Testosterone-related steroid hormones are associated with various types of diseases, including prostate cancer and androgenetic alopecia (AGA). The testosterone or dihydroxy testosterone (DHT) circulates through the blood, binds to the androgen receptor (AR) in the cytoplasm, and finally enters the nucleus to activate downstream target genes. We previously found that immortalized dermal papilla cells (DPCs) lost AR expression, which may be explained by the repeated cell passages of DPCs. To compensate for the AR expression, DPCs that express AR exogenously were established. In this study, we performed an RNA-Seq analysis of the AR-expressing and non-AR-expressing DPCs in the presence or absence of DHT to identify the downstream target genes regulated by AR signalling. Furthermore, we treated DPCs with minoxidil sulphate, which has the potential to treat AGA. This is the first comprehensive analysis to identify the downstream genes involved in testosterone signalling in DPCs. Our manuscript provides high-priority data for the discovery of molecular targets for prostate cancer and AGA.  This metabolic process is predominantly mediated by 5-alpha-reductase (5α-R)2. DHT has a much stronger affinity for the androgen receptor (AR) than the precursor form of testosterone. Due to the high affinity of DHT, understanding the pathogenesis of prostate cancer or androgenetic alopecia (AGA) may be important. However, the gene networks controlled by testosterone, especially in dermal papilla cells (DPCs) are not fully understood. Interestingly, we accidentally found that the expression of AR is strongly suppressed even in the early passage of human DPCs3. The suppression of AR gene expression under the cultured cell conditions of DPCs has been previously reported in rat-derived DPCs, which is consistent with our finding4. Therefore, our established immortalized DPCs are almost completely negative for AR expression. These situations led us to build the hypothesis that if we exogenously introduce AR via retrovirus, re-constitution of the AR signalling pathway might be possible in immortalized DPCs. We previously carried out the comparison between AR positive or negative DPCs with RNA-Seq, and identified that expression of caveolin-1 is suppressed in AR-expressing DPCs5. However, the downregulated caveolin-1 recovered after the DHT treatment, and this response was observed regardless of whether AR is expressed or not. Based on our previous experience trying to identify the downstream genes of AR, in this study, we set up the experimental design again to cover the gene expression change after DHT stimulation. We set up the AR negative and positive cells of DPCs, and no treated and treated cells with DHT. Furthermore, we classified AR downstream genes which are commonly upregulated or downregulated in AR-expressing cells and DHT-treated cells with AR expression. We then narrowed down the AR downstream genes that were not affected after DHT treatment under the absence of AR expression. Our high quality data would help scientists study the effect of AR signaling.The metabolic form of testosterone, dihydroxy testosterone (DHT) has greater androgenic activity than the parental form of androgen3. Based on the characteristics of the introduced genes , we named this immortalized cell line HFDPC_K4DT. Furthermore, we introduced AR-expressing retrovirus into immortalized DPCs as described in our previous papers5.We obtained human primary DPCs from Promocell  through the local distributor, Takara Bio . The primary cells were maintained in a Follicle Dermal Papilla Cell Growth Medium (Promocell) as per the manufacturer’s instructions. Cells immortalized by the expression of R24C-mutant CDK4, cyclin D1, and TERT have been described in our previous paperWe set up 15 experimental groups to cover all aspects of the biological response to AR signalling activation. Each experimental group contains triplicate samples to ensure reproducibility.Sample 1, HFDPC_K4DT_rep1, K4DT, intact immortalized DPCs.Sample 2, HFDPC_K4DT_rep2, K4DT, intact immortalized DPCs.Sample 3, HFDPC_K4DT_rep3, K4DT, intact immortalized DPCs.Sample 4, HFDPC_K4DT_DHT_rep1, DHT, immortalized DPCs treated with 50 nM dihydroxy testosterone (DHT). No AR expression.Sample 5, HFDPC_K4DT_DHT_rep2, DHT, immortalized DPCs treated with 50 nM dihydroxy testosterone (DHT). No AR expression.Sample 6, HFDPC_K4DT_DHT_rep3, DHT, immortalized DPCs treated with 50 nM dihydroxy testosterone (DHT). No AR expression.Sample 7, HFDPC_K4DT_AR_rep1, AR, immortalized DPCs with retroviral AR expression, No ligand treatment.Sample 8, HFDPC_K4DT_AR_rep2, AR, immortalized DPCs with retroviral AR expression, No ligand treatment.Sample 9, HFDPC_K4DT_AR_rep3, AR, immortalized DPCs with retroviral AR expression, No ligand treatment.Sample 10, HFDPC_K4DT_AR_DHT_rep1, ARDHT, immortalized DPCs with retroviral AR expression, treated with 50 nM DHT.Sample 11, HFDPC_K4DT_AR_DHT_rep2, ARDHT, immortalized DPCs with retroviral AR expression, treated with 50 nM of DHT.Sample 12, HFDPC_K4DT_AR_DHT_rep3, ARDHT, immortalized DPCs with retroviral AR expression, treated with 50 nM of DHT.Sample 13, HFDPC_K4DT_AR_DHT_mino_rep1, mino, immortalized DPCs with retroviral AR expression, treated with 50 nM of DHT and 30 nM of Minoxidil sulphate (MXS)Sample 14, HFDPC_K4DT_AR_DHT_mino_rep2, mino, immortalized DPCs with retroviral AR expression, treated with 50 nM of DHT and 30 nM of MXSSample 15, HFDPC_K4DT_AR_DHT_mino_rep3, mino, immortalized DPCs with retroviral AR expression, treated with 50 nM of DHT and 30 nM of MXSThe cells were treated with DHT or MXS for 24 h at approximately 70% confluence in 35 mm diameter cell culture dish. The total cellular RNA was extracted using the Nucleopsin RNA kit (Takara Bio), according to the manufacturer’s instructions. DNase I treatment was performed with the enzyme included in the RNA extraction kit. The quality of Total RNA was assessed using tapestation, and cDNA libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs). We confirmed that the RIN value of extracted RNA from all samples was 10.0.6. The low-quality reads were removed with the FASTP program7. The filtered reads were mapped to the NCBI reference human genome sequence (CRCh38) using the STAR program8. The mapped data were processed into the expression count data using featureCounts9 included in the Subread package. To extract differentially expressed genes (DE genes), we first performed a pairwise comparison with iDep10. The DE genes were detected with DESeq211 with FDR cut off of 0.050 and a minimum fold change of 3. We also processed the data using TCC-GUI12 for Principal component analysis (PCA), heatmap analysis, and bar plot analysis  was used to obtain pair-end 150 bp sequencing of poly-A tailed RNA. All sequencing reads were processed with PEAT to remove the adaptor sequencesis Fig. .Fig. 1Th13. The number of sample reads is summarised in Fig. 14. The mapping ratio to the reference genome was approximately 95 to 96.3%, which is sufficient for quantifying gene expression15. We compared the expression pattern to the correlation matrix, as shown in Fig. 16. Furthermore, the difference in expression profiling between Samples 10–12: HFDPC-K4DT with AR expression, treated with 50 nM DHT and Samples 13–15: HFDPC-K4DT with AR expression, treated with 50 nM DHT and 30 nM MXS was also very limited, indicating that MXS does not significantly influence the signal activation of AR.The quality score of reads is shown in Figures We performed a pairwise comparison of Samples 1–3: HFDPC-K4DT and Samples 7–9: HFDPC-K4DT with AR expression with no DHT treatment with Dseq2 using iDep. As shown in the left panel of Fig. 17, experimental groups were primarily classified into two clusters: cell cluster with DHT treatment and cell cluster without DHT treatment (Fig. We performed PCA on gene expression profiling. Due to software limitation, the top 25,000 genes were the maximum number of genes involved in the analysis. As demonstrated by the results of 3D-PCA in Fig. As shown in Fig. Furthermore, we analysed the 130 upregulated genes. As shown in Fig. The bar plots of the downregulated 27 candidates are listed in Figures Non-coding RNAs such as lncRNA was reported to control the transcriptional activity of promoter region and gene expression. To elucidate the differentially expressed lncRNA in our study, we identified differentially expressed lnc RNAs. As shown in Fig. 18. The RIN value of all the RNA was 10.0, indicating that the sample we obtained was in near-perfect condition.The RNA quality data was submitted to FigshareTo evaluate the reproducibility of the data, triple biological replicates were established. The setup of experimental groups is described in the method section. We established AR negative cells and positive cells. Similarly, we established DHT ligand-treated cells and untreated cells. In addition to these groups, immortalized DPCs without AR expression and treated by DHT ligands were established. The upregulated or downregulated genes in these groups suggest that detected gene expression change is mediated by a pathway other than the AR pathway.Fig_S1_S24


Cardiovascular diseases are the most common causes of death, in both Poland and the world. Their development and progression are largely influenced by the lifestyle with the presence/occurrence of classic, modifiable risk factors. Among them, low physical activity plays a significant role. The aim of the study was to evaluate the expression of the endothelin-1 gene and its type A receptor, taking into account physical activity  among patients with acute myocardial infarction. A total of 234 patients with acute myocardial infarction were examined, including 167 patients undergoing early post-hospital cardiac rehabilitation and 67 not participating in it. All of them were assessed with the IPAQ questionnaire and the quantitative real-time polymerase reaction method (QRT-PCR). Physical activity in the group of patients after early post-hospital cardiac rehabilitation increased after rehabilitation. Transcriptional activity of the endothelin-1 (ET-1) gene in both studied group of patients increased significantly, but in a group of patients not participating in early post-hospital cardiac rehabilitation more than in a group of patients participating in it. In our study, the expression of ET-1 was also significantly higher in the group of patients with acute myocardial infarction with ST-segment elevation, without diabetes, with lipid disorders, smoking, with normal body weight. Expression of the ENDRA (Endothelin receptor A) gene increased with age. These results prove the beneficial effect of rehabilitation and may indicate another pathomechanism of pro-atherogenic activity of above-mentioned factors. Cardiovascular diseases are the most common causes of death, in both Poland and the world. Their development and progression are largely influenced by the lifestyle with the presence/occurrence of classic, modifiable risk factors. Among them, low physical activity plays a significant role ,2,3.Scientific research emphasizes the beneficial effect of exercise on the human body ,5,6. RegThe vascular endothelium plays an important role in the development of cardiovascular diseases, especially coronary artery disease, heart failure, hypertension, as well as diabetes, and hyperlipidemia. Numerous scientific studies suggest that endothelial dysfunction is the initial stage of atherosclerosis development, preceding the occurrence of morphological vascular changes and clinical disease symptoms ,10,11.The risk factors may lead to the endothelial functions’ failure and intensification of the pro-inflammatory, pro-adhesive and pro-thrombotic reactions and consequently to the development of coronary atherosclerosis. The progression of atherosclerotic lesions is associated with the accumulation of cholesterol particles, migration of monocytes and their differentiation into macrophages, foam cells creation, LDL cholesterol particles oxidation, the presence of proinflammatory cytokines, migration of smooth muscle cells to the inner vessel wall and aggregation of platelets. All the above-mentioned processes lead to the formation of atherosclerotic plaques, their growth, rupture, and consequently—A heart attack . RestoriEndothelin-1  is a 21 amino acid peptide and under physiological conditions, secreted by endothelial cells in small amounts, acting as an auto and/or paracrine mediator. It is produced from preproendothelin and big endothelin with the use of endothelin converting enzyme (ECE) . The factors stimulating the secretion of endothelin-1 include: reduction in endothelial shear stress, adrenaline, thrombin, angiotensin II, hypoxia, vasopressin, interleukin-6, insulin, transforming growth factor-beta, homocysteine, calcium ions, and free radicals. On the other hand, the factors that inhibit its release include: Nitric oxide, prostacyclin, atrial natriuretic peptide, heparin, and prostaglandins ,16.2−, and a reduction in the bioavailability of nitric oxide. As a result of the reduction in NO concentration, the vasomotor functions are impaired with impaired vasodilating functions of the endothelium, which is manifested by excessive vasoconstriction  = 8 MET × number of days in the week × amount of time [min.]Abbreviations explanation: AF1—Hard work factor; MET—Equivalent metabolic; min.—Minutes.Moderate effort factor (AF2) such as cycling at a normal pace, playing volleyball or brisk walking, assigned a value of 4 MET, was calculated by the formula:AF2 [MET] = 4 MET × number of days in the week × amount of time [min.]Abbreviations explanation: AF2—Moderate work factor; MET—Equivalent metabolic; min.—Minutes.The third part of the questionnaire concerned the time spent walking during the day (AF3). Efforts of nature such as walking or walking to work or shopping, and lasting at least 10 min at a time, were assigned with a value of 3.3 MET. The factor was calculated according to the formula:AF3 [MET] = 3.3 MET × number of days in the week × amount of time [min.]Abbreviations explanation: AF3—Light Work Factor; Met—Equivalent metabolic; min.—Minutes.In the last part of the questionnaire, the respondents determined how much time during the day they spent sitting, e.g., at work, watching TV, taking into account weekdays. Based on the above data, patients were classified into a group with a high, sufficient, or insufficient level of physical activity .Examined patients (group A) participated in nationwide comprehensive cardiac rehabilitation program (KOS-ZAWAŁ—Comprehensive Patient Care after a Heart Attack). Other patients (group B) did not participate in it due to a serious health condition, numerous comorbidities that prevent the patient from functioning independently and lack of consent to participate in early post-hospital cardiac rehabilitation. Hospitalization after myocardial infarction lasted approximately 4–5 days. During above-mentioned hospitalization all patients underwent rehabilitation in a hospital ward consisting of simple breathing and circulatory exercises, as well as standing up and walking. On average, 7–14 days after discharge from the hospital, patients participating in early post-hospital cardiac rehabilitation (group A) performed an exercise test on a treadmill, on the basis of which they were qualified for the appropriate model of cardiac rehabilitation:A model—Patients with good exercise tolerance (≥7 METs); recommended training: continuous endurance training, resistance training and general fitness exercises with an intensity of 60 to 80% of the heart rate reserve for 5 days a week, the exercise duration is 60 to 90 min a day,B model—Patients with good and moderate exercise tolerance (≥5 METs); recommended training: Continuous or interval endurance training with an intensity of 50–60% of the heart rate reserve, 45–60 min a day, resistance training for 2–3 days a week, and general fitness exercises for 5 days a week,C model—Patients with low exercise tolerance (3–5 METs); recommended training: interval endurance training and general fitness exercises (5 days a week), elements of resistance training , 45 min a day, and its intensity should be between 40 and 50% of the heart rate reserve,D model—Patients with very low exercise tolerance (<3 MET); recommended training: individual exercises ranging from 30 to 45 min, the intensity of the exercise should be below 20% of the heart rate reserve or cause the heart rate to accelerate by no more than 15–20%.The characteristics of the studied group of patients after myocardial infarction participating in early post-hospital cardiac rehabilitation (A), including the rehabilitation model, is presented in Mentioned patients participated in training sessions 5 times a week. Each session consisted of two parts: 1. Training on a bicycle ergometer—Continuous (A model) or interval (B or C model), depending on the patient’s exercise tolerance, and the training duration ranged from 15 to 30 min, depending on the rehabilitation model.2. Group exercises in the gym, lasting 30 min. This training began with a warm-up, which the purpose was to prepare the movement, circulation and respiratory systems for physical exertion. In the main part, which lasted from 15 to 20 min, the patients performed slow active exercises, with resistance, general fitness and breathing exercises. In order calm down the body after the effort, low-load, stretching, relaxation and breathing exercises were performed. Statistical analysis was performed with the use of MedCalc and Statistica v.12.0 programs. For general characteristics of the studied group of patients after myocardial infarction, mean, minimum and maximum values, standard deviations and percentages were calculated.t-test for dependent samples. However, if the distribution of the results was different from normal, the statistical analysis was performed using the Rank sum tests (Mann–Whitney U test and Wilcoxon test). The level of statistical significance was p ≤ 0.05.In order to assess changes in the examined parameters, in the first stage, the distribution of variables was checked using the Shapiro–Wilk test. In the case of the normal distribution, a parametric test was used to assess the significance of changes in a given group—Student’s The characteristics of the studied group of patients after myocardial infarction before (A) and after early post-hospital cardiac rehabilitation (A1), including physical activity assessed by the International Physical Activity Questionnaire, are presented in Characteristics of the studied patients after myocardial infarction who did not participate in early post-hospital cardiac rehabilitation (B) and after 3 weeks without rehabilitation (B1) taking into account physical activity assessed by the International Physical Activity Questionnaire is presented in The level of physical activity of the studied patients after myocardial infarction assessed by the International Physical Activity Questionnaire is presented in In the group of patients after myocardial infarction qualified for early post-hospital cardiac rehabilitation (A) and after its completion (A1), a decrease in the number of people with insufficient physical activity (from 10.2% to 4.2%) was observed, with a simultaneous increase in the number of patients with high levels of physical activity (from 9.6% to 28.7%). On the other hand, in the group of patients after myocardial infarction (B) and after 3 weeks without rehabilitation (B1), none of the respondents achieved a high level of physical activity, whereas the number of people with insufficient physical activity increased (from 11.9% to 40.3%). The transcriptional activity of the studied genes: endothelin-1 (ET-1) and endothelin-1 receptor type A (ENDRA) among patients after myocardial infarction before (A) and after early post-hospital cardiac rehabilitation (A1) are presented in p = 0.0064).Transcriptional activity of the endothelin-1 gene in the studied group of patients after myocardial infarction (A) and after early post-hospital cardiac rehabilitation (A1) increased significantly (p = 0.1410).Comparing the results of the studied patients after myocardial infarction before (A) and after early post-hospital cardiac rehabilitation (A1), no statistically significant differences were observed in the transcriptional activity of the endothelin-1 receptor type A gene  gene and endothelin-1 receptor type A (ENDRA) in the studied group of patients after myocardial infarction who did not participate in early post-hospital cardiac rehabilitation (B) and after 3 weeks without rehabilitation (B’) are presented in p = 0.0268).Comparing the results of patients after acute myocardial infarction who did not participate in rehabilitation (B) and after 3 weeks without rehabilitation (B1), a statistically significant increase in the expression of the endothelin-1 gene was observed (p = 0.1439). In the studied group of patients after acute myocardial infarction who did not participate in early post-hospital cardiac rehabilitation (B) and after 3 weeks without rehabilitation (B1), no statistically significant differences in the transcriptional activity of the endothelin-1 receptor type A were observed . Moreover, comparing patients with STEMI infarction after rehabilitation to patients after MI with NSTEMI and 3 weeks without rehabilitation, despite the lack of statistically significant difference, the expression of the ET-1 gene was higher among patients after STEMI MI.In the studied group of patients after myocardial infarction with ST-segment elevation before and after early post-hospital cardiac rehabilitation, a statistically significant increase in the expression of the endothelin-1 gene was observed (p = 0.0091). It was also observed that the transcriptional activity of the ET-1 gene among patients after acute STEMI infarction after 3 weeks without rehabilitation was significantly higher compared to patients with STEMI after early rehabilitation  and after early post-hospital cardiac rehabilitation (A1), including the presence of risk factors, is presented in p = 0.0310).In the studied group of men after myocardial infarction (A) and early post-hospital cardiac rehabilitation (A1), a significant increase in ET-1 expression was observed (p = 0.0001), which means that that expression of the ENDRA genes increased with age. A statistically significant correlation was observed between the transcriptional activity of the endothelin-1 receptor gene among patients after myocardial infarction before (A) and after early post-hospital cardiac rehabilitation (A1), and the age of the respondents. The correlation was positive (p = 0.0458), but with impaired lipid metabolism (p = 0.0107). Statistically significantly higher transcriptional activity of the ET-1 gene was observed among patients after myocardial infarction and early post-hospital cardiac rehabilitation without diabetes (p = 0.0403).Statistically significantly higher expression of the ET-1 gene was also found among patients after myocardial infarction and post-hospital rehabilitation, compared to non-smokers (p = 0.0005) and after rehabilitation (p = 0.0001) in the group of patients with acute myocardial infarction and normal body weight, compared to obesity.On the other hand, the transcriptional activity of the endothelin-1 receptor type A was statistically significantly higher both before rehabilitation  .Physical activity reduction among patients after myocardial infarction increases the risk of cardiovascular disease progression and, consequently, morbidity and mortality. Early post-hospital cardiac rehabilitation enables patients after a heart attack to carry out a controlled effort under the supervision of qualified medical personnel, to learn about the possibilities and limitations of their body in the conditions of the existing disease, and to recognize the disturbing symptoms associated with it . ConductIn the presented studies, the transcriptional genes activity of selected vascular endothelial factors among patients after myocardial infarction before and after early post-hospital cardiac rehabilitation and in the group of post-infarction patients not participating in rehabilitation was assessed.For the study of transcriptional activity, the genes of endothelin-1 (ET-1) and its A-type receptor (ENDRA) acting pro-inflammatory, pro-coagulatory, pro-thrombotic, atherosclerotic and as endothelial vasoconstrictors were selected. Other researchers, based on the results of the conducted studies, suggested an increased expression of the endothelin-1 gene, both among patients with coronary artery disease, hypertension, and acute coronary syndrome, compared to healthy subjects ,30.In the conducted study, both in the group of patients after myocardial infarction and early post-hospital cardiac rehabilitation, as well as in the group of people not participating in rehabilitation, significantly increased expression of the endothelin-1 gene was demonstrated. It is worth noticing that despite the increased expression of this gene in the studied group of patients after myocardial infarction and early post-hospital cardiac rehabilitation, its transcriptional activity among patients without rehabilitation was even greater. Therefore, it can be concluded that early post-hospital cardiac rehabilitation among patients after myocardial infarction has a positive effect on the transcriptional activity of the ET-1 gene, the reduction in which contributes to the improvement of the vascular endothelial function. In addition, it is worth noticing that in the group of patients after myocardial infarction and after early post-hospital cardiac rehabilitation, despite the lack of statistical significance, a decrease in the expression of the endothelin-1 receptor type A (ENDRA) gene was shown, while in the group without rehabilitation it showed an increase. This may be important in explaining the positive effect of physical activity in the studied patients after a heart attack.Increased transcriptional activity of the endothelin-1 receptor type A gene enhances the unfavorable effect of the cytokine in question on endothelial function. It contributes not only to the increased tension of the blood vessel walls but also, according to some authors, it causes ENDRA mediated growth and proliferation of smooth muscle cells and the triggering of oxidative stress in endothelial cells . In the It should be emphasized that patients after myocardial infarction, despite the applied revascularization and pharmacological treatment, are not fully cured patients. They often progress to coronary artery disease, develop complications related to it, or recurrent cardiovascular events. It is worth mentioning that the duration of early post-hospital cardiac rehabilitation was on average 3 weeks, so it was relatively short. Therefore, it is extremely important to conduct a multi-stage, comprehensive cardiac rehabilitation which lasts to the end of the patient’s life, provides better treatment results, and contributes to reducing the risk of cardiovascular events and mortality among patients with coronary heart disease and after myocardial infarction.Confirmation of the sense of the need to conduct the above-mentioned rehabilitation and treatment measures is indicated by, for example, the comparative results of endothelin-1 gene expression among patients after STEMI and early post-hospital rehabilitation with the results of similar patients not participating in rehabilitation, with significantly higher transcriptional activity of the gene mentioned. It is also worth noting that the type of myocardial infarction (STEMI vs. NSTEMI) may play a role in the transcriptional activity of the ET-1 gene under study. Comparing patients after STEMI and rehabilitation with patients with NSTEMI after 3 weeks without rehabilitation, despite the lack of a statistically significant difference, the transcriptional activity of the ET-1 gene was higher among patients with STEMI. Therefore, it can be assumed that the type of myocardial infarction may be a significant factor affecting the success of the rehabilitation process, but it may also result from the pathophysiological basis of myocardial infarction with and without ST-segment elevation.In their research, Yang et al. have shown that regular physical activity of healthy elderly people, despite the ongoing aging processes, has a beneficial effect on blood vessels and has a protective effect. It should therefore be emphasized that regular physical activity of elderly people, not only healthy people, but most of all those burdened with cardiovascular diseases, is very important . On the other hand, A. Chalghouma et al. analyzed the activity of endothelin-1 depending on the presence of risk factors among patients with the diagnosed acute coronary syndrome. They observed that the concentration of ET-1 was significantly higher in the group of men, patients with arterial hypertension, smokers, with normal body weight, and without lipid disorders. Moreover, they found that the level of ET-1 in the patients of the above-mentioned group did not differ between those leading a sedentary lifestyle and those with active and diabetic and non-diabetic patients . In our The dysfunction of the vascular endothelium precedes the development of coronary disease and may be a prognostic factor for future heart attacks. Understanding the mechanisms of the influence of risk factors on endothelial dysfunction emphasizes their important role in the pathophysiology of many cardiovascular diseases developing on the bases of atherosclerosis. Moreover, each of the risk factors may have a variety of effects on the severity of endothelial dysfunction.p < 0.0001), which means that the expression of the ENDRA gene increased with age.Age over 65 is considered a high-risk factor for cardiovascular diseases. However, it should be added that the increased risk of cardiovascular diseases is associated it is also dependent on gender with age. Literature data indicate that the risk increases in women over 50 years of age and in men over 40 years of age. It is also noted that the definition of “risk age” is not always consistent with the actual age. When determining the “age of risk”, it should be associated with the presence of other risk factors. In the group of men after myocardial infarction and early post-hospital cardiac rehabilitation, a significantly higher transcriptional activity of the endothelin-1 gene was found. This may be due to the unequal size of the groups taking into account the sex of the respondents (19.2% of women and 80.8% of men), which is epidemiologically justified. However, according to gender-specific statistical data, MI is diagnosed more often among men . In the It is known that increased body weight (overweight and obesity) is a risk factor for cardiovascular diseases, including hypertension, lipid metabolism disorders, or diabetes, and has been recognized by the WHO as a civilization disease ,36. One In the studied group of patients after MI with normal body weight, both before and after rehabilitation, significantly higher expression of the ENDRA gene was observed compared to obese patients. An interesting observation concerning the above results is the so-called “obesity paradox” described in the literature. Researchers have shown a better prognosis among patients with coronary artery disease and obesity compared to patients with normal body weight ,39. It wp = 0.0403). Moreover, it was found that early post-hospital cardiac rehabilitation did not change the transcriptional activity of genes of the studied vascular endothelial factors in diabetic patients. A study by L. J. Reynolds et al. showed a correlation between elevated blood glucose concentration and increased endothelin-1 activity. Thus, the lack of changes in the activity of the ET-1 gene among patients with diabetes could have resulted from the use of prescribed medications and/or diet [The negative effect of nicotine on the increase in ET-1 gene expression in various disease entities has been confirmed by scientific studies ,42. As a/or diet .A smaller increase in the expression of the endothelin-1 gene with a simultaneous increase in physical activity in the group of patients after early post-hospital cardiac rehabilitation, compared to patients not participating in it, proves the beneficial effect of rehabilitation and indicates the need for its use throughout life. The increase in the transcriptional activity of the endothelin-1 gene in the group of men, smokers, and people with lipid metabolism disorders, and among patients after myocardial infarction with ST-segment elevation (STEMI), as well as endothelin-1 receptor type A (ENDRA) with the age of the respondents may indicate another pathomechanism of pro-atherogenic activity of the above-mentioned factors.


Many epidemiological studies have shown that anovulatory polycystic ovary syndrome (PCOS) is accompanied by hyperandrogenism. However, the exact mechanism of hyperandrogen-induced anovulation remains to be elucidated. In this study, we aimed to investigate the potential mechanism of anovulation in PCOS. To investigate the role of klotho as a key factor in the androgen receptor (AR)-mediated development of PCOS, we investigated the effects of testosterone on ovarian klotho expression in vivo and in vitro.Testosterone propionate (TP)-induced rats showed cycle irregularity, hyperandrogenism, polycystic ovarian changes, dyslipidemia. However, inhibition of AR expression could relieve PCOS traits. We also found that AR and klotho showed relatively high expression in PCOS rat ovarian tissue and in TP-induced granulosa cells (GCs), which was inhibited by the addition of flutamide. TP-induced GCs apoptosis was suppressed by AR antagonist, as well as silencing klotho expression in human GCs. Chromatin immunoprecipitation assay demonstrated that AR indirectly binds to the klotho promoter.Our results demonstrated TP mediates the expression of klotho via androgen receptor and klotho alterations could be a reason for ovarian dysfunction in PCOS. PCOS is a heterogeneous disease that is mainly characterized by hyperandrogenism, ovulation changes, infertility and polycystic ovaries . AbnormaExcess androgen not only causes hairy and acne but also it increases over-recruitment of follicles, which leads to the absence of dominant follicles, ultimately resulting in anovulation . In factAR is a steroid receptor in the nuclear receptor superfamily. AR expression has been identified in ovarian follicles in different stages but exhibits differential expression among the different stages , 12. HowKlotho, a recently discovered aging suppressor gene, is expressed only in the brain, kidney, reproductive organs, pituitary gland, and parathyroid gland . PreviouIn the present study, we aim to investigate whether TP changes the expression of klotho via AR-mediated pathway and is able to change reproductive functions in female rats and human ovarian granulosa cells. Thus, we evaluated several peripheral biomarkers, as well as the expression of AR and klotho, between TP group and control group. To further confirm the roles of AR and klotho, we observed the changes in KGN cell apoptosis induced by using androgen receptor antagonists and klotho silencing, and the ChIP experiment verified the binding between AR and klotho. This study provides a more scientific basis for establishing AR and Klotho as new targets affecting GCs apoptosis.P < 0.01 vs. control), lower ratio of ovary/body weight (P < 0.01 vs. control) and higher androgen level than control group (P < 0.001 vs. control). HE staining showed that ovaries of TP rats generally abundant corpus lutea, indicating a polycystic state. In contrast, a complete follicular cycle was observed in the control group. Taken together, these findings indicated that chronic androgen exposure is a negative regulator of ovarian growth and ovulation in rats, and can induce the PCOS model.We started using vaginal smear to monitor the estrous cycle after TP injection for 21 days. The results showed that TP-induced rats did not observe the complete estrus cycle and most of them stayed at diestrus . In the blood, the T level was significantly reduced (**** P < 0.001 vs. control) and dyslipidemia was improved. HE staining revealed the corpus luteum and increased number of GCs . Klotho also showed high expression in PCOS . However, flutamide could converse this situation  and Western blot in TP-induced PCOS rats. In IHC, the expression of AR and klotho in the ovaries of TP-stimulated rats were up-regulated compared with the control group . Hoechst detected apoptosis in TP-stimulated GCs. Cells were seeded in 6-well plates in DMEM with FBS. TP  was added to incubate cells for 24 h. Subsequently, we combined with TP- and flutamide-treated GCs for 24 h. We found that TP increased GCs apoptosis, but the specific AR antagonist flutamide decreased TP-induced GCs apoptosis . These results suggested that klotho affects TP-induced apoptosis of GCs mediated through AR.To explore the relationship among TP-induced GCs apoptosis, AR, and klotho. The expression of AR and klotho were detected by immunohistochemistry after TP treatment. The results showed that different concentrations of TP all promoted the expression of AR and klotho Fig. . The expTo clarify whether AR and klotho can be directly combined, we used the ChIP experiment for verification. According to the results of the primer pre-experiment, the primer klotho-115 was selected for subsequent formal experiments. The bands of the positive control group were clear and free of miscellaneous bands, and the negative control group had no bands, indicating that the ChIP experiment results were reliable and the operation was correct. The bands in the input group were clearly visible, indicating that the PCR amplification primers could be used to detect this indicator. The KGN experimental group had no bands at the corresponding positions, indicating that the promoter region of the klotho gene did not bind to AR in KGN cells, suggesting that TP indirectly regulates klotho expression through AR Fig. .Fig. 6ARHyperandrogenism affects the apoptosis of GCs, which determines the fate of follicles. Androgen acts via the AR to regulate the development of follicular. Since AR can also be regulated by a variety of factors, the androgen/AR signaling that regulates follicular development in PCOS remains to be fully elucidated. In this study, we investigated that the mechanism of AR-mediated apoptosis of GCs. After successful construction of the PCOS model in this experiment, we used the AR-specific antagonist flutamide to interfere with AR expression. Compared with normal group, WB and IHC showed that upregulation AR and klotho expression in PCOS, conversely, the androgen-induced high expression of AR and klotho were attenuated after treatment with flutamide. In vitro experiments further confirmed that androgen induced GCs apoptosis is positively correlated with upregulation of AR and klotho expression, whereas in CHIP experiments it was found that AR does not directly act on klotho, suggesting that we can further explore the upstream and downstream targets of klotho.Results have been inconsistent in a limited number of investigations klotho levels in women with PCOS. In our study, klotho expression was upregulated in the ovaries of PCOS, and some investigators have also found higher levels of klotho in women with PCOS than in heathy women , 22, 23.To reveal the exact role of the androgen/AR pathway in PCOS, it is necessary to replicate an animal model similar to human PCOS-like symptoms. According to the literature, several methods for constructing PCOS rat models have been explored, such as the use of androgen, letrozole, and HCG hormone modeling methods –26. The Although we used TP-reduced rat model to study the relationship between GCs apoptosis and klotho, this study still had the limitation that the rat sample size was not large enough. Relevant clinical research is lacking, limited to animal and cell experiments.In conclusion, our study demonstrated that the chronic androgenized rat model evaluated is useful for studying the long-term effects of androgens on follicular apoptosis. TP might induce both AR expression and klotho expression in the ovary in vivo and in vitro. Exogenous TP induces indirect binding of AR to the klotho promoter to occur abnormal GCs development. Based on the above experimental results, this study proposes that klotho is involved in the apoptosis of GCs and provides a candidate target for the treatment of PCOS.The main reagents are described. Testosterone propionate was produced by AbMole (lot number: T7260); and flutamide was produced by Tokyo Chemical Industry, . Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco. Androgen receptor antibody was purchased from Abcam (ab108341), and Klotho antibody was obtained from ABclonal (A12028). The SimpleChIP® Enzymatic Chromatin IP Kit was obtained from CST (#9003).The PCOS model was established using TP . Fifty fTwenty-one days old SD rats were randomly divided into three groups . The vehicle group was treated with corn oil (0.1 ml/day for 35 days). In the PCOS group, the PCOS model was established with daily injections of TP . From treatment for 21 days, the estrous cycle was monitored by vaginal smears for 2 weeks. After confirming successful establishment of a PCOS model, they were randomly divided into 4 groups . The PCOS-flutamide groups are divided into low-dose group and high-dose group. Flutamide was administered at a dose of 10 mg/kg BW s.c. for the next 15 consecutive days as low dose group and 30 mg/kg BW s.c. is high dose group. All the rats were anesthetized with 0.2 ml/10 g chloral hydrate 24 h after the last treatment. Blood and ovarian samples were collected from all animals. The bilateral ovaries were photographed. Immunohistochemistry, Western blot and TUNEL staining were used for subsequent experiments.2 in air. Confluent cells were detached by incubation with 0.05% trypsin for 2 min and inoculated in 96-well plates or 6-well plates containing 104 cells for experiments.Ovarian granulosa cells were obtained from BNCC. Cells were cultured in 10% FBS DMEM. Cells were incubated at 37.0 °C and 5% COTotal protein was extracted, and Western blotting was performed as described previously . Equal qBriefly, cells were fixed in 4% paraformaldehyde for 30 min at room temperature (RT), subsequently, blocking with endogenous peroxidase solution for 10 min after washing with phosphate-buffered saline (PBS) three times. Cells were incubated overnight with AR antibody (1:100 dilution) in a 4 °C refrigerator, followed by enhancement of the response with an enhancer for 20 min at 37 °C after three washes with PBS. Then, cells were incubated with goat anti-mouse IgG secondary antibody for 20 min at 37 °C after washing with PBS three times, followed by staining for 3 min at RT. Cells were observed under a stereomicroscope.4 cells/100 ml per well with five replicate wells for each sample. Cells were incubated for 24 h after added 5 nmol/L, 25 nmol/L, 50 nmol/L, 75 nmol/L, or 100 nmol/L TP. Cell Counting Kit-8 (CCK-8)  was used to detect cell viability. 10 μl CCK-8 solution was added to each well under aphotic conditions and further incubated for 2 h. The optical density was measured at 450 nm using a microplate reader . The experiment was repeated 3 times under the best conditions.GCs cells were seeded into 96-well plate at the density of 1 × 10Cells were seeded in 12-well plates in DMEM with FBS for 24 h. After drug treatment, cells were fixed in 4% paraformaldehyde for 30 min at room temperature and subsequently washed with PBS three times. Then, cells were incubated with Hoechst for 5 min at room temperature after washing with PBS three times. Washing with PBS three times for 30 min. Finally, cells were observed under the microscope after adding the antifade mounting medium.5 cells/well were reseeded in a 6-well plate. After drug treatment for 24 h or 48 h, we prepared experiments. According to the manufacturer's instructions for apoptosis analysis, cells were separated from 6-well plates with trypsin without EDTA and stained with FITC Annexin v and propidium iodide (PI) positive.Cell apoptosis was assessed by an FITC Annexin v Apoptosis Detection Kit . Approximately 1.8 × 10In order to cross-link proteins with DNA, each 15 cm Petri dish containing 20 ml of culture medium was added with 1% formaldehyde and incubated for 10 min, and terminated by 2 ml of 10 × glycine. Washing the cells twice with cold PBS and add 2 ml cold PBS + Protease Inhibitor Cocktail (PIC) to obtain the cell pellet. For each IP preparation, resuspend the cells in 1 ml 1 × Buffer A (250 μl 4 × Buffer A # 7006 + 750 μl water) + 0.5 μl 1 M DTT + 5 μl 200 × PIC and place on ice. After 10 min incubation on ice, the cell pellet was obtained and resuspended in 1 ml ice-cold 1 × buffer B (275 μl 4 × Buffer B # 7007 + 825 μl water) + 0.55 μl 1 M DTT + DTT. The centrifugation step was repeated, the supernatant was removed, and the pellet was resuspended in 100 μl of 1 × Buffer B + DTT. Then, 0.5 μl of Micrococcal Nuclease (# 10011) was added and incubated at 37 °C for 20 min. To obtain the precipitate, 10 μl of 0.5 M EDTA was added, and samples were resuspended in 100 μl of 1 × ChIP buffer + PIC and incubated on ice for 10 min. Each immunoprecipitation uses 400 μl 1 × ChIP buffer (40 μl 10 × ChIP buffer + 360 μl water) + 2 μl 200 × PIC. When determining the number of immunoprecipitations, include the positive control sample Histone H3 (D2B12) XP® Rabbit mAb and the negative control sample Normal Rabbit IgG Antibody. The IP samples were incubated at 4 °C for 4 h to overnight. ChIP-grade Protein G Magnetic Beads (# 9006) were resuspended, and 30 μl of Protein G Magnetic Beads was immediately added to each IP reaction and incubated at 4 °C for 2 h with rotation. After washing, the supernatant was obtained, the chromatin was eluted from the antibody/Protein G magnetic beads, and the cross-linking was reversed. After DNA was purified using a spin column, it was quantified by PCR. The primer pairs klotho 115, 5'-tttccctccacagctcagat-3' (forward) and 5'-gtgttacctttcggctctg-3' (reverse), and klotho 177, 5'- ccgagtgggagaaaagtgag-3' (forward) and 5'- ggacgctcaggttcattctc-3' (reverse) were used to amplify the klotho promotor region.P value < 0.01 and < 0.05.Data were expressed as means ± SD. Statistical differences between groups were applied using Graph Pad Prism software version 7.0 . All the data were analyzed by T test, data were considered to be statistically significant at


Advanced prostate cancer development is associated with androgen-independent AR signaling. TR3 overexpression alters AR expression, splicing process, and transactivation towards increasing the androgen independence of AR signaling in prostate cancer cells. These results suggest that TR3 is a pivotal factor in the progression of prostate cancer to advanced form.The pro-oncogenic function of TR3, an orphan nuclear receptor, has been reported in prostate cancer. However, the roles of TR3 in androgen receptor (AR) expression and signaling in prostate cancer cells are poorly understood. Database analysis revealed that TR3 expression level is elevated in prostate tumors, and is positively, although weakly, correlated with that of AR. TR3 overexpression increased the production of AR splice variants in addition to general upregulation of AR expression. TR3 interacted with some spliceosomal complex components and AR precursor mRNA, altering the splice junction rates between exons. TR3 also enhanced androgen-independent AR function. Furthermore, TR3 overexpression increased cell proliferation and mobility of AR-positive prostate cancer cells and stimulated tumorigenesis of androgen-independent prostate cancer cells in mouse xenograft models. This is the first study to report that TR3 is a multifunctional regulator of AR signaling in prostate cancer cells. TR3 alters AR expression, splicing process, and activity in prostate cancer cells, increasing the androgen independence of AR signaling. Therefore, TR3 may play a crucial role in the progression of prostate cancer to an advanced castration-resistant form. Prostate cancer is one of the leading lethal malignancies in males, and androgen receptor (AR), a ligand-dependent transcription factor, plays a pivotal role in the development, progression, and metastasis of prostate cancers . AR has Several splicing factors have been found to be associated with alternative splicing events in the AR precursor mRNA (pre-mRNA) in prostate cancer cells. Among these, heterogeneous nuclear ribonucleoprotein (hnRNP) A2B1, hnRNP E1/E2, and HUR  are RNA-binding proteins (RBPs) that bind to AR pre-mRNA and regulate the expression of AR-Vs in advanced CRPCs ,8. hnRNPβ, which induces cell invasion as well as epithelial-mesenchymal-transition in many cancers, promotes the phosphorylation and nuclear export of TR3 to inhibit pro-apoptosis in cancer cells, while TR3 antagonists block TGF-β-induced nuclear export of TR3  overnight at 4 °C. RNA and protein degradation was minimized by adding RNase (RNasin) and protease inhibitors, respectively. Reactions were analyzed on a 3% horizontal agarose gel in TBE buffer containing 10 µg/mL EtBr. Gels were cooled and run in cooled TBE buffer. Free RNA and RNA-protein complex signals were detected using a UV transilluminator. ssRNA sequence is listed in Thirty-mer ssRNA labeled with biotin at the 5′ end (ssRNA oligo) was designed for RNA-protein interaction assays. ssRNA oligo sequence was identical to that found within Reg4, which contains one hnRNP A2B1 binding site overlapping with one HUB/HUR and two other HUB binding sites but has no hnRNP E1/E2 binding site. ssRNA oligo was incubated with or without the purified protein (FLAG-TR3 or splicing factors hnRNP A2B1 and HUB/HUR) in a binding buffer . Mice were then warmed on a veterinary warming system. Inducible TR3-overexpressing  or control (EV) CWR22Rv1 cell clones (2 × 106 cells/site mixed 1:1 with Matrigel) were subcutaneously injected (s.c.) into the shoulders of each mouse. The following day, mice were provided distilled water with or without 2 µg/mL doxycycline. Tumor size and volume were monitored and measured three times a week. Two days after the last tumor measurement, the animals were subjected to CO2-induced euthanasia and sacrificed, and the tumors were extracted and weighed. Animals were maintained inside a cleaned bench in an animal room with a 12-h light/dark cycle and controlled temperature during all processes. The sterilized mouse cages, water, food, and bedding used for the animal study were replaced once a week. Ultra-fine II short needles (U-100 INSULIN 30 gauge 5/16′’ (8 mm) needle) were used for all injections to minimize any pain and lesions. Tumor volumes were calculated using the following formula: length × width × height × 0.5236 (V = π × L × W × H/6 = L × W × H × 0.5236) [t-test analysis. p-value < 0.05 was considered statistically significant. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Chonnam National University (permit number: 2012-44). Animal experiments have been performed in accordance with the ARRIVE/NC3R guidelines.Healthy and microbiologically monitored 4-week-old male NOD.CB17-Prkdc 0.5236) . Statistt-test analysis. p-value < 0.05 was considered statistically significant.Animal experiments were performed using six mice in each group. Data, which were obtained from more than three independent experiments, are presented as the mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey’s post-hoc test, two-way ANOVA with Bonferroni’s post hoc test, and two-tailed α-dihydrotestosterone (DHT)  and evaluated the correlation between TR3 and AR expression levels in prostate tumor patients (The Cancer Genome Atlas database). We found that TR3 expression is upregulated in primary prostate tumors A and thane (DHT) , indicatAs TR3 and AR expression levels are positively correlated in prostate tumors, we investigated whether TR3 regulates AR expression. Overexpression of TR3 enhanced the protein levels of AR-Vs including AR-V7 in prostate cancer cells which were treated with DHT . We alsoThe coupling of transcription and splicing processes is well-known ,21,22. Ty and exon 2 increased (~30%) , a short transcript that lacks exon 1, which is produced by utilizing an alternative transcription start site located between −3 and +5 of exon 1by [To investigate the alteration of AR splicing events in TR3-overexpressing prostate cancer cells, we performed next-generation sequencing (NGS) high-throughput RNA-Seq analysis of RNA obtained from TR3-overexpressing and control CWR22Rv1 cells. The INDEL coupling with Integrative Genomic Viewer (IGV) analysis of RNA-Seq data of AR transcripts revealed that splice junction rates between exons within the pre-mRNA of full-length AR gene were generally decreased (up to 50%) when TR3 was overexpressed in CWR22Rv1 cells, while the splice junction rate between exon 1bd (~30%) C. The spexon 1by ,24. Overy, exon 2, exon 3, cryptic exon CE1, cryptic exon CE3, and exon 4 (y and exon 2 (found in AR45), exon 3 and CE1 (found in AR-V1~4), and exon 3 and CE3 (found in AR-V7 and AR-8) was higher than the general increase in total AR mRNA containing the exon 3–exon 4 junction, which was upregulated by TR3 activation of the AR promoter , and other splicing factors involved in alternative splicing of AR pre-mRNA, such as hnRNP A2B1, hnRNP E1/E2, and HUR. RNA-Seq analysis revealed ~6.4-fold induction of NOVA-2 and ~1.7-fold induction of HUB in TR3-overexpressing cells compared with the control A. The inhnRNP A2B1 plays a critical role in the control of the AR splicing process , and it We further investigated whether TR3 altered the recruitment of splicing factors near splice sites. We chose 8 regions, designated Reg1–8, located ~1 kb upstream and/or downstream of the exon/intron junction of AR pre-mRNA and covering exons 1–4 A. This iTo examine whether TR3 is associated with the spliceosomal complex at Reg4, we performed in vitro RNA-protein interaction assays involving single-strand RNA (ssRNA) protection and ssRNA oligo pull-down assays. A 30-mer ssRNA labeled with biotin at the 5′ end (ssRNA oligo) was designed to include the sequence found within Reg4, which contains one hnRNP A2B1 binding site overlapping with one HUB/HUR and two other HUB binding sites, but it has no hnRNP E1/E2 binding site. ssRNA protection assays, which were performed using the ssRNA oligo and either purified TR3 or splicing factors (hnRNP A2B1 and HUB/HUR), showed that both TR3 and the splicing factors bound to and protected this ssRNA oligo from degradation C. The ssWe then examined the effect of TR3 on the transcriptional activity of ARs  based on luciferase reporter assays. The AR-NTD lacking LBD was used to represent the ligand-independent nature of AR, and AR-V7 was used to mimic AR-V behavior in CWR22Rv1 cells. Overexpression of TR3 markedly enhanced both androgen-dependent and -independent transactivation of exogenous AR-FL, AR-NTD, and AR-V7 overexpressed in PPC-1 cells in a dose-dependent manner . In addiThe subcellular localization of ARs and recruitment of coactivators to AR target genes are important for AR transactivation. TR3 overexpression promoted the nuclear translocation of ARs even in the absence of androgen G. In addRNA-Seq analysis showed the upregulation of genes that are involved in cell proliferation, migration, and invasion as well as PI3K/AKT and MAPK/ERK signaling pathways when TR3 is overexpressed A,B. The Overexpression of TR3 enhanced the proliferation of androgen-independent CWR22Rv1 cells C, left aTo examine the effect of TR3 on cell mobility, we performed cell migration and invasion assays using CWR22Rv1 cells. TR3 overexpression markedly increased cell migration when examined by the Boyden chamber migration E and scrThe effect of TR3 on prostate tumorigenesis was investigated using the inducible TR3-overexpressing (TR3) or control (EV) CWR22Rv1 xenograft mouse models A,B. The With the xenograft mouse models, we observed that the average volume of TR3 tumors was approximately 10-fold larger than that of EV tumors after ~7-week treatment with DOX E,F. The We further evaluated the protein expression levels of AR and AR-Vs as well as AR-V7 in xenograft tumors I,J. UnliConstitutive transcriptional activity of AR-Vs is important for prostate cancer progression. However, the molecular mechanisms underlying the production of AR-Vs remain unclear. In the present study, for the first time, we found a novel function of TR3, which controls AR splicing events in prostate cancer cells. Overexpression of TR3 upregulated the expression of mRNAs and proteins of smaller AR-Vs. Consistent with this finding, TR3 overexpression altered the splicing junction rates of AR pre-mRNA, resulting in the enhancement of mRNA levels of AR-Vs, such as AR45, AR-V7, and AR-V1~4, in advanced CRPC cells (CWR22Rv1 and C4-2). We also demonstrated that TR3 overexpression alters the expression of certain splicing factors and that TR3 interacts with several known AR splicing factors (hnRNP A2B1 and hnRNP E1/E2) as well as the intron regions in AR pre-mRNA. Interestingly, TR3 functions in the RNA splicing process, while also regulating gene expression as a transcription factor. The association of TR3 with splicing factors and AR pre-mRNA might control the recruitment of spliceosomal complex components or identification of intron/exon boundaries.Many splicing factors, including hnRNP A family  , hnRNP EThe coupling of transcription and splicing processes is well-documented ,21,22. TRNA-Seq analysis in the present study revealed that TR3 overexpression in CWR22Rv1 cells enhanced the expression of immune tolerance-related genes  while deTR3 expression is upregulated in both primary prostate tumor and tissue adjacent to the tumor, and high expression level of TR3 in the tumor is associated with the short-term survival of patients. In addition, the overexpression of TR3 upregulated the expression of genes that are involved in cell proliferation, migration, invasion, angiogenesis, and pathways in cancer as well as PI3K/AKT and MAPK/ERK signaling pathways that are amplified as prostate cancer progresses into CRPC. Therefore, TR3 could be a therapeutic target to improve the outcome of prostate cancer treatment.TR3 increases the androgen independence of AR signaling in prostate cancer cells by controlling AR expression, splicing events, and androgen-independent function and also stimulates tumorigenesis of prostate cancer cells. These results suggest that TR3 is a pivotal factor in the progression of prostate cancer to advanced castration-resistant form. Therefore, TR3 could be an alternative therapeutic target for the treatment of advanced prostate cancer.


The Pica, ARFID, and Rumination Disorder Interview (PARDI) is a structured interview that can be used to determine diagnosis, presenting characteristics, and severity across three disorders, including avoidant/restrictive food intake disorder (ARFID). The purpose of this study was to evaluate the psychometric properties of a questionnaire focused specifically on ARFID (PARDI-AR-Q), which has the potential to provide related information with less participant burden.n = 71, ages 14–40 years) with ARFID (n = 42) and healthy control participants  completed the PARDI-AR-Q and other measures. A subset of the ARFID group (n = 27) also completed the PARDI interview.Adolescents and adults  with the corresponding subscales on the interview. The ARFID group scored significantly higher than HC on all subscales. Furthermore, 90% of the ARFID group scored positive on the PARDI-AR-Q diagnostic algorithm while 93% of the HC scored negative.An exploratory factor analysis of proposed subscale items identified three factors corresponding to the ARFID phenotypes of avoidance based on the sensory characteristics of food, lack of interest in eating or food, and concern about aversive consequences of eating. Further analyses supported the internal consistency and convergent validity of the PARDI-AR-Q subscales, and subscale ratings on the questionnaire showed large and significant correlations  in clinics or research settings. This makes it difficult for individuals with the disorder to be identified and offered appropriate care. We developed the Pica, ARFID, and Rumination Disorder Interview ARFID Questionnaire (PARDI-AR-Q), a brief self-report measure of possible ARFID, and evaluated how well it worked in the current study. In our preliminary study of 71 adolescents and adults, most individuals with ARFID (90%) scored positive on this measure, whereas most healthy control participants (93%) scored negative. Our findings provide early support for the PARDI-AR-Q as a promising new measure for detecting possible ARFID when a full clinical interview is not possible.The online version contains supplementary material available at 10.1186/s40337-022-00706-7. DSM-5  ARFID and 26 [90% of] HC) completed the NIAS , a 9-itelescents . In the A subset of participants (28 [66% of] ARFID and 26 [90% of] HC) completed the FNS , a self-All participants with ARFID and 26 (90% of) HC completed the CIA , a self-All participants in both the ARFID and HC groups completed the EDE-Q , which mX2 [36] = 250.33, p < 0.001), suggesting that EFA was appropriate. To evaluate the internal consistency of each subscale, we calculated Cronbach alphas for each of the ARFID-only group, the HC-only group, and the full sample.We conducted all analyses in R . First, Second, to evaluate convergent validity, we evaluated the strength of correlations between PARDI-AR-Q subscales and related constructs, including the corresponding NIAS subscales, the FNS, and the CIA. To evaluate divergent validity, we evaluated the strength of correlation with EDE-Q, which should not be conceptually related to PARDI-AR-Q scores as weight and shape concerns are not central to the ARFID diagnosis. To evaluate concurrent validity, in the subset of individuals with ARFID for whom PARDI interview data were also available, we evaluated the strength of intraclass correlation coefficients between subscale scores on the PARDI-AR-Q and their analogous scores on the PARDI interview.r values to determine effect size [r effect sizes range from 0 to 1, with r < 0.3 indicating a small effect, 0.3–0.5 indicating a moderate effect, and > 0.5 indicating a large effect.Third, to test our hypothesis that individuals with ARFID would score significantly higher than HC on all PARDI-AR-Q subscales, we conducted a Mann–Whitney-Wilcoxon test . We also calculated Wilcoxon ect size . WilcoxoLastly, we evaluated the percentage of participants with ARFID as well as HC who scored positive versus negative on the PARDI-AR-Q diagnostic algorithm. We then qualitatively reviewed responses of ARFID participants who scored negatively as well as HC who scored positively to evaluate the reasons for any discrepancies.The EFA identified either a three-factor solution (explaining 75% of the variance) or four-factor solution (explaining 76% of the variance) as optimal. Additional file Cronbach alphas for all subscales were good to excellent for the ARFID group (but lower for the HC group due to low variance in HC responses) for sensory-based avoidance , concern about aversive consequences , and lack of interest in eating or food .Table n = 27), intraclass correlations were large and significant across subscales, supporting concurrent validity .Furthermore, as hypothesized, none of the PARDI-AR-Q subscales were significantly correlated with EDE-Q global score . In the subset of ARFID participants who also completed the PARDI interview  scored positive on the PARDI-AR-Q diagnostic algorithm. However, the remaining 10% (n = 4) scored negative on the diagnostic algorithm, despite receiving an ARFID diagnosis via PARDI or clinical interview. Of the four who scored negative, one was an adolescent male who met criteria via interview based on having an underweight BMI percentile and psychosocial impairment . This participant was also undergoing psychological treatment for ARFID at the time. However, this participant did not endorse any items on the PARDI-AR-Q. The other three false negative cases met criteria for ARFID during the interview based on psychosocial impairment only (diagnostic criterion A4). However, all three scored negative on the PARDI-AR-Q because they did not endorse item 21 , though two of the three did endorse items 6 (thinking they had a problem with eating) and 7 (other people saying they had a problem with eating).In contrast, of the 29 of participants in the HC group, only 7% (n = 2) scored positive on the PARDI-AR-Q diagnostic algorithm, while 93% (n = 27) scored negative. Both HC participants who scored positive on the diagnostic algorithm endorsed item 6 (thinking they had a problem with eating) and 9 (weight loss due to avoidance or restriction). Both participants were adults with BMIs in the overweight range, based on the heights and weights they provided on the PARDI-AR-Q.DSM-5 ARFID phenotypes. Furthermore, medium to large correlations between PARDI-AR-Q subscales and related constructs  provided preliminary support for convergent validity, and high intraclass correlations between PARDI-AR-Q subscales and their corresponding subscales on the PARDI-AR-Q provided preliminary evidence of concurrent validity. In contrast, low and non-significant correlations between PARDI-AR-Q subscales and EDE-Q global scores provided preliminary support for divergent validity. Finally, as expected, the majority of participants with ARFID scored positive on the PARDI-AR-Q whereas the majority of the HC scored negative.Though replication in larger and more diverse samples is needed, findings provide early support for the validity and reliability of the PARDI-AR-Q as a self-report measure of possible ARFID in clinical or research settings. Specifically, our exploratory factor analysis provided preliminary support for a three-factor structure consistent with PARDI-AR-Q subscale scores correlated highly with one another, but still loaded onto distinct factors. This was also true of NIAS subscale scores in the current study, as well as in prior studies . Taken tOur qualitative analysis of discordant cases highlights the challenges inherent to the self-report assessment of ARFID diagnostic criteria. In the case of the four participants diagnosed with ARFID who scored negative on the PARDI-AR-Q diagnostic algorithm, one participant was underweight and concurrently undergoing ARFID treatment. This patient may have exhibited a minimizing response style on the questionnaire, which is also common in other eating disorders, such as anorexia nervosa . In the Our findings should be interpreted in light of limitations. Our sample size was modest, and our sample was from a single country (United States) and identified primarily as non-Hispanic white, thus limiting generalizability. This limitation is particularly important in the context of assessment, which may play a gate-keeping function for treatment access. Future research on the PARDI-AR-Q and other eating-disorder assessments should recruit more racially, ethnically, and geographically diverse samples . SimilarThe study also had strengths. Specifically, our entire ARFID sample included participants whose diagnosis had been confirmed via PARDI or structured clinical interview. This is particularly important as past studies of self-report measures of ARFID have largely relied on analogue or college samples with picky eating . FurtherDSM-5 criteria. Compared to clinical interviews , the PARDI-AR-Q is quick to administer and does not require a trained interviewer or clinician for scoring. Our findings suggest that the PARDI-AR-Q can be used to elucidate clinical characteristics  when a clinical interview is not practical or desirable. Future research should use the PARDI as part of a two-stage screening process with which to evaluate the diagnostic properties of the PARDI-AR-Q against a gold standard interview.In summary, the PARDI-AR-Q showed preliminary evidence of reliability and validity in the current study. The PARDI-AR-Q has advantages over existing self-report and interview measures of ARFID symptoms. For example, while the PARDI-AR-Q and NIAS both measure ARFID psychopathology via self-report, only the PARDI-AR-Q has a severity of impact scale and diagnostic algorithm for screening purposes. Indeed, in the current study, the PARDI-AR-Q correctly identified 90% of ARFID cases, regardless of specific ARFID profile, based on Portions of these findings were presented at the 2020 Eating Disorders Research Society meeting and 2022 International Conference on Eating Disorders .Additional file 1. Supplemental tables and supplemental figure.Additional file 2. PARDI-AR-Q Self 14+.Additional file 3. PARDI-AR-Q instructions for administrators.