous researches demonstrated that very early mobilization rehabilitation could increase the risk of bleeding again and cause worse outcomes.–16 It is still controversial whether the early exercise is beneficial and safe for ICH. Therefore, this systematic review aims to explore whether early physical rehabilitation has a better effect for the patient with ICH than standard rehabilitation care.Early functional training is recommended to facility rehabilitation after the stroke.2https://inplasy.com/) on November 17, 2020 . This review will develop following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement guidelines.This protocol was registered with the International Platform of Registered Systematic Review and Meta-Analysis Protocols , China National Knowledge Infrastructure (CNKI), Wanfang and Chongqing VIP. No restrictions in language, publication date or publication year are applied. Additional source including WHO clinical trial registry website, clinicaltrail.gov, conference abstracts, will also be searched. Further, the references of included trails will also be checked for more potential studies. The search strategy will use keywords and mesh term including “stroke”, “early physical rehabilitation”, “intracerebral hemorrhage”, and “early mobilization”, etc. The selection process will be presented in a PRISMA flow diagram Fig. .2.2The RCTs which investigate the rehabilitation effective of the early mobilization for ICH compared with routine nursing or standard care will be eligible for this systematic review.2.2.1Adult patients with ICH.2.2.2Early physical rehabilitation therapy starting within 72 hours of stroke onset.2.2.3Standard rehabilitation care.2.2.4Only randomized controlled trials (RCTs) will be included.2.2.5The primary outcomes include the mortality, the functional performance and quality of life evaluated by the Functional Independence Measure (FIM-motor), Postural Assessment Scale for Stroke Patients, and Functional Ambulation Category (FAC), Modified Barthel Index, Short Form-36 (SF-36), and other validated scales. The secondary outcomes include the length of stay, recurrent stroke, and adverse effects.2.3The retrieved records will be imported into the EndNote X9 software and the duplicate publications were excluded. Two reviewers independently read the titles and abstracts of all identified records to exclude those that are clearly not relevant. Then the full texts of the articles retained are reviewed to further determine their eligibility. Differences opinions were resolved by consensus.The data are extracted by 2 reviewers independently using a pre-defined form. The following characteristics of included studies are collected: the first author, publication year, country, number of included patients, age, the detail of the intervention, treatment duration, follow-up time, and adverse events. Any discrepancies are resolved by consensus.2.4The risk of bias (ROB) for included studies will be assessed using the Cochrane collaborations tool by 2 independent reviewers form the following dominants: the assessment includes sequence generation; allocation concealment; blinding of participants, personnel, and outcome assessors; incomplete outcome data; selective outcome reporting; and other sources of bias. Any differences between the authors on the data extraction and quality assessment will be resolves by discussion.2.5(I2) and Chi-Squared will be calculated for heterogeneity detection between studies. When assessing the difference in outcome, heterogeneity involving all trials will be examined. A value of P < .05 will be considered statistically significant. Egger test will be calculated to detect publication bias if there are at least ten studies included.Rev-Man version 5.3 will be used to conduct all calculations related to the meta-analysis. Dichotomous data will be calculated in terms of a fixed or random effect model and expressed by the relative risk (RR) with 95% confidence interval (CI). Continuous data will be presented as mean difference and 95% CI. The inconsistency index 2.6The subgroup will be performed based on the several participants characters (including the various severity of stroke and different age), and therapy duration and start time of intervention, and the studies qualities according to the assessment results of risk of bias, etc.2.7Sensitivity analysis will be carried out for primary outcomes by removing 1 study a time to investigate the robustness of the meta-analysis result.3ICH is an important public health problem leading to high rates of death and disability in adults. It is crucial that commencing functional rehabilitation interventions as soon as possible to decrease disability caused by disease, meanwhile making sure the safety to avoid potential further damage for the ICH. More high-quality evidence focusing on the special schedule of early physical rehabilitation are needed to provide relevant reliable evidence to support the clinical practice.Conceptualization: Fang Zhang.Data curation: Yibao Zhang, Xiaoyun Peng, Bo Wan, Lulu Wang.Formal analysis: Shunqin Mao, Bo Wan, Lulu Wang.Investigation: Shunqin Mao, Mei Liu, Dengjuan Sun.Methodology: Xiaorong Cheng, Shunqin Mao, Rui Ma, Yingcun Bao, Qun Li, Mei Liu.Project administration: Rui Ma, Qun Li.Software: Xiaoyun Peng, Yingcun Bao, Dengjuan Sun.Supervision: Fang Zhang.Visualization: Yibao Zhang.Writing – original draft: Xiaorong Cheng, Yibao Zhang.Writing – review & editing: Yingcun Bao.


Thus, AR technology could be an effective way to enhance student learning about medicines.There is increasing use of augmented reality (AR) technology, which combines the virtual and real world, in the tertiary education sector. AR enables flexibility in student learning, since this technology may be used in the face to face setting and may also be accessed by students at any time outside of this setting. The purpose of this study was to develop an AR tool and investigate its effectiveness for learning about the medication naloxone using AR in a MagicBook; and determine student opinions on its acceptability and usability. Using a sequential explanatory, mixed method design, 25 undergraduate pharmacy students were recruited to participate in the study. Pre- and post-tests were used to measure changes in knowledge and a survey was used to collect information on the usability and acceptability of AR for learning. The findings of the study indicated that AR technology was able to support student learning on the chosen topic, showing 42% improvement in quiz score  In the health and education sectors, the growing use of immersive technologies, such as Augmented Reality (AR) and Virtual Reality (VR), have generated new opportunities to enhance the development of clinical and communication skills of students as well as the way in which they learn ,2. WhilePharmacy Times developed an AR application that reveals extra content when readers scan the pages [The use of AR as a teaching resource represents a student-centred approach to learning. AR tools may be used to augment traditional teaching modalities such as face to face learning. These resources also provide students with the flexibility to easily access and revisit content and concepts at any time or location outside of the classroom setting. It is suggested that students who actively participate in an authentic learning environment focus their attention , not onlhe pages . HoweverA key learning area for pharmacy students is the use of medicines and students must acquire comprehensive knowledge about an extensive range of medicines and devices. Students must also become well versed in the application of this knowledge so that, when practising as a pharmacist, they will be able to optimise patient care through provision of appropriate medication and device counselling. The learning and appropriate development of such skills can be enhanced when students actively participate in the learning process . TertiarThese limitations provided the motivation to explore the use of AR in pharmacy teaching. AR has the ability to provide visual images of products and devices for use inside and outside the classroom setting. By purchasing only one product or device, images such as 360-degree photos or videos of the product or device may be produced, while images of foil strips of tablets or other components contained within the packaging may also be captured for use in AR resources. If the presentation of a product or the design changes only one new product needs to be purchased to update the AR resource. Devices may be disassembled so students can view the various components. Using AR enables students to see products, replacing the need to purchase multiple medications and devices for use once or twice a year. For products with restricted access, photos or videos can be produced by accessing these on only one occasion. While AR may be used to produce standalone resources where a picture or other “trigger” is scanned to provide a video, picture or other information, it may also be incorporated into interactive learning modules that provide visualisations and augmented information about medicines to the student, delivered using a flexible online platform that would otherwise not be routinely available. It was this approach of developing a training module which incorporated AR elements which was evaluated in this study.Pharmacist Only medicine, making it more widely available over the counter from Australian community pharmacies without requiring a prescription from a medical practitioner. For all other purposes, naloxone remains classified as Prescription Only. There are no data which indicate that there has been increased naloxone usage since its rescheduling, or whether expanding the provision of naloxone is associated with greater risk-taking by patients or any increase in drug use [Naloxone, an opioid antagonist, was selected as the subject of this AR study. From 1 February 2016, naloxone, when used for the treatment of opioid overdose, became classified as a drug use . HoweverThis study was approved by the Human Research Ethics Committee at the University of Newcastle, Australia, approval number, H-2013-0151.This study was conducted between March and October 2018.https://www.wikitude.com/), HP Reveal® and Blippar (https://www.blippar.com/). The advantages of these options are summarised in Before commencing the design and application research phase, three AR systems were evaluated to determine their suitability for use in this study. Feasibility was established using the following principles: affordability, user friendliness, design and availability. Initially, a web-based user portal was identified as an efficient and cost-effective method of creating an AR application. This system has the ability to create an AR experience without prior knowledge of programming code . Three w® is a popular AR system which provides a robust platform for AR production and that produces web-based AR content without coding knowledge. Content produced is stored on the cloud [® was able to perform many sophisticated tasks, in terms of ease of use for creating content required for this study, it was more difficult to use than the other options.Wikitudehe cloud . While t® provided an easily accessible web-based AR platform that linked to a cloud database. Access to the platform to create AR resources was free. Once created, AR trigger images, videos, audio or 3D models, which are immediately available and can be visualised using the camera of any mobile device [® software was that all videos had to be under 100 MB in size [HP Reveale device . The tim in size .® is a user-friendly web-based AR browser that enables the user to interact with tangible objects through the camera or touchscreen of a mobile device [®, it can visualise images, audio, video and 3D models over the trigger images, but there is a cost involved [Blippare device . As withinvolved , which m® appeared to be the most suitable for this study and was chosen as the platform to develop an AR MagicBook to build student learning about the medication supply process for naloxone.Overall, HP RevealThree currently practicing and academic pharmacists were individually consulted to determine the learning objectives for the AR activity and the therapeutic content to include within the AR MagicBook, as presented in ®. The trigger image could then be used to conceal a linked item known as an overlay which provides more information, for example the medications inside the box or a video of a pharmacist providing over the counter information on the use of the medication to a customer. An example page can be seen in ® downloaded, is held over the image of the medication box it triggers the appearance of the overlay.A MagicBook is like a traditional book in appearance; pages can be turned, pictures looked at and the text read without any additional technology. However, each page also contains an AR image relevant to the content . Known aThrough an iterative process of consultation, the final AR images for including in the 14-page book were selected with appropriate information included with the images. The MagicBook was made available to students as a hard copy version for first time use of the module. Subsequently it was made available online for students to access at any time and in any place through the learning management system Blackboard.The AR MagicBook was initially used with undergraduate students in the Bachelor of Pharmacy (Honours) degree at the University of Newcastle, Australia. A convenience sampling method was used and students from all four years of the Bachelor of Pharmacy (Honours) degree were invited to participate in the study which was conducted during a separate session outside of student’s usual course requirements, in their own time. Students who consented to participate individually completed the AR activity on a single occasion by attending a session which included a pharmacy academic who was able to provide clinical context and a researcher who managed the completion of the AR research activity sequentially as outlined below. A sequential explanatory, mixed method design  was used.® had been downloaded and were shown how to proceed with the module. Working with a copy of the MagicBook, one page at a time, the students held the tablet over the trigger image to reveal the overlay(s). Guided by the AR markers, students worked at their own pace so that when they had worked through the information presented, they moved on to the page(s) that followed (Step 2). Students then completed a post-test, which included the same questions as the pre-test. Lastly, students were asked to complete a survey to collect their opinions on the usability and acceptability of the AR module for learning (Step 3).Students were asked to complete a demographic survey followed by a pre-test to collect baseline knowledge of the topic (Step 1). Students were then provided with a tablet or iPad on to which HP RevealThe questions contained in the pre- and post-tests were identical to determine whether participants had acquired content knowledge after completing the AR MagicBook activity. The pre- and post-tests contained four multiple choice questions, each with four optional answers, and one short-answer question to assess the students’ knowledge of the process of naloxone supply .t-tests were conducted to determine significant differences for each question as well as for the overall results.Student results from the pre- and post-tests were analysed using SPSS . The total of correct responses for each question pre- and post-test were calculated, and two-tailed paired Student acceptance of using the MagicBook for learning was measured with a usability/acceptability survey comprising six statements using a five-point Likert scale, with scoring from 1 (strongly disagree) to 5 (strongly agree). The survey also included two open-ended questions for collection of qualitative information from the students on what they liked about using AR and possible improvements for future medicines learning. Thematic analysis was used to group and analyse the responsesA total of 25 students completed the AR module, 14 females and 11 males. Most of the students (n = 23) were studying the fourth year of the Bachelor of Pharmacy (Honours) degree, with two students in the third year of the program. Half of the participating students had no prior experience using AR technology, 40% had limited use of 10 h or less per year, with only one student using AR technology for more than 5 h per week.p < 0.001, sd = 1.42, df = 24)  .Results for user acceptance of the AR MagicBook scored highly in all areas, as seen in We identified recurring key words and phrases in the qualitative data that aligned with the aims of the study. Emerging themes described student experiences using the AR tool.It consolidated my knowledge by providing helpful videos of patient-pharmacy interaction, which greatly improved my knowledge and understanding of naloxone”, and “The animation/videos were interesting and made it easier for me to understand and remember the context”.Theme 1 was centred on knowledge acquisition, e.g., “It was interactive and engaging”, and “It was very stimulating”, and “The animation videos were interesting”.Theme 2 was based on emotional responses and illustrated positive feelings about using the tool, e.g., “It was easy to follow”.Theme 3 was related to usability of the tool, e.g., “It could incorporate 3D models and models that can be moved”.Theme 4 related to AR MagicBook content and identified that additional content such as 3D images and subtitles could enhance the utility of the AR tool, e.g., “This study was designed to develop a tool which would enable pharmacy students more flexible opportunities for engaging in learning about medicines beyond their limited formal class time, with a focus on taking a student-centred approach to learning. An interactive learning module using AR technology was developed for upskilling pharmacy students in the supply of naloxone, and presented in a MagicBook format. As this was the first time the students had experienced learning with the use of AR technology in their program this pilot study provides important information about their perceptions on its acceptability and usability as well as its effectiveness for learning. It is envisaged that future applications of this work would enable students to engage with the AR learning experience at any place and at any time throughout their pharmacy program.In the user acceptance survey, all students agreed/strongly agreed that the AR MagicBook enabled the presentation of information on medicines as a practical patient-pharmacist interaction which enhanced and stimulated their learning, and that it could be appropriately used in a range of learning activities throughout their program. To maintain and build on these positive responses from students, it was important to consider the positive impact of AR on learning. One suggestion is that interactive technology can improve student achievement levels through an increased level of motivation ,22,23,24p < 0.001. With question one, however, most of the students (92%) obtained the correct response at baseline. This is possible given that students have prior foundation knowledge based on previous study or experience.Although all students agreed/strongly agreed that they thought their knowledge of medicines improved after using AR for learning, establishing the extent of these gains to determine the effectiveness of this learning strategy was important. Overall, there appeared to be a significant improvement in student knowledge about the medication naloxone after using the AR MagicBook for learning. As the results show, for four of the five test questions, there was a significant improvement between the pre- and post-test results, with an increase in the overall mean score from the pre-test (57%) to the post-test (99%), which was statistically significant at p = 0.03) [p < 0.05) [Similar learning outcomes were reported in a study by Albrecht et al., with a small group of 10 medical students in Germany learning about forensic medicine. However, in this study, the students were divided into an experimental group (n = 6) using AR for learning, as well as a control group (n = 4), with the experimental group showing a statistically significant gain in knowledge compared to the control ( = 0.03) . Another < 0.05) . Similar < 0.05)  or in co < 0.05) . These f < 0.05) .There is considerable evidence to support the observation that AR produces good learning outcomes because students who are actively involved in the learning process are better able to retain knowledge resulting in a higher level of achievement ,25,27. M® was the most appropriate AR tool to use at the time of the study, with rapid changes in technology HP Reveal® has now been superseded with similar software.A limitation of this study is the small sample size of 25 university students and the lack of a control group for the comparison of using AR for learning. However, the results gained from this pilot study provides baseline information on which to build for the use of augmented and virtual reality technologies within pharmacy education in Australia. It should also be noted that while HP RevealAR technology can position the learner within a real-world context whilst enabling participatory learning practices. While the results of this study require further investigation, it is clear that AR technology has the potential to engage and stimulate students in a way that could create a more inclusive learning environment. This study has identified the benefits that AR can bring to tertiary pharmacy education. AR technology can be implemented at minimal cost to the creator and the learner as the vast majority of students own mobile devices. Web-based AR platforms are easy to learn and implement and can provide educators with a simple introduction to the world of augmented and virtual reality technology. Further research is required to investigate the use of AR for learning about a range of different medications and build on the implementation of AR in tertiary pharmacy education.


We aimed to investigate the association of long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (lnc-MALAT1) with acute ischemic stroke (AIS), and its association with disease severity, inflammation, and recurrence-free survival (RFS) in AIS patients. One hundred and twenty AIS patients and 120 controls were recruited. Venous blood samples from AIS patients (within 24 h after symptoms onset) and controls (at entry to study) were collected to detect plasma lnc-MALAT1 expression by real-time quantitative polymerase chain reaction. AIS severity was assessed by the National Institutes of Health Stroke Scale (NIHSS) score. Plasma concentrations of inflammation factors (including C-reactive protein (CRP), tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-8, IL-10, IL-17, and IL-22) were measured and RFS was calculated. lnc-MALAT1 expression was decreased in AIS patients compared to controls, and it had a close correlation with AIS . For disease condition, lnc-MALAT1 expression negatively correlated with NIHSS score and pro-inflammatory factor expression , while it positively correlated with anti-inflammatory factor IL-10 expression. Furthermore, lnc-MALAT1 expression was elevated in AIS patients with diabetes. For prognosis, no statistical correlation of lnc-MALAT1 expression with RFS was found, while a trend for longer RFS was observed in patients with lnc-MALAT1 high expression compared to those with lnc-MALAT1 low expression. Acute ischemic stroke (AIS), a common disorder caused by embolic or thromboembolic occlusion in an artery supplying the brain, results in irreversible infarction of brain tissue and functional impairment, which causes economic and social burden for families –3. CurrLong non-coding RNAs (lncRNAs) are RNA transcripts more than 200 nucleotides and rarely encode proteins, which play essential roles in regulation of protein-coding genes and signaling pathways related to development of diseases . lncRNA In total, 120 AIS patients and 120 controls with high stroke risk factors were recruited from our hospital between July 2014 and June 2017. For the AIS patients, screening criteria were as follows: i) primary diagnosis of AIS by clinical presentation, brain non-contrast computed tomography (CT), or diffusion-weighted magnetic resonance imaging, according to the “Guidelines for the Early Management of Patients with Acute Ischemic Stroke: A Guideline for Healthcare Professionals from the American Heart Association/American Stroke Association (AHA/ASA)”; ii) morAt entry to the study, baseline data of AIS patients were obtained through interviews and medical records, including age, gender, body mass index (BMI), smoking, hypertension, diabetes mellitus, hyperlipidemia, hyperuricemia, and chronic kidney disease (CKD). Hypertension was defined as a history of high blood pressure ≥140/90 mmHg) reported by the respondent or current use of antihypertensive medication; diabetes mellitus was defined as a previous diagnosis, treatment with insulin or oral hypoglycemic medications, fasting plasma glucose ≥126 mg/dL, or glycosylated hemoglobin ≥6.5%; hyperlipidemia was defined as current use of antilipidemic medication, total cholesterol ≥5.70 mmol/L, serum triglycerides ≥1.70 mmol/L, or low-density lipoprotein cholesterol ≥3.10 mmol/L; hyperuricemia was defined as serum uric acid ≥420 μmol/L for men and ≥360 μmol/L for women 0/90 mmHg. Severitg for 10 min at 4°C (within 30 min) to acquire supernatant. The supernatant was further centrifuged at 16,000 g for 10 min at 4°C to obtain the plasma, which was stored at -80°C for further analysis. lnc-MALAT1 relative expression in the plasma of AIS patients and controls was determined by real-time quantitative polymerase chain reaction (RT-qPCR). AIS patients' CRP concentration in plasma was detected using fully automatic POCT fluorescence immunoassay analyzer , and the plasma level of inflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), IL-8, IL-10, IL-17, and IL-22 were measured by human enzyme-linked immunosorbent assay (ELISA) kits  according to the manufacturer's recommendations.Venous blood samples were collected from AIS patients (within 24 h after symptoms onset) and controls using ethylene diamine tetraacetic acid (EDTA) tubes, and were subsequently centrifuged at 1600 Using QIAamp RNA Blood Mini kit , total RNA was extracted from plasma samples. Then, reverse transcription to cDNA was performed using PrimeScript™ RT reagent kit  , and qPCR was performed using TB Green™ Fast qPCR Mix (Takara). GAPDH was applied as the internal reference. Primers used in the RT-qPCR were as follows: lnc-MALAT1, forward (5′→3′): TCCTAAGGTCAAGAGAAGTGTCAG, reverse (5′→3′): GTGGCGATGTGGCAGAGAA; GAPDH, forward (5′→3′): TGACCACAGTCCATGCCATCAC, reverse (5′→3′): GCCTGCTTCACCACCTTCTTGA.All AIS patients received appropriate treatments as recommended by the 2013 AIS Guidelines , and were followed-up regularly or as clinically indicated. The last follow-up date was June 30, 2018, and the median follow-up duration was 25.0 months (range: 1.0-42.0 months). RFS was calculated from the date of hospital admission to the date of recurrence or death.t-test, Wilcoxon rank-sum test, or chi-squared test. Correlation between variables was analyzed by Spearman rank test. Diagnostic value of variables was assessed by receiver operating characteristic (ROC) curve analysis and the derived area under the curve (AUC) as well as 95% confidence interval (CI). RFS profile is shown by plotting the Kaplan-Meier (K-M) curve, and the difference of RFS between groups was determined by the log-rank test. Factors affecting RFS were analyzed by univariate and multivariate Cox's proportional hazards regression models. All tests were two-sided, and a P value <0.05 was considered statistically significant.SPSS 24.0 statistical software  was used for statistical data processing, and GraphPad Prism 6.01  was applied for graph plotting. Continuous variables are reported as means±SD or median and interquartile range (IQR), and the categorical variables are reported as number (percentage). Differences between groups were determined by Student's No difference of age (P=0.363), gender (P=0.479), and BMI (P=0.159) between AIS patients and controls was observed. Also, the percentage of smoking (P=0.796), hypertension (P=0.421), diabetes mellitus (P=0.182), hyperlipidemia (P=0.296), hyperuricemia (P=0.888), as well as CKD (P=0.360) was similar between the two groups. The detailed information about other baseline characteristics are shown in Mean NIHSS score and mean values of inflammatory factors are reported in lnc-MALAT1 expression was decreased in AIS patients compared to controls (P<0.001) . The ROClnc-MALAT1 expression was negatively correlated with NIHSS score in AIS patients  .Lnc-MALAT1 expression was negatively associated with expression of inflammatory factors including CRP  , TNF-α , IL-6 P, IL-8 P, and IL-To investigate the correlation of lnc-MALAT1 expression with some risk factors for AIS, all patients were categorized into different subgroups according to the risk factors . Lnc-MALPatients were divided into lnc-MALAT1 high expression group and lnc-MALAT1 low expression group according to the median value of lnc-MALAT1 expression. No difference of RFS was found between the groups, while there was a trend for longer RFS in lnc-MALAT1 high expression (P=0.053) compared to lnc-MALAT1 low expression .vs <29.0 mg/L) was associated with worse RFS (P=0.006) (vs <29.0 mg/L) independently predicted reduced RFS (P=0.024).Univariate Cox's regression analysis showed that there was a trend for better RFS in lnc-MALAT1 high expression patients compared to lnc-MALAT1 low expression patients  (P=0.063), while CRP (≥29.0 P=0.006) . MoreoveOur results indicated that: 1) lnc-MALAT1 expression was decreased in AIS patients compared to controls, and it had a close correlation with AIS; 2) lnc-MALAT1 high expression was correlated with decreased NIHSS score as well as reduced inflammatory factors levels in AIS patients; and 3) lnc-MALAT1 high expression showed a trend for prolonged RFS compared to lnc-MALAT1 low expression in AIS patients, but without statistical significance.in vitro mimic of ischemic stroke conditions  –19. Asnditions . Anothernditions . These sPrevious clinical trials focus mainly on the exploration of lnc-MALAT1 in cancer patients, showing that lnc-MALAT1 plays a tumor-promotive role, while information about the role of lnc-MALAT1 in neurological diseases or cerebrovascular diseases is still limited –24. OnlIn our study, we assessed the association of lnc-MALAT1 with AIS, and observed that lnc-MALAT1 expression was lower in AIS patients compared to controls, and lnc-MALAT1 expression presented good diagnostic value for AIS, which might be due to its protective effect via repressing cell apoptosis of BMECs in these diseases. Moreover, we investigated the correlation of lnc-MALAT1 expression with disease severity as well as inflammation in AIS patients. We observed that lnc-MALAT1 high expression was associated with decreased NIHSS score and reduced level of inflammatory factors . In addition, there was a trend for an increased proportion of diabetes  in AIS patients with lnc-MALAT1 high expression. The possible reasons for these results might be as follows: 1) lnc-MALAT1 had a protective effect on brain microvascular endothelial cells through inhibiting cell apoptosis, thereby contributed to maintaining healthy brain endothelium that was essential for normal cerebrovascular physiology, therefore the severity of AIS was attenuated and a decreased NIHSS score was observed ,20; 2) lAs to the prognostic value of lnc-MALAT1 in human diseases, it has been identified as a valuable biomarker for disease prognosis in several cancers, while limited clinical studies have been found in AIS. Only one study reports that lnc-MALAT1 high expression is associated with prolonged main adverse cardiovascular and cerebrovascular events (MACCE)-free survival in atherosclerosis patients . To our Some limitations existed in our study: 1) the sample size (120 AIS patients) was relatively small, thus the statistical power might be low; 2) the median follow-up duration (25.0 months (range: 1.0-42.0 months)) was relatively short, thus the correlation of lnc-MALAT1 expression with long-term prognosis remains to be further investigated; and 3) detailed mechanism of lnc-MALAT1 in AIS is still unclear, and further study is still needed.In conclusion, lnc-MALAT1 was downregulated and presented a close association with AIS, and its high expression correlated with decreased NIHSS score as well as reduced inflammation in AIS patients. Moreover, there was a trend for better RFS in AIS patients with lnc-MALAT1 high expression compared to those with lnc-MALAT1 low expression, despite the lack of statistical correlation.


Despite the improvement of relapse-free survival mediated by anti-angiogenic drugs like sunitinib (Sutent®), or by combinations of anti-angiogenic drugs with immunotherapy, metastatic clear cell Renal Cell Carcinoma (mccRCC) remain incurable. Hence, new relevant treatments are urgently needed. The VEGFs coreceptors, Neuropilins 1, 2  are expressed on several tumor cells including ccRCC. We analyzed the role of the VEGFs/NRPs signaling in ccRCC aggressiveness and evaluated the relevance to target this pathway.NRP1 and NRP2 genes by a CRISPR/Cas9 method. The number of metabolically active cells was evaluated by XTT assays. Migration ability was determined by wound closure experiments and invasion ability by using Boyden chamber coated with collagen. Production of VEGFA and VEGFC was evaluated by ELISA. Experimental ccRCC were generated in immuno-competent/deficient mice. The effects of a competitive inhibitor of NRP1, 2, NRPa-308, was tested in vitro and in vivo with the above-mentioned tests and on experimental ccRCC. NRPa-308 docking was performed on both NRPs.We correlated the NRP1, 2 levels to patients’ survival using online available data base. Human and mouse ccRCC cells were knocked-out for the NRP1 and NRP2 genes inhibited cell metabolism and migration and stimulated the expression of VEGFA or VEGFC, respectively. NRPa-308 presented a higher affinity for NRP2 than for NRP1. It decreased cell metabolism and migration/invasion more efficiently than sunitinib and the commercially available NRP inhibitor EG00229. NRPa-308 presented a robust inhibition of experimental ccRCC growth in immunocompetent and immunodeficient mice. Such inhibition was associated with decreased expression of several pro-tumoral factors. Analysis of the TCGA database showed that the NRP2 pathway, more than the NRP1 pathway correlates with tumor aggressiveness only in metastatic patients.Knock-out of the Our study strongly suggests that inhibiting NRPs is a relevant treatment for mccRCC patients in therapeutic impasses and NRPa-308 represents a relevant hit.The online version contains supplementary material available at 10.1186/s13046-021-01832-x. Presented data are the mean ± SEM of two or three independent experiments, each performed in triplicate.https://www.ncbi.nlm.nih.gov/pubmed/19399780. (Accessed: 1st February 2019). Three-dimensional conformations of NRPa-308 were generated using iCon, the LigandScout v.4.3 conformer generator [NRP1 (PDB ID: 6FMF) and NRP2 (PDB ID: 5DN2) structures were retrieved from the Protein Data Bank (PDB) . The NRPenerator  (default786-O cells expressing luciferase (Luc 1) or RENCA cells expressing luciferase (Luc 2) were injected subcutaneously into the flanks of 5 weeks old nude female mice or Balb-C mice. Treatment by NRPa-308 in carboxymethyl cellulose was carried out by oral gavage 3 days a week; the control group was treated with carboxymethyl cellulose. Tumors measurements were carried out once a week with a caliper and by luciferase measurements with IVIS chamber as previously described . All aniTumor sections (5 μm cryostat sections) were fixed in 4% paraformaldehyde for 20 min at room temperature and blocked in 1% donkey serum in tris-buffered saline (TBS) for 2 h. Sections were then incubated overnight with anti-rabbit LYVE-1 polyclonal  or rat monoclonal anti-mouse CD31  and monoclonal anti- mouse α-smooth muscle actin  antibodies. Preparations were mounted and analyzed with a Leica microscope, and counted at a 10x magnification.www.cbioportal.org, TCGA Provisional; RNA-Seq V2). Data were available for 534 RCC tumor samples or from 1020 different cell lines. The results published here are in whole or in part based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/ [Normalized RNA sequencing (RNA-Seq) data of The Cancer Genome Atlas (TCGA) were downloaded from cBioportal  and migration (NRP2). These results were surprising since they described that NRP1 knock-down decreased the AKT activity, a major pathway involved in cell proliferation/survival. Moreover, the NRPs-mediated signaling pathways were associated with cell proliferation and migration in several cancers [NRP1 knock-down decreased NRP2 expression, an observation that was not described in the Cao Y et al papers. This observation, confirmed with two independent shRNA, eliminates an unspecific effect, and suggests a crosstalk between the NRP1 and NRP2 signaling pathways , NRPs’ KO decreased the migration velocity of 786-O cells which is consistent with the results obtained by down-regulating NRP1 and NRP2  or VEGFA (NRP2 KO) has to be paralleled with variable up- or down-regulation of NRP2 or NRP1 which reflects a clonal specificity  as compared to the control group, in nude mice  aggressiveness in comparison to its effect on normal dermal fibroblasts (HDF). The NRPa-308 effects were compared to those of the reference treatment for ccRCC, sunitinib, and to the commercially available NRPs’ inhibitor EG00229. EG00229 was poorly efficient in inhibiting the metabolic activity of ccRCC cell lines  as compared to tumor cells  served as the reference value. The SI was below 1 which indicates that NRPa-308 is more efficient on tumor cells and that its general toxicity is low. The SI of NRPa-308 was below those of sunitinib suggesting a higher efficacy and a lower toxicity of NRPa-308  as compared to the parental cells. 786R cells presented a four-fold and a nine-fold reduction of the NRP1 and NRP2 mRNA levels , had no50 . NRPa-302 was high in the control group and increased in a dose-dependent manner  and immune tolerance. The modifications to their mRNAs, analyzed by qPCR, were compiled in Fig. We were puzzled by confronting our previous results on breast cancers with those enclosed in this manuscript; high dose of NRPa-308 was the most efficient on models of breast cancers whereas it has no effect in models of ccRCC [For M0 ccRCC patients, expression of VEGFR2, NRP2, VEGFC, VEGFR3, PLXA2 above their respective best cut off was of good prognosis for DFS  for NRP2, VEGFC) and significant (S) for VEGFR2, VEGFR3, PLXA2). Expression above the best cut off for Sema3A and Prox1 was of poor prognosis for DFS. For M0 patients OS, NRP1, VEGFR1, VEGFR2, NRP2, VEGFC, VEGFR3, Sema3F and PLXA2 . For M1 ccRCC patients, only Sema3F (T) was correlated to a longer PFS whereas eight parameters were correlated to a worse prognosis (NRP1 (S), VEGFR2 (S), Sema3A (S), NRP2 (S), VEGFC (S), VEGFR (S)3, PLXA2 (T) and Prox1 (S)). NRP1 (S), VEGFR1 (S), VEGFR2 (S), Sema3F (S) and PLXA2 (S) were correlated with a longer OS while Sema3A (S), PLXA1 (S), NRP2 (S), VEGFC (S) and Prox1 (S) were correlated to a shorter one.For TNBC, VEGFA was curiously associated with a longer PFS (T) but NRP1 (S), VEGFR1 (S), PLXNA1 (S), NRP2 (T), VEGFC (T), Sema3F (S) and PLXNA2 (T) were correlated with a shorter one. NRP1 (S), VEGFR1 (S), VEGFR2 (T), PLXNA1 (S), VEGFC (T), Sema3F (S) and PLXNA2 (T) were correlated with a shorter OS. NRP2 was not correlated to survival in that case.We defined a score by attributing a relative weight of − 2 for a gene associated with a significant poor prognosis and a relative weight of − 1 for a trend. Inversely, a relative weight of 2 was given for a gene associated with a significant good prognosis and 1 for a trend. NRP1 and NRP2 pathways were considered separately. For the NRP1 pathway, a − 1 score was obtained for the DFS and OS of M0 ccRCC patients, − 6 and 2 scores for PFS and OS of M1 ccRCC patients, and − 5 and − 7 scores for the PFS and OS of TNBC patients. For the NRP2 pathway, positive score of 4 and 6 were obtained for the DFS and OS of ccRCC patients, − 8 and − 2 scores for M1 ccRCC patients and − 5 and − 7 scores for the PFS and OS of TNBC patients.These results showed that NRP1 and NRP2 signaling pathways, in general, strongly correlate with shorter survival for the most aggressive cancers, M1 ccRCC and TNBC. However, NRP2 is correlated with shorter DFS and OS in ccRCC while NRP1 is more involved in TNBC patients’ survival. These results suggest that NRP1 targeting is more adapted for TNBC while NRP2 targeting is more adapted for ccRCC.. We also showed that inflammatory cytokines compensate for the inhibition of NRPs’ pathways. These compensatory mechanisms are key for an optimized targeting of NRPs in the context of ccRCC treatment. Our results had to be compared to those of Cao Y et al who showed that inhibition of experimental tumor growth generated with cells downregulated for NRPs only relies on microenvironment shaping [NRPs, through their direct effect on tumor cells (stimulation of metabolic activity and migration/invasion) and on cells of the microenvironment (angio/lymphangiogenesis and immune tolerance) are key signaling molecules stimulating ccRCC growth and metastasis. However, the multi partnerships of NRPs render difficult the determination of the relative importance of each pathway. Moreover, we discovered that NRP1 and NRP2 signaling cross-talked to establish a steady state depending on the production of VEGFA and VEGFC. This phenomenon was strikingly observed in NRP1 KO clones in which compensatory expression of NRP2 was insufficient to inhibit cell migration .Additional file 2: Fig. S1. Study of down-regulation of NRPs by shRNA in 786-O cells. (A-B) Effects of the downregulation of NRP by shRNA on NRP1 and NRP2 mRNA expression measured by qPCR. (C) Effects on cell metabolic activity measured by MTT assays. (D) Down-regulation of NRPs decreased cell migration. Bevacizumab increased this effect for NRP1 down-regulation. (E) Down-regulation of NRPs had no effect on VEGFA and VEGFC production measured by ELISA. *p < 0.05; **p < 0.01; *** p < 0.001.Additional file 3: Fig. S2. Effects of NRP1 or NRP2 gene invalidation in RENCA cells. (A) NRP1 and NRP2 protein levels were evaluated by flow cytometry in control (RENCA), in #NRP1 4.1.7 and #NRP2 5.1.8 clones. (B) Effects of NRPs KO on RENCA cell metabolic activity measured by MTT assays. (C) Effects of NRPs KO in RENCA cells on the VEGFA and VEGFC protein levels measured by ELISA. *p < 0.05; **p < 0.01; *** p < 0.001.Additional file 4: Fig. S3. NRPs KO in 786-O tumor cells inhibited experimental RCC growth in immunodeficient mice. (A) Experimental tumors in nude mice (5 mice per condition) were obtained after injection of 3 × 106 wildtype (Ctrl) or NRPs KO 786-O cells. One NRP1 (#NRP1 2.7) clone and one NRP2 (#NRP2 2.3) clone were injected. Tumor volume is presented. *p < 0.05; **p < 0.01; *** p < 0.001.Additional file 5: Fig. S4. NRPs KO in RENCA tumor cells inhibited experimental RCC growth in immunodeficient mice. (A) Experimental tumors in nude mice were obtained after injection of 3 × 105 control  or NRPs KO RENCA cells (5 mice for each condition). One NRP1 KO clone (4.1 7) and one NRP2 KO clone (5.1 8) were injected. Tumor volume at the indicated times is presented. Each curve stands for an individual mouse.Additional file 6: Fig. S5. In-vivo effects of NRPa-308 on mice weight. The weight of nude mice xenografted with 786-O cells and treated with increasing doses of NRPa-308 was evaluated once a week.


The FDA-approved anti-androgen Enzalutamide (Enz) has been used successfully as the last line therapy to extend castration-resistant prostate cancer (CRPC) patients’ survival by an extra 4.8 months. However, CRPC patients eventually develop Enz-resistance that may involve the induction of the androgen receptor (AR) splicing variant ARv7. Here we found that Cisplatin (Cis) or Carboplatin, currently used in chemotherapy/radiation therapy to suppress tumor progression, could restore the Enz sensitivity in multiple Enz-resistant (EnzR) CRPC cells via directly degrading/suppressing the ARv7. Combining Cis or Carboplatin with Enz therapy can also delay the development of Enz-resistance in CRPC C4-2 cells. Mechanism dissection found that Cis or Carboplatin might decrease the ARv7 expression via multiple mechanisms including targeting the lncRNA-Malat1/SF2 RNA splicing complex and increasing ARv7 degradation via altering ubiquitination. Preclinical studies using in vivo mouse model with implanted EnzR1-C4-2 cells also demonstrated that Cis plus Enz therapy resulted in better suppression of EnzR CRPC progression than Enz treatment alone. These results not only unveil the previously unrecognized Cis mechanism to degrade ARv7 via targeting the Malat1/SF2 complex and ubiquitination signals, it may also provide a novel and ready therapy to further suppress the EnzR CRPC progression in the near future. It is estimated that there will be 191,930 new cases of PCa and 33,330 associated deaths worldwide in 20202. Androgen deprivation therapy (ADT), the current standard treatment for advanced PCa via reducing androgen synthesis or preventing androgens from binding to the androgen receptor (AR), has little effect to reduce AR expression6.Prostate cancer (PCa) is the second leading cause of death among men in the United States7. However, patients still eventually develop Enz-resistance8. Clinical studies indicated the failure of ADT with Enz (ADT-Enz) treatment might be linked to the AR splicing variant ARv79, a process that involves the splicing of full-length AR pre-mRNA10 and altering the RNA splicing pattern11. While other mechanistic studies also indicated that the development of Enz-resistance could also be due to Glucocorticoid receptor (GR) activation, AR gain, ligand binding domain mutations, or alternative AR variants14, the emergence of the ARv7 splice variant remains the most interesting explanation, supported by clear and strong evidence from human clinical sample surveys15.Enzalutamide (Enz) could suppress the castration resistant PCa (CRPC) and might extend patients overall survival by 4.8 months16 and is widely used as a chemotherapeutic reagent to suppress many solid tumors17. Early mechanistic studies aimed at understanding how Cis might suppress tumor progression focused on its ability to alter the DNA damage-repair (DDR) cellular functions17, yet its linkage to alter AR function remains unclear.Cisplatin (Cis) is the first FDA-approved platinum compound for cancer treatmentHere we found Enz combined with Cis could to restore/increase Enz sensitivity via targeting the Malat1/SF2 RNA splicing signals to suppress the generation of the ARv7 mRNA or via altering the ubiquitination of ARv7 protein. These unexpected findings for the new Cis mechanism may help us to quickly develop a novel and ready therapy to suppress progression due to Enz-resistance to further extend CRPC patients’ survivals.C4-2 and CWR22Rv1, were obtained from the American Type Culture Collection (ATCC) and maintained in RPMI 1640 media  supplemented with 10% fetal bovine serum. EnzR1_C4-2 clone was selected by culturing cells with Enz in a dose-escalation manner with the initial culture at 10 μM Enz, and then gradually increased to 20 μM Enz. Cells proliferation rates were analyzed by MTT assays monthly. The process of acquiring drug resistance took around 12 months. EnzS4_C4-2B and EnzR4_C4-2B were obtained from from Dr. Allen Gao’s lab.3 cells per well) and cultured for 0, 2, 4, and 6 days. Cells were harvested, and absorbance (at 570 nM O.D.) were calculated and recorded after incubating with yellow tetrazolium MTT -2,5-diphenyltetrazolium bromide agent at 37 °C for 30 min and dissolving in DMSO.Cells were seeded in 24-well plates , and AdnaTest ProstateCancerDetect  instructions. The total mRNA was amplified by cDNA synthesis kit . The samples were obtained after patients signed Informed Consent Agreements.GAPDH or RPL13A (for CTCs samples).Total RNAs were isolated using Trizol reagent , or adna kit (for CTC samples) and 2 µg of total RNA was subjected to reverse transcription using Superscript III transcriptase (Invitrogen). 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. Expression levels were normalized to the expression of Cells were lysed in lysis buffer and proteins (50 µg) were separated on 10% SDS/PAGE gel and then transferred onto PVDF membranes . After blocking membranes with 5% non-fat milk, they were incubated with appropriate dilutions of specific primary antibodies, anti-AR , anti-GFP , anti-GAPDH , anti-α-tubulin , the blots were incubated with HRP-conjugated secondary antibodies and visualized using ECL system .6 parental EnzR3_22Rv-1 cells were subcutaneously injected with matrigel (1:1) into the right hip of nude mice. After tumors grew to ~400 mm3, the mice were randomly separated to 4 groups, and then treated with DMSO, Cis , Enz  or Cis plus Enz for 20 days. Tumor sizes were measured with calipers every 5 days, and the volume of tumor calculated as follows: [(short axis2 × long axis)/2]. Based on preliminary data (3 mice data), if p < 0.05 and power = 90%, each group needs at least 4 mice. A sample will be considered to be excluded when it is 1.5 times greater than the quartile interval. Day 20 results were analyzed by One-way ANOVA. The entire Animal procedure followed UCAR regulations and was accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).1 × 10t test. Multiple comparison was analyzed by One-way ANOVA, p ≤ 0.05 was considered statistically significant.All experiments were performed at least 3 times with data points in triplicate. All statistical analyses were carried out with GraphPad Prism . The data values were presented as the mean ± S.D. (except QPCR is presented as mean ± SEM). Differences in mean values between two groups were analyzed by two-tailed unpaired Student’s 18. However, most patients may still develop Enz-resistance (after an average of 4.8 months treatment)18, and recent studies indicated that the development of Enz-resistance might involve the induction of the AR splicing mutant/variant, ARv7, which could still transactivate AR at the castration level of androgens19. Knowing how to target this Enz-induced ARv7 may help in the development of a novel therapy to overcome the Enz-resistance to further improve CRPC patients’ survival rates.After PCa patients developed CRPC, the current standard therapy includes either docetaxel (Doc) chemotherapy or ADT-Enz to prevent androgens from binding to AR (or with ABI to prevent androgen biosynthesis)21. The linkage of Cis or Carboplatin to alterations of the ARv7 expression, however, remains unclear.The Cis (and its derivative Carboplatin with fewer side effects) is currently used widely to suppress various tumors due to its capacity to cross-link the DNA to trigger apoptosis and/or alter the DNA damage-repair (DDR) signalsWe first established the EnzR CRPC cell line from C4-2 cells  Fig. . We then17 in the mutated PC3-AR F876L cells  Fig. , before Together, the results shown in Fig. We then applied the MTT proliferation assay to examine the consequences of Cis-degraded ARv7 on altering the Enz-sensitivity, and results revealed that treating with 20 μM Enz resulted in little suppression of the EnzR1_C4-2 growth Fig. . In contImportantly, we found those low doses of Cis or Carboplatin had little effect on activating the cleaved PARP-1, the marker of the apoptotic pathway Fig. , suggestFinally, results from the interruption assay also revealed that overexpressing (OE) ARv7 (OEARv7) back into the EnzR1_C4-2 cells also led these cells to become more resistant to Cis treatment compared with the control group .Together, the results shown in Fig. Fig. .To dissect the mechanisms why Cis can decrease ARv7 (and AR-F876L) protein expression, we first assayed the Cis effects on ARv7 (and AR876) protein stability by treating with proteasome inhibitor MG132, in EnzR3_CWR22Rv1 cells. The results revealed that Cis could degrade ARv7 after 3 hr treatment Fig.  left, anTogether, the results in Fig. 22, we were interested to see if Cis may also function through altering this Malat1/SF2 RNA splicing complex to decrease the ARv7 mRNA biosynthesis/expression. As expected, the results revealed in EnzR1_C4-2 cells, treating with Cis led to decrease the mRNA expression of lncRNA-Malat1 and ARv7 in a dose-dependent manner  to increase ARv7 biosynthesis/expressionner Fig. . In contner Fig. .Fig. 5CiFurthermore, we found that treating EnzS1_C4-2 cells with 5 μM Enz alone for 2 weeks led to increase the expression of Malat1 and ARv7 Fig.  cells.Together, the results shown in Fig. AR promoter into pGL3 luciferase vector (pAR-luc) and transfected pAR-luc into EnzR3_CWR22Rv1 cells for luciferase assay. The results revealed that 1 μg/ml Cis could also decrease AR mRNA expression at the transcriptional level via altering AR promoter activity  for mRNA purification. The quantitative PCR results revealed that Cis treatment in 3 out of 6 samples resulted in decreased ARv7 expression Fig. .3 the mice were then i.p. injected with Enz (30 mM/kg/every other day) and with or without i.p. injections of Cis  for 20 days23 and tumors measured weekly by calipers.Finally, to study the in vitro results in an in vivo model, we first confirmed most in vitro conclusions in the EnzR3_CWR22Rv-1 (EnzR_22Rv-1) cells that have better tumor growth rate in mice Fig. . Mice weThe results revealed that treating with Cis  of AR, named AR-F876L)25 that is no longer sensitive to Enz treatment25. Early clinical data revealed that 3 of 29 CRPC patients receiving ARN509 treatment had the AR-F876L mutant26. Balbas et al.26 further confirmed these findings by showing that AR-F876L could convert Enz into an AR agonist and reverse the growth inhibition of Enz treatment.Most CRPC patients who receive Enz may develop the Enz-resistance, which might involve multiple mechanisms. For example, Enz or its 212 in a subset of PCa cells due to relief of AR-mediated feedback repression of GR expression27. GR and AR are closely related members of the nuclear receptor superfamily with a similar DNA-binding-domain, and GR could replace part of AR roles during development of Enz-resistance28. Importantly, Arora et al.12 found the GR agonist dexamethasone was sufficient to confer Enz-resistance, whereas a GR antagonist could partially restore sensitivity.Enz might also increase the glucocorticoid receptor (GR) signals8, and these ARv7 positive patients had lower PSA response rates than ARv7 negative patients with shorter PSA progression–free survival , suggesting CRPC patients with ARv7 might have poor response to Enz treatment, and Enz treatment might enhance ARv7 expression8. ARv7 is constitutively active and reported to regulate a transcriptional program that is similar, but not identical, to that of AR in CRPC32, and treating with Enz could increase the expression of constitutively active ARv79 that might transactivate AR target genes to promote CRPC progression in an androgen-independent manner33.However, the emergence of the AR splicing variant ARv7 may represent the key factor for the development of Enz-resistance as recent clinical studies from CRPC patients demonstrated that 39% of metastatic CRPC patients treated with Enz had detectable ARv7 in their circulating tumor cells®, that was shown to selectively degrade AR protein in some, but not all cell types34, with fewer adverse effects in all in vivo mice studies40. Importantly, ASC-J9®, but not the anti-androgens Enz or Casodex, could degrade both full-length AR and the AR variant ARv7 or AR mutants including AR-F876L33.At least 2 newly developed compounds have been demonstrated to be able to target the ARv7. The first one is the AR degradation enhancer ASC-J9nd compound identified as an ARv7 inhibitor to suppress the PCa progression41. Liu et al. found niclosamide could suppress ARv7 protein expression via a proteasome-dependent pathway to suppress the PCa cell growth in vitro and in vivo41.Niclosamide, an anti-helminthic compound, is the 242, and miR-124 could also suppress ARv7 along with EZH2 and Src signals42.The microRNA (miR), miR-124, has recently been identified as a tumor suppressor to suppress the PCa progression44, since its approval in 1978 by the U.S. Food and Drug Administration. Currently, medical oncologists apply Cis chemotherapy in common clinical practice to treat various tumors with either 70 mg/m2 every 3 weeks or 20 mg/m2 daily × 5 days every 3 weeks or 20 mg/m2 weekly 47. Our effective Cis dose used in the in vivo model here is 3.5 mg/kg mouse that is less than the allowed (extrapolated to human) dose used to treat human tumor patients.While all 3 compounds mentioned above may have the capacity to target the ARv7 in various in vitro PCa cell lines and in vivo models, none of these compounds are ready to be used in CRPC patients that already developed Enz-resistance. In contrast, Cis or Carboplatin have been widely used as chemotherapy in various tumors, including PCa17. Its linkage to alter the AR signals, especially the degradation of AR mutant ARv7, however, remains unclear. Our results showing Cis may function via degrading ARv7 to restore/increase Enz sensitivity to further suppress EnzR cell growth and to delay the Enz-induced Enz-resistance in CRPC may have clinical relevance to be immediately used to treat those CRPC patients who received Enz treatment and developed Enz-resistance.Interestingly, all previous studies for the Cis effect to suppress tumor progression focused on its capacity to cross-link to DNA that resulted in triggering apoptosis and interfering with the process of cell division49. Here, we demonstrate there are therapeutic windows that can suppress lncRNA-Malat1 function and enhance ARv7 degradation before cell apoptosis, which means it is possibly able to increase Enz therapeutic efficacy via ARv7 degradation without carrying those unwanted adverse effects.The potential adverse effects of Cis treatment include myelosuppression, asthenia and gastrointestinal disorders, as well as long-term cardiac, renal and neurological consequences, which may result in its discontinuation and limit its therapeutic efficacy50. Importantly, Carboplatin like Cis, can also target ARv7 (see Fig. Alternatively, we may be able to use Carboplatin to replace Cis since Carboplatin shares similar anti-tumor effects with Cis, but has less adverse effects than Cissee Fig.  as well see Fig. .In conclusion, our results from using multiple CRPC cell lines, a mouse model, and human CTC samples, not only reveal unrecognized mechanisms by which Cis can degrade ARv7/AR-F876L via suppressing the lncRNA-Malat1/ARv7 and boost ubiquitination signals (Fig. Supplementary legendsFig S1Fig S2Fig S3Fig S4Fig S5Fig S6Fig S7


The androgen receptor (AR) plays a predominant role in prostate cancer (PCa) pathology. It consists of an N-terminal domain (NTD), a DNA-binding domain (DBD), a hinge region (HR), and a ligand-binding domain (LBD) that binds androgens, including testosterone (T) and dihydrotestosterone (DHT). Ligand binding at the LBD promotes AR dimerization and translocation to the nucleus where the DBD binds target DNA. In PCa, AR signaling is perturbed by excessive androgen synthesis, AR amplification, mutation, or the formation of AR alternatively spliced variants (AR-V) that lack the LBD. Current therapies for advanced PCa include androgen synthesis inhibitors that suppress T and/or DHT synthesis, and AR inhibitors that prevent ligand binding at the LBD. However, AR mutations and AR-Vs render LBD-specific therapeutics ineffective. The DBD and NTD are novel targets for inhibition as both perform necessary roles in AR transcriptional activity and are less susceptible to AR alternative splicing compared to the LBD. DBD and NTD inhibition can potentially extend patient survival, improve quality of life, and overcome predominant mechanisms of resistance to current therapies. This review discusses various small molecule and other inhibitors developed against the DBD and NTD—and the current state of the available compounds in clinical development. The prostate gland, located beneath the bladder, secretes alkaline prostatic fluid that constitutes 30% of the semen . The proAmong men worldwide and in the United States, PCa is a leading contributor to overall cancer incidence . In 2020PSA gene transcription, making serum PSA levels a strong indicator of AR transcriptional activity and subsequently of disease state. Specifically, PSA levels are positively associated with disease stage and clinical grade . Th. ThAR gein (LBD) ; of thes23FQNLF27 motif at the N-terminal interacts both with activation function 2 (AF2) on the C-terminus and with coactivators in a ligand-dependent manner to form an AR dimer  located between amino acids 142 and 485, is the main region responsible for mediating AR transcription . The NTDAR dimer . Additio the LBD . Co-regu the LBD .The DBD is critical for AR function. It plays a role in AR dimerization and binding of dimerized AR to select motifs on target DNA . Contrib629RKLKKL634motif of the hinge region allows nuclear translocation through possible interactions with nuclear import machinery  domain (amino acids 718–741) . The LBDCYP11A1), a rate-limiting enzyme that begins T production from cholesterol . UIn the nucleus, new intermolecular interactions arise, and subsequent steps allow intermolecular interactions to occur between the DBDs of two ligand-bound AR monomers. The D-box regions bind to the partner monomer (DBD–DBD interaction), and the P-box regions of each AR monomer binds to the target DNA D 42]. T. T42]. TCo-regulators activate (co-activator) or repress (co-repressor) transcription by altering the 3D structure of chromatin, influencing the stability of the receptor, facilitating nuclear transportation, affecting DNA binding, and regulating molecular interactions ,47. In CPost-translational modifications influence both AR genomic activity and non-genomic protein dynamics in the cytoplasm or nucleus. The AR can acquire modifications throughout all domains, with phosphorylation and SUMOylation sites concentrated in the NTD, acetylation and methylation sites in the DBD, and ubiquitination sites localized mostly to the LBD . Such moIn the normal environment, production of T and conversion to DHT regulate AR activation through the LBD. Co-regulatory proteins and post-translational modifications aid in defining the AR transcriptional repertoire and tuning AR activity. In PCa, these pathways may become dysregulated, subverted, or hijacked to cause outlaw AR activation and the production of an oncogenic transcriptional program. This implicates the AR as a major promoter of cancer growth and disease progression. Indeed, the genes most often altered in hormone-naïve PCa, which is characterized by no prior treatment with hormone therapies, are those involved in the AR signaling axis . Often, for his discoveries concerning hormonal treatment of prostatic cancer” [castration”, remains key to the treatment of advanced and metastatic PCa. ADT is achieved through surgical, or more commonly chemical, castration [In a 1941 landmark study, prominent Urologists Drs. Charles Huggins and Clarence Hodges reported on the benefits of castration to aid in the reduction of advanced and metastatic PCa burden and the worsening of clinical condition when androgens were administered. This study highlighted the importance of androgen in driving disease state. Based on these observations, Dr. Huggins received a Nobel Prize in Physiology or Medicine in 1966 “ cancer” ,60,61,62stration . ADT stastration .ADT frequently uses GnRH agonists that prevent androgen production ,64,65. CDuring CRPC progression, subversions within the cell allow a continued oncogenic transcriptional program and a rise in serum PSA despite low serum androgen levels ,15,21,41Additionally, in CRPC, increased androgen synthesis may occur due to upregulated key enzymes used in the steroidogenesis pathway. Upregulation of enzymes that convert androstenedione to T, including aldo-keto reductase family 1, member C3 (AKR1C3), also known as 17β-HSD, may lead to elevated androgen levels in this context . In thesAR mutations can also function as a ligand-dependent mechanism of resistance, because in many cases, mutant AR can bind to ligands other than androgens in order to be activated . AdditioCYP17A1 inhibitor abiraterone acetate (ABI) prevents steroidogenesis and subsequently halts, although sometimes incompletely, adrenal production of T [While ADT inhibits production of testicular testosterone, it does not impact production of adrenal DHEA or intratumoral androgen, which can also drive CRPC. The ion of T ,17. Inition of T , the useion of T , and morion of T . Patiention of T .In addition to ABI, AR inhibitors have been developed and investigated as both first- and second-line therapies to address some mechanisms of resistance utilized by CRPC ,19. NextLigand-independent resistance mechanisms can result from altered co-regulator repertoire that modulates the various steps of AR activation, increased growth factor-AR crosstalk, altered post-translational modifications to AR, and alternative splicing of AR mRNA . CertainAR alternative splicing produces AR variants (AR-Vs) that are contributors to ligand- and LBD- specific AR treatment resistance. Many known, and most clinically relevant, AR-Vs lack the LBD, making them naturally resistant to ADT , LBD-speAR-Vs can dimerize with each other to work independent of the full-length AR, or they can dimerize and work synergistically with the full-length AR ,83. ThisV567es currently clinically identifiable from blood or tissue samples associated with CRPC [At least 22 AR-Vs have been described  with the latter resulting in an addition of 16 amino-acids to the C-terminal side of exon 3. AR-V7 mRNA can be found both in hormone naïve PCa and in CRPC . AR-V7 pv567es expresses the very C-terminal end of the LBD, encoded by exon 8, and lacks exons 5, 6, and 7 [V567es transcript was detected in 23% of CRPC bone metastases [v567es were detected in whole blood samples from CRPC patients, but only AR-V7 was detected in blood from patients who were treatment-naïve [v567es, was prognostic for resistance to taxanes in CRPC patients [v567es expression resulted in epithelial hyperplasia by 16 weeks, and invasive adenocarcinoma is evident by 1 year of age [v567es levels were upregulated in xenograft tumors that had acquired ENZA resistance [v567es homodimerize and heterodimerize with each other and also heterodimerize with full-length androgen receptor (AR-FL) in a ligand-independent manner [Unlike AR-V7, AR6, and 7 . Another6, and 7 ,98. The 6, and 7 . ARV567etastases ,97. Bothnt-naïve . AR-V7, patients . A genetr of age . Similarsistance . AR-V7 at manner .Most known AR-Vs lack the LBD and retain the NTD. However, not all AR-Vs follow this profile—a unique 45 kDa AR variant (AR45) is detected in normal tissue that lacks the NTD and expresses the LBD preceded by a novel seven amino-acid long N-terminal extension . No tumoIn most variants, the DBD is intact, reflecting the significant role played by this domain in AR transcriptional activity. Only one known AR-V, the cell membrane-localized and inactive AR8, splices out exon 2, thus disrupting the DBD ,106. BecNo clinical therapy to date completely addresses androgen-independent pro-tumor activity. The production, activity, and enrichment of AR-Vs during disease progression emphasize a clinical need for AR inhibitors that target non-LBD sites. As of 2013, all identified gain-of-function AR-Vs expressed in CRPC contain both the full NTD and DBD ; hence, The NTD emerges as a potential target for AR inhibition; it is implicated as a promoter of ligand-independent AR activity, has a role in nuclear localization, binds co-regulatory molecules, appears necessary for AR function in tumorigenic proliferation, and is conserved in all gain-of-function AR-Vs ,90,108. Beyond EPI-001 and derivative compounds, NTD targeting therapies have been tested. Chlorinated peptides, called sintokamides, extracted from a marine sponge, were shown to target the AR-NTD. Sintokamide A inhibited LNCaP proliferation while reducing AR transcriptional activity ,114,115.The DBD is an easier domain to target based on rational design as docking programs have the ability to identify candidate inhibitors and improve such inhibitors for specificity . The DBDA surface-exposed pocket on the AR-DBD was identified as an alternative drug-target site for AR inhibition, and small molecules designed to selectively bind the pocket effectively block transcriptional activity of full-length AR and AR-V forms at low- to sub-micromolar concentrations . The comAnother way to target the DBD is by using a polyamide designed to bind to the consensus sequence of ARE sites and disrupt AR binding . The maiSalts of pyrvinium (Viprynium), an anti-helmintic therapeutic effective against pinworms, was shown to inhibit the growth of cancer cells; in particular, this inhibition of growth occurred during glucose starvation . It was Other anti-helminthic drugs that target the AR-Vs have been identified, including niclosamide . This drIn summary, NTD and DBD are attractive targets for AR inhibition. Such inhibitors may extend survival and improve quality of life, especially when ADT or current ASIs fail. However, predictable challenges will arise due to the highly mutagenic nature of cancer. The NTD and DBD, like LBD, may acquire point mutations , potenti


Moreotatic BC –11. Alth−/AR+ BCs , which express genes commonly detected in luminal tumors, such as XPB-1, SCUBE2, SPDEF, and FOXA1  has provided a proof-of-principle for its application in BC therapy. Specifically, the development of new, more effective, and safer AR-targeted agents has once again opened up the debate on whether or not to use these compounds in BCs and especially in TNBCs, for which no specific treatment guidelines are available and systemic chemotherapy remains almost the only option in both early and advanced stages of the disease. Anti-androgen therapies showed promising results in preclinical models of BCs –18 and spatients –21. Of nThe complexity of AR action is further underscored by studies on non-genomic actions mediated by the hormone receptor in BCs. The ligand-bound receptor interacts with various signaling effectors in the extranuclear compartment of target cells. Depending on cell type and hormone concentration, androgen-triggered association of AR with the tyrosine kinase Src activates Src-dependent signaling pathway, leading to cell proliferation, while hormone-stimulated interaction between AR and filamin A controls machinery driving cell motility and invasiveness . Of noteBleach and Mcllroy discuss the complexity of AR-driven mechanisms within the context of our current knowledge of steroid intracrinology in BC. The paper describes the action of key mediators of transcriptional or non-transcriptional AR-induced events in BC, with particular emphasis on the role of intracrine ligands and their fluctuations. Interestingly, the review outlines the role of AR/ERα crosstalk occurring at transcriptional level in BCs, together with factors that modulate their interaction. The simultaneous targeting of AR and ERα is an intriguing proposition and might provide a valid approach to overcome therapeutic resistance in BC.In this Research Topic, we have invited leading groups to contribute review and original research articles on the role of AR in BC. Three reviews within this Research Topic address some of the questions still pending and identify challenges in the field, with the aim of re-examining the intricate molecular landscape of AR and offering new clues as to its prognostic and therapeutic value in BCs. Giovannelli et al. review major findings on the biology of AR in BC, with a focus on its prognostic and predictive role in TNBCs. The authors discuss the development of future therapeutic strategies in this BC subtype in light of the group’s most recent discoveries obtained using new site-specific compounds that specifically perturb the non-genomic mechanism mediated by AR in preclinical models of TNBCs (Over the last decade, conflicting evidence has been reported on the role of AR in BCs. of TNBCs . InhibitChen et al. explore the role of AR and AR-related mechanisms in various BC subtypes, with a focus on experimental limitations and issues encountered in translating research from bench to bedside. The review spotlights the need to develop more appropriate AR detection methods and to identify new markers of AR responsiveness for better stratification of patients who might benefit from AR-targeted treatments.Lastly, Michmerhuizen et al. reports promising data on the effects of seviteronel, a non-steroidal selective CYP17-lyase inhibitor that blocks AR activation. Although the compound exhibits a limited effect in monotherapy, it shows great promise in radiosensitizing AR+ TNBCs in preclinical models. These findings may help expand the toolkit of radiotherapists and oncologists.BC therapy has evolved over the years from surgery, chemotherapy, radiation therapy, and hormone therapy to combinatorial treatments , 25. HowBravaccini et al. examines the role of AR as a prognostic marker in a Sub-Saharan African setting, where access to healthcare and cancer prevention services is extremely poor. The paper reports that TNBCs analyzed among the Tanzanian population expressed very high AR levels, opening up new therapeutic perspectives in this low-income country.Differences in BC outcome among specific patient groups based on age, ethnicity, disability, and other factors are widely recognized. By comparing the biological characteristics of Tanzanian and Italian BC patients matched for date of and age at diagnosis, the research article by In recent years, precision medicine strategies have yielded very encouraging results that lay the groundwork for more personalized diagnosis and treatment of AR-expressing BCs. However, much work is still needed to further explore the molecular and clinical aspects of this disease. The efforts of multidisciplinary teams combining molecular endocrinologists, oncologists, clinicians, and radiotherapists are expected to deliver high-quality research and therapeutic advances in the global battle against BC.AT and GC wrote, reviewed, and approved the content of this editorial. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AR and MAP3K1 plasmid. Cell lysates were used to analyze androgen receptor (AR) production. A novel hemizygous AR variant  and a rare heterozygous MAP3K1 variant  were identified by WES in the proband and her mother. Bioinformatic analysis predicted these two variants to be pathogenic. Multiple amino acid sequence alignments showed that p. His690 and p. Arg260 are conserved among various species. His690Glu is a mutation that decreased the AR production, whereas the Arg260Cys mutation increased the AR production. The novel compound variants of the AR and MAP3K1 genes also increased the production of AR protein. Thus, the phenotype of the patient may be caused by defects in both the AR and MAP3K1 signaling pathways. Compound variants of the AR and MAP3K1 genes resulted in a specific phenotype in this patient with AIS. WES might reveal genetic variants that explain the heterogeneity of AIS.Androgen insensitivity syndrome  is the most frequent cause of 46, XY disorders of sex development (DSD). However, the correlation between genotype and phenotype has not been determined. We conducted a systematic analysis of the clinical characteristics, hormone levels, ultrasonography data and histopathology of a 46, XY Chinese patient with AIS. The family was followed up for nearly 8 years. We applied whole-exome sequencing (WES) for genetic analysis of the pedigree and performed bioinformatic analysis of the identified variants. Human embryonic kidney 293T/17 (HEK293T/17) cells were transiently transfected with wild-type or mutant  Furthermore, the exact cause of this variation is not well understood. With the development of next-generation sequencing, useful information for explaining this heterogeneity, such as unidentified coactivator proteins, is expected to be forthcoming.Androgen insensitivity syndrome  is the most frequent cause of 46, XY disorders of sex development (DSD). AIS is characterized by a patient exhibiting a 46, XY karyotype and a normal amount of testosterone secretion but with symptoms and signs of androgen deficiency or reduction, such as female external genitalia, blind vagina, and uterine absence ,2. It isAR) gene . More thpatients . The samAR gene, there are many other genes that cause disorders of sex development, such as the MAP3K1 gene. MAP3K1 variants were first described in two large families and two of 11 sporadic cases of 46, XY DSD in 2010 [MAP3K1 is one of the more common genes mutated in 46, XY DSD, with 13–18% of these patients showing variants of this gene [MAP3K1 gene is located at 5q11.2 and encodes a key serine/threonine protein kinase (MAP3K1) of approximately 196 kD in the MAPK pathway that activates the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) pathways and plays an important role in cell proliferation, differentiation, and apoptosis [MAP3K1 and AR variants can cause 46, XY DSD, the question of whether a correlation exists between the two molecules needs to be addressed.AIS exhibits overlapping clinical phenotypes with other DSDs, such as ovotesticular DSD and 5α-reductase 2 deficiency ,9. Mülle in 2010 . Recent his gene . The MAPpoptosis . As bothMAP3K1 and AR in a 46, XY Chinese patient with AIS. The patient presented with bilateral inguinal masses , female external genitalia (underdeveloped labia and clitoris), and the presence of a uterus, which was indicative of incomplete Müllerian regression and did not match the typical characteristics of AIS. A novel hemizygous AR variant  and a rare heterozygous MAP3K1 variant  were identified by whole-exome sequencing (WES) in the proband and her mother. Bioinformatic analysis and in vitro experiments showed that these two variants are pathogenic. Our study emphasizes the heterogeneity of AIS, which has significance for research on the heterogeneity of diseases.Interestingly, we detected a compound variant of The Chinese patient with AIS in our study, in whom bilateral inguinal hernias were discovered, was 2.6 years of age in November 2011. We collected her clinical characteristics, physical examination results, hormone levels, ultrasonography data, and pathological examination results to determine the location and nature of the gonads and the appropriate surgical intervention. Peripheral blood samples were obtained from the patient and her parents.We followed this family for nearly 8 years. We closely tracked the patient’s urogenital development, intellectual development, drug treatment regimens and potential complications, and developments such as psychological and cognitive impairment, gender anxiety disorder, and the presence of gonadal tumors.Genomic DNA was isolated from peripheral blood leukocytes using QIAamp DNA Mini Kit  following the manufacturer’s instructions. WES was performed using DNA from peripheral blood. After genomic DNA fragmentation, paired-end adaptor ligation, amplification and purification, human exons were captured by using SeqCap EZ Med Exome Enrichment Kit . A DNA library was generated by postcapture amplification and purification and then sequenced with the Illumina HiSeq sequencing platform. Sequence data alignment to the human genome reference (hg19) and variant calling were performed with NextGene V2.3.4 software to obtain the coverage and mean read depth of target regions. The average coverage of the exome was >100×, which permitted examination of the target region with sufficient depth to exactly match >99% of the target exome. To ensure the accuracy of data analysis, mutations with low coverage in the target area were filtered.http://www.ncbi.nlm.nih.gov/snp/), in the exome variant server (http://evs.gs.washington.edu/EVS/), in the ensemble database and in 500 Chinese controls or when the allele frequency was found to be <0.001 in the database. Pathogenic variants were determined according to Standards and Guidelines for the Interpretation of Sequence Variants published by the American College of Medical Genetics and Genomics (ACMG) in 2015 with Human Genome Variation Society (HGVS) nomenclature.Additionally, annotation information, including the conservation of nucleotide bases and amino acids, predictions of biological functions, frequency in normal populations (Genome Aggregation Database (GnomAD), Trans-Omics for Precision Medicine (TOPMED), Exome Aggregation Consortium (ExAC), and data from Human Gene Mutation Database (HGMD), Clinvar and Online Mendelian Inheritance in Man (OMIM) databases, was performed by using NextGene V2.3.4 and our in-house scripts. A variant was recognized as a mutation when it was not found in dbSNP (AR and MAP3K1 were designed using Primer3 version 1.1.4 (http://www.sourceforge.net) and GeneDistiller 2014 (http://www.genedistiller.org/), as follows: Forward primer (AR): 5′-GCATTCAGGCAGAGCTGGAT-3′; Reverse primer (AR): 5′-CTGCTGTGCATCAATGTGGG-3′; Forward primer (MAP3K1): 5′-GTTGATATATTGTCTGGTATACTGG-3′; Reverse primer (MAP3K1): 5′-AGGGGTCTGCAGATCATATTTTG-3′. Polymerase chain reaction (PCR) was performed in a 50-μl reaction system including 4 μl genomic DNA, 1 μl forward and reverse primers, 5 μl 10 × PCR buffer, 4 μl dNTPs, and 0.3 μl Taq Hot Start . The PCR conditions were as follows: an initial denaturation step (95°C for 5 min), followed by 40 cycles of denaturation (95°C for 30 s), annealing (65°C for 30 s), and elongation (72°C for 30 s). Amplicons were sequenced using an ABI 3730 system , and sequence analysis was performed using the autoassembler software Chromas 2.6.6 , Mutation Taster (http://www.mutationtaster.org/) and PROVEAN (http://provean.jcvi.org/index.php). Modeling of wild-type and mutant proteins was performed with I-TASSER software (https://zhanglab.ccmb.med.umich.edu/I-TASSER/), and PyMOL Viewer was employed to observe the effect of the two variants on the protein.We applied Clustal W  software to compare 20 species to confirm the conservation of amino acids at mutated positions. To predict the potential pathogenic effects of the two variants, we used online software such as PolyPhen-2 . AR variants (pcDNA3.1-AR-MU) and MAP3K1 variants (pcDNA3.1-MAP3K1-MU) were generated by site-directed mutagenesis. To obtain the AR His690Glu amino acidic substitution (from cytosine to adenine at nucleotide 2070), we used the corresponding primers: primer forward ACGGGCCCTCTAGACTCGAGCGCCACCATGGAAGTGCAGTTAGGGCTGGGAAGG, primer reverse AGTCACTTAAGCTTGGTACCGACTGGGTGTGGAAATAGATGGGCTTGACTTTC. To obtain the MAP3K1 Arg260Cys amino acidic substitution (from cytosine to thymine at nucleotide 778), the following primers were used: primer forward ACGGGCCCTCTAGACTCGAGCGCCACCATGGCGGCGGCGGCGGGGAATCGCGCC, primer reverse ACGGGCCCTCTAGACTCGAGCGCCACCATGGCGGCGGCGGCGGGGAATCGCGCC. The protocol used for site-directed mutagenesis is available upon request. All the primers were purchased from GenScript . The presence and the construct fidelity of pcDNA3.1-AR-MU and pcDNA3.1-MAP3K1-MU were confirmed by an ABI 3730 system .Wild-type AR (pcDNA3.1-AR-WT) and MAP3K1(pcDNA3.1-MAP3K1-WT) or pcDNA3.1-AR-MU and pcDNA3.1-MAP3K1-MU using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). The transfection concentration of a single plasmid was 2.5 μg/ml. When the cotransfection of the two plasmids was conducted, the concentration of each plasmid is 1.5 μg/ml.Then, we used the high glucose Dulbecco’s Modified Eagle’s Medium containing 10% fetal bovine serum (FBS), 5% penicillin-streptomycin and 2 mM L-glutamine to culture the human embryonic kidney 293T/17 (HEK293T/17) cells . HEK293T/17 cells were transfected with plasmids containing the wild-type After 48 h, cells were collected and lysed by mammalian protein extraction reagent (ThermoFisher Scientific) and protease inhibitor cocktail (Sigma Aldrich). The cells lysates were used to analyze the AR protein expression by Western blot.The cells lysates were mixed with Laemmli buffer containing 2-mercaptoethanol. Then, the mixtures were boiled at 95°C for 5 min. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes . After membranes were incubated overnight with primary antibodies, they were washed two times for 10 min with 0.2% tris-buffered saline Tween. Next, membranes had been incubated with secondary antibodies for 1 h. Before 5-min incubation with chemiluminescent HRP substrate , membranes were washed three times for 10 min with 0.2% tris-buffered saline Tween. Finally, bands were visualized by Chemidoc XRS System . The following antibodies were used: rabbit anti-AR (Abcam) and mouse anti-Actin (Servicebio).The patient’s lineage is shown in The patient’s last follow-up was at 10.3 years of age, and the patient identified as female. Gynecological examination confirmed mild clitoromegaly, an existing vagina, no significant labia majora, and the absence of pubic hair or crest for the female external genitalia E. The paHer hormone levels at the last follow-up, including testosterone, 17α-hydroxyprogesterone, follicle stimulating hormone, adrenocorticotropic hormone and cortisol, were normal for a prepubertal patient. Conversely, the patient’s estradiol, prolactin and luteinizing hormone levels were low for a prepubertal patient .AR gene (c.2070C>A) of I-2 and II-1, resulting in a change in the 690th amino acid of the encoded protein from histidine to glutamine  that results in a change in the 260th amino acid of the encoded protein from arginine to cysteine , T = 0.00001  and T = 0.00002 . The serine/threonine kinase domain is located at the C-terminus of MAP3K1, upstream of which there is a conserved caspase-3 cleavage site and a ubiquitin interaction motif (UIM). The N-terminus of MAP3K1 contains two zinc finger structures, including one RING structure and one SWIM region. The heterozygous variant occurs at position 260 (Arg260Cys)  B. Polyphof 0.791 C, and PR species D. HoweveAR variant and the rare heterozygous MAP3K1 variant, we did not find any other DSD gene variants.Other than the novel hemizygous AR and MAP3K1 variants at the protein level, plasmids containing the wild-type AR and MAP3K1 cDNA or carrying AR and MAP3K1 variants were transfected into HEK293T/17 cells. The AR production of HEK293T/17 cells transfected with the pcDNA3.1-AR-MU decreased significantly by about 50% compared with that of cells transfected with pcDNA3.1-AR-WT. Cells transfected with both wild-type plasmids showed a significantly increase in AR protein production, compared to cells with pcDNA3.1-AR-WT or pcDNA3.1- MAP3K1-WT . This result indicates that pcDNA3.1- MAP3K1-WT can increase the expression of AR. The AR production of HEK293T/17 cells transfected with pcDNA3.1- MAP3K1-MU was 6.73 times higher than that of cells with pcDNA3.1- MAP3K1-WT transfection, indicating that the effect of increasing AR production became stronger after MAP3K1 was mutated. Compared to cells transfected with the pcDNA3.1-AR-MU, cells transfected with both mutant plasmids showed a significantly increase in AR protein production (approximately 14 times), which also indicated that the effect of increasing AR production became stronger after MAP3K1 was mutated  and a rare heterozygous variant in the MAP3K1 gene . Bioinformatics analysis predicted these two variants to be pathogenic. And we characterized at a molecular level the pathogenic variants in the AR and MAP3K1 genes that affect the production of AR protein. The rare heterozygous variant in the MAP3K1 gene might act as a genetic modifier of the phenotype, as the patient has rudimentary Müllerian structures, which is uncommon in AIS. Therefore, we consider that the two pathogenic variants are the cause of AIS and that they are the exclusive causative agents of the phenotype of the described patient. Moreover, the confirmation of an AIS diagnosis by WES can reveal additional genetic variants to explain the heterogeneity of the disease.In the present study, we describe a patient with AIS with a novel hemizygous variant in the AIS is highly heterogeneous and divided into three categories according to the degree of genital masculinization: mild androgen insensitivity syndrome (MAIS), partial androgen insensitivity syndrome (PAIS) and complete androgen insensitivity syndrome (CAIS). The clinical phenotypes range from a typical male habitus with mild spermatogenic defects and reduced secondary terminal hair to a full female habitus despite the presence of a Y chromosome. The correlation of genotype and phenotype has not yet been established.AR gene leads to cellular resistance to androgens. More than 1000 AIS-causing mutations in the AR gene have been identified [AR gene variants include gene deletions, splice site mutations, premature stop codons and missense mutations, among others. The most common genetic abnormalities are missense mutations, which often occur in two important segments of the receptor protein: the DBD and LDB regions. Missense mutations that result in a single amino acid substitution are known to produce the most phenotypic diversity.AIS is a congenital disorder in which a defect in the entified . The typAR variants reduce or abolish AR function  and a ubiquitin interaction motif (UIM). As mentioned above, two zinc finger structures, including one RING structure and one SWIM region, are present at the N-terminus of MAP3K1 [MAP3K1 gene variants result in altered binding of cofactors and increase phosphorylation of the downstream MAP kinase pathway targets MAPK11, MAP3K and MAPK1, thereby promoting ERR1/2 and p38 phosphorylation and ultimately leading to 46, XY DSD. Increased phosphorylation of ERK1/2 and p38 may result in decreased expression of SOX9 and increased activity of β-catenin, respectively, which are important signaling molecules in testicular and ovarian-promoting pathways  and a rare heterozygous variant of MAP3K1 . A missense mutation in the AR gene  has been reported in the same codon [MAP3K1 gene might be related to the clinical heterogeneity of AIS.Interestingly, we detected a compound variant of the with AIS ,50–54. Tme codon . Rosa etin vitro experiments of the AR and MAP3K1 variants. His690Glu is an AR mutation that decreased the AR production, whereas the Arg260Cys mutation of MAP3K1 increased the AR production. The novel compound variants of the AR and MAP3K1 genes also increased the production of AR protein. Taken together, these findings suggest that the AR and MAP3K1 variants severely affect the AR protein production, explaining the uncommon phenotype in the patient carrying these variants. Furthermore, we predicted the relationship between AR and MAP3K1. MAP3K1 is expected to activate the transcription factor AP-1 [AR cistrome [MAP3K1 promoter regulation [To explain the molecular mechanism underlying our genetic findings, we carried out tor AP-1 , which mcistrome , leadingcistrome , other sgulation . Casalingulation . Overallgulation . Furthergulation . These rAR and MAP3K1 gene. Our findings extend the gene variant profile of AR and MAP3K1. However, the association between the phenotype and genotype of AIS remains unclear, and more patients need to be studied to provide more comprehensive data.It is undeniable that the present study has some limitations. The study included a 46, XY Chinese female patient with AIS whose grandparents had died, and we were unable to trace potential genetic variation. In addition, there is a lack of basic experiments to validate regulatory mechanism between the In summary, our study contributes to the known heterogeneity of AIS, which has significance for research on the heterogeneity of diseases. The widespread use of WES may reveal further genetic variants, and some genetic variants may help to explain genotype–phenotype associations.