MicroRNA‐205 is associated with diabetes mellitus‐induced erectile dysfunction via down‐regulating the androgen receptor

Abstract As a major class of regulatory genes in majority metazoans, microRNAs (miRs) play an important role in various diseases including diabetes mellitus (DM). Lack of androgens has previously been associated with DM‐induced erectile dysfunction (DMED). In addition, the biological functioning of androgen is mediated by androgen receptor (AR). Herein, we sought to investigate whether miRs participate in AR‐associated DMED. Sprague‐Dawlay rats were employed to establish DMED models. After modelling, levels of miR‐205 and AR in their cavernous bodies were measured. The relationship between miR‐205 and AR was verified using a dual‐luciferase reporter gene assay. The underlying regulatory mechanisms of miR‐205 were investigated in concert with the treatment of mimics or inhibitors of miR‐205, or AR overexpression in the cavernous smooth muscle cells (CSMCs) isolated from rats with DMED. Meanwhile, the effects of miR‐205 and AR on cell proliferation and apoptosis were evaluated using MTT assay and flow cytometry respectively. Rats with DMED presented with increased miR‐205 and decreased AR levels in the cavernous bodies. AR was identified as a target gene of miR‐205. Down‐regulation of miR‐205 or up‐regulation of AR could increase proliferation and inhibits apoptosis of CSMCs in addition to improvements in the erectile functioning of rats with DMED. In summary, miR‐205 may contribute to the pathogenesis of DMED via down‐regulation of AR expressions.

cellular processes, such as proliferation, development and apoptosis by regulating the expression of approximately 60% of human genes. 9 Although the role of miRs in DMED remains to be unclear, growing evidence has indicated that miRs are involved in the pathogenesis of diabetes. 10 For instance, increased miR-503 was reported to contribute to DM-induced impairment of endothelial functioning and reparative angiogenesis after limb ischemia. 11 Moreover, significantly high levels of miR-146a were expressed in patients with type 2 DM, 12 indicating the possible involvement of miR-146 in type 2 DM progression.
Androgen receptor (AR) belongs to a kind of nuclear transcription factors that are activated by the steroid hormone receptor family of ligands, and contains four functional regions including a DNA-binding domain, an amino terminal regulatory domain, a carboxy-terminal ligand-binding domain, and a hinge region containing a nuclear localization signal. 13 In addition, AR mediates a wide range of cellular processes, such as proliferation, differentiation and apoptosis. 14 Interestingly, multiple studies have indicated that AR may prevent visceral fat accumulation, while low levels of AR may serve as a potential risk factor for DM in elderly men. 15,16 Similarly, AR is highly expressed in pancreatic beta-cell cytoplasm of control mice, and is known to gradually decrease with the progression of type 1 DM. Furthermore, the decrement of AR expression in diabetic mice was closely related to beta-cell proliferation as well as beta-cell apoptosis inhibition. 13,14 Interestingly, miR-205 was previously found to be associated with the poor prognoses in patients with prostate cancer (PC) partly by negatively regulating AR. [14][15][16][17] However, the underlying mechanism between the involvement of miR-205 and DMED remains to be largely unknown. Therefore, the current study aims to establish DMED rat models in order to investigate the involvement of miR-205 in the disease development, and its further association with AR regulation.

| Establishment of DMED rat models
A total of 120 Sprague-Dawley male rats (weighing 210-240 g and aged 6-9 weeks) were purchased from Experimental Animal Center, Sun Yat-sen University (Guangzhou, Guangdong, China), and raised in the animal house of the China-Japan Union Hospital of Jilin University using a 12/12 light-dark schedule with water and food available ad libitum. The mating test showed that all rats presented with normal sexual functioning. All detailed accommodation and care procedures complied with Chinese recommendations and legislations. In addition, all efforts were made to minimize the number and suffering of the included animals. Next, the rats were randomly divided into the normal group (n = 10) and the DMED group (n = 110). After being fasted for a duration of 8-12 hours, the rats in the DMED group were intraperitoneally injected with streptozotocin (STZ, 40 mg/kg, Sigma-Aldrich Chemical Company, St Louis, MO, USA) in citrate buffer solution (0.1 mol/L, pH = 4.5). Simultaneously, the rats in the normal group were intraperitoneally injected with equal amounts of 0.1 mol/L citric acid-sodium citrate buffer solution. All symptoms after the administration of STZ injection were recorded, in addition to measurement of blood glucose levels in caudal blood using the tail-clipping method with a blood glucose meter (Roche Diagnostics, Indianapolis, IN, USA) from the 4th day. The blood glucose levels were measured once in a week. Rats presenting with random blood glucose level >16.67 mmol/L were considered to be diabetic. Protamine zinc insulin would be injected subcutaneously to the neck of rats with blood glucose >25 mmol/L, and in coma. A total of 96 rats were found to be successfully established into DM rat models. After 10 weeks of feeding, DM rats were screened, and the animals were weighed and placed into test cages. Under quiet conditions, the light in the room was diminished, so that the rats could adapt to the environment for 10 minutes. Next, a total of 100 µg/ kg apomorphine (APO, Shenyang No.1 Pharmaceutical Factory, Shenyang, Liaoning, China) was subcutaneously injected into the soft skin of the neck. After administering injections, the rats were observed using a video recorder from the bottom of the cage for 30 minutes, and the erection times were recorded. The standards of penile erection in rats were as follows: the prepuce was receded, and the penis was enlarged and the glans was exposed. Abnormal erectile function was defined as no erection. 18 Our statistical results showed that 70 DMED rats were successfully established. All aforementioned experiments were approved by the Ethics Committee of the China-Japan Union Hospital of Jilin University.

| Observation of erectile function in rats
One week after intervention, observations were conducted during the night in a quiet environment, and the rats were injected with 100 g/kg APO in the neck. After the rats were allowed to adapt for 5 minutes, the number of erections in rats in each group was recorded for 30 min.

| Animal grouping and transfection
A total of 60 rats from the DMED groups were selected randomly, and divided into the following six groups (10 rats in each group): the DMED group (without any transfection), the negative control group (NC, transfected with empty adenovirus), the miR-205 mimic group (transfected with miR-205 mimic lentivirus), the miR-205 inhibitor group (transfected with miR-205 inhibitor lentivirus), the AR overexpression group (transfected with AR overexpression lentivirus) and the miR-205 mimic +AR overexpression group (transfected with miR-205 mimic and AR overexpression lentiviruses). All lentiviruses were constructed by Shanghai Genechem Co., Ltd. (Shanghai, China).
Next, the rats were administered intraperitoneal injections with 0.4% nembutal by 35 mg/kg, and immediately fixed on fixed trays in the supine position after anaesthetization. The corpora penis was fully exposed and ligated at the root using elastic in order to obstruct injecting fluid into the systemic circulation. Then, 50 µL of virus diluent (1 × 10 7 virus) was injected into cavernosa with an insulin syringe within 1 minute. The insulin needle was placed on the cavernosa for 5 minutes, and then extracted carefully, and a tampon was used for hemostasia. After the elastic was loosened, the rats were placed in cages for feeding. The times of penile erection, body weight and blood glucose levels of rats were recorded again.

| Measurement of intracavernosal pressure and mean arterial blood pressure
After transfection, the rats from each group were anaesthetized, and fixed again according to the aforementioned procedures, the median skin of the neck and muscle were cut-off, and the common carotid artery was isolated to avoid vagal nerve destruction. Next, the distal end of the isolated common carotid artery was ligated (the proximal end was not ligated), and a V-shaped incision was carefully made through the common carotid artery under an anatomical microscope. The PE50 tube (which was flushed with heparin and connected to the PowerLab/4sp A/D converter) was rapidly inserted into the proximal suture along the incision. The vessel and PE50 tube were clamped using tweezers, and the common carotid artery and PE50 tube were fixed in order to determine the mean arterial blood pressure (MAP). Skin overlying the median abdomen was incised to isolate muscles and fascia, and to fully expose the prostate gland and carefully dissected the surrounding fat tissues. Pelvic ganglion and cavernous body nerve were found under an anatomic microscope, and the skin and fascia of the penis were cut to fully expose the penile tissues. Under the anatomic microscope, a 25-gauge intravenous infusion needle (which was flushed with heparin and connected to the three-way tube and PowerLab/4sp A/D converter) was carefully inserted into the right cavernous body of the penis, accompanied by 250 U/mL heparin injections to observe whether it was smooth or not (if not smooth, the needle was adjusted until it was smooth).
With 15 HZ, 1.2 ms serving as the electrical stimulation parameter, a bipolar hook electrode was used to stimulate the cavernous nerve of the penis, and 5. 0 V voltage was for stimulation. The stimulation was carried out for 1 minute (three times at an interval of 10 minutes between two stimulations). The two channels were connected with the pressure transducer respectively and directly connected to a pressure transducer BL-410 biologic function experiment system (Chengdu Taimeng Technology Co., Ltd., Chengdu, Sichuan, China), and accordingly, the MAP and intracavernosal pressure (ICP) values were recorded. The ratio of ICP to MAP was used as the evaluation standard of erectile function in rats as follows: erectile function (%) = ICP/MAP ×100%. After the measurement was carried out, blood samples were obtained from the caudal artery, and the serum content was separated. In addition, radioimmunoassay was employed in order to determine the serum testosterone levels. Then, the rats were sacrificed with excessive pentobarbital sodium. The fascia surrounding the penile tissues was rapidly dissociated, and the whole penis (including the cavernosum) was cut-off and rinsed two times with phosphate buffer saline (PBS). The cavernosa tissues were fixed with 4% paraformaldehyde overnight, embedded in paraffin and sectioned continuously. The remaining corpus cavernosa tissues were placed in a cryopreserved tube, and preserved in liquid nitrogen pot at −80℃. Referring to the quantitative real-time polymerase chain reaction (qRT-PCR) procedure of cell experiment, the expression of miR-205 and AR in the corpus cavernosum of normal rats and DMED rats was detected. Each experiment was repeated three times to obtain the mean value.

| Hematoxylin and eosin staining
Cavernosa tissues were fixed in 4% formaldehyde for 24 hours, washed under tap water and sliced into 5-μm-thick sections after convention dehydration, clearing two times with xylene (5 minutes each time) and cooled on the cold table of the paraffin-embedded machine. Next, the paraffin sections were heated for 1 hour at 70°C, and then heated for 5 hours at 60°C. Then, the sections were de-

| Masson staining
The paraffin sections were heated at 65°C for 3 hours, dewaxed and dehydrated conventionally, rinsed for 40 minutes in 10% trichloroacetic acid and 10% kalium dichromicum, and washed under tap water. Next, the sections were stained using haematoxylin (Shanghai Bogoo Co., Ltd., Shanghai, China) for 8 minutes and washed under tap water. Then, the sections were rinsed using a mixture of 1% ponceau (HL12202, Shanghai Haling Biotechnology Co., Ltd., Shanghai, China) and 1% magenta (HPBIO-SJ820, Hepeng Biotechnology Co., Ltd., Shanghai, China). The reaction was terminated with 1% ice vinegar and 1% molybdic acid, followed by treatment of 1% brilliant green, 1% phosphomolybdic acid and tap water. Subsequently, conventional dehydration, clearing and mounting with resinene were carried out. Twenty high-power visual fields of each slice were randomly selected, and the staining results of smooth muscle and connective tissue were observed under an optical microscope.

| Immunohistochemistry
Paraformaldehyde-fixed cavernosa tissues were dehydrated, cleared, embedded in paraffin and sliced into 4-μm-thick sections. The sections were supplemented with 10% normal goat serum (C-0005, Shanghai Haoran Bio Technologies Co., Ltd., Shanghai, China), incubated at room temperature for 20 minutes. Next, the sections were incubated with the addition of primary rabbit anti-rat antibody to AR (dilution ratio China) for 5-10 minutes. After being washed, the sections were counterstained with haematoxylin (PT001, Shanghai Bogoo Co., Ltd., Shanghai, China) for 1 minute and then rinsed with water. Finally, the sections were soaked with 1% ammonia water for 10 seconds, washed and sealed using resinene. Subsequently, the sections were observed under a 100-fold microscope with PBS instead of the primary antibody serving as the negative control. A total of five high-power visual fields with 100 cells in each field were randomly selected and observed. The positive cells were identified as those with staining degree greater than 25% with brown or tan granules in the nucleus or cytoplasm. 19 When the positive area was located, the image was obtained using a colour camera, and input in the 'Video Pro32' colour image analysis system.
The grey value of the area was measured after the positive area was segmented accurately. 20

| Quantitative real-time polymerase chain reaction
Total RNA content of cavernosa tissues from each group was extracted in strict accordance with the instructions of the Trizol kit (15596-018, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and subsequently, its concentration was measured. All primers employed in the current study were synthesized by Takara Biotechnology Ltd. (Dalian, Liaoning, China) and shown in Table 1.
Reverse transcription was performed using a cDNA reverse transcription system (K1622, Beijing Reanta Company, Beijing, China), with the reaction condition set as 30-50 minutes at 42°C and 5 seconds at 85°C. The obtained cDNA was diluted to 50 ng/μL, and 2 μL was used each time for 25 μL of amplification system. qRT-PCR was then performed with 2 μg cDNA serving as the templet using a fluo-

| Western blot analysis
Extracted cavernosa tissues from each group were treated with protein lysis at 4°C for 30 minutes, vibrated once every 10 minutes, and then centrifuged at 2000 × g for 20 minutes at 4°C. The fat layer was discarded, and the supernatant was collected as the protein extract.

| Cell culture and transfection
Cavernous smooth muscle cells (CSMCs) of the penis were cul-

| Dual-luciferase reporter gene assay
The target relationship between miR-205 and AR was predicted

| Pull-down assay
Cavernous smooth muscle cells were transfected with wide-type  cells. Next, the mixture was incubated for 15 minutes at room temperature, added with 1 mL of HEPES and mixed. Subsequently, the cells were excited at 488 nm, and FITC fluorescence was detected using a 520 nm band-pass filter, while PI was detected using a 610 nm band-pass filter.

| Statistical analysis
Statistical analyses were conducted using the SPSS 21.0 software (IBM Corp. Armonk, NY, USA). Measurement data were presented as mean values ± SD. The one-way analysis of variance (ANOVA) was used to compare values among groups, followed by the Student's ttest to compare values between groups. The Pearson correlation analysis was carried out for the analysis of the correlation of two variable quantities. P < 0.05 indicated that the difference was statistically significant.

| Successful establishment of DMED rat models
A total of 110 rats were included in the current study, and five rats died; and nine rats presented with blood glucose levels were not more than the results showed ( Figure 1A) that the times of erection of rats with DMED were significantly reduced compared with the normal rats. In addition, the expression of miR-205 and AR in cavernosa tissues of rats with DMED and normal rats using qRT-PCR. Interestingly, the expression of miR-205 in rats with DMED was found to be significantly higher than those in the normal rats, while the expressions of AR mRNA were significantly lower respectively (both P < 0.05) ( Figure 1B). The above results indicated that rats suffering from DMED exhibited high expression of miR-205, in addition to reduced AR expressions.
F I G U R E 1 The DMED rat models are successfully established. A, the times of erection; B, the mRNA levels of miR-205 and AR measured by qRT-PCR; *, P < 0.05 vs the normal group; measurement data were expressed as mean ± SD and analysed by unpaired t test; the sample size of each group was 10; the experiment was repeated three times; DMED, erectile dysfunction rats with diabetic mellitus; miR-205, microRNA-205; AR, androgen receptor; qRT-PCR, quantitative real-time polymerase chain reaction.

| MiR-205 down-regulation or AR up-regulation improves the erectile functioning of rats with DMED
Body weight and blood glucose level changes are shown in Table 2.
After STZ administration, the body weight and blood glucose level were found to be dramatically elevated (P < 0.05). There were no significant differences in the body weight and blood glucose levels among the NC, DMED and miR-205 mimic +AR overexpression groups (P > 0.05). In comparison with the DMED group and the NC group, the body weight and the blood glucose levels of rats in the miR-205 mimic group were found to be significantly increased after STZ administration respectively (both P < 0.05); whereas the body weight and the blood glucose levels of rats in the miR-205 inhibitor group and the AR overexpression group were decreased (both P < 0.05).
In comparison with the normal group, decreased times of erection of rats were recorded in all other groups (P < 0.05). There were no significant differences in erection times among the NC, DMED and miR-205 mimic +AR overexpression groups (P > 0.05). Compared with the DMED group and the NC group, the times of erection of rats in the miR-205 mimic group were found to be markedly reduced, while those in the miR-205 inhibitor group and the AR overexpression group were significantly increased (P < 0.05) (Figure 2).
The results of electrical stimulation testing are shown in Table 3 as well as in the Figures S1 and S2 After ICP/MAP recording and prior to rat sacrifice, blood samples were obtained from the caudal artery, and serum was isolated.
In addition, serum testosterone concentration was measured using radioimmunoassay. Results as shown in Table S1 demonstrate that the serum testosterone concentration of rats in each group was consistent with that of electrical stimulation, indicating that testosterone concentration was directly correlated to the progression of ED. The findings also indicate that testosterone and AR may be TA B L E 2 STZ-inducement leads to statistically significant difference in body weight and blood sugar level of rats Measurement data was expressed as mean ± SD; data in each group were analysed with ANOVA; the sample size of each group was 10; the experiment was repeated three times. a P < 0.05 vs. the normal group. b P < 0.05 vs. the DMED group and the NC group.
F I G U R E 2 miR-205 down-regulation enhances the times of erection in rats with DMED. *, P < 0.05 vs the normal group; #, P < 0.05 vs the DMED group and the NC group; measurement data were expressed as mean ± SD; data in each group were analysed with ANOVA; the sample size of each group was 10; the experiment was repeated three times; DMED, erectile dysfunction rats with diabetic mellitus; miR-205, microRNA-205; AR, androgen receptor; NC, negative control.

| MiR-205 downregulation or AR overexpression alleviates the pathology of cavernous bodies
In the following experiments, the current study evaluated the ef-

| MiR-205 down-regulation or AR overexpression inhibits apoptosis of CSMCs in rats with DMED
QRT-PCR and Western blot analysis were employed to determine the expression of apoptosis-related genes, including Caspase-3, Bax and Bcl-2 in order to explore the mechanism of down-regula-  The level of MAP, ICP and ICP/MAP were measurement data, which was expressed as mean ± SD; data in each group was analysed with ANOVA; the sample size of each group was 10; the experiment was repeated for three times. a P < 0.05 vs. the normal group. b P < 0.05 vs. the DMED group and the NC group.

| MiR-205 targets and negatively regulates AR
According to analyses by the online target gene prediction software, microRNA.org, a specific binding area exists between the AR gene sequence and miR-205, indicating that AR is a potential target gene of miR-205 ( Figure 5A). We further confirmed the relationship between miR-205 and AR by using a dual-luciferase reporter gene assay. In comparison with the NC group, the luciferase activity was found to be significantly decreased in Wt-smiR-205/AR co-transfection of the miR-205 mimic transfection group (P < 0.05), while there were no significant differences in terms of the luciferase activity of MUT 3'-UTR (P > 0.05) ( Figure 5B), suggesting that miR-205 could specifically bind to the AR gene.
As shown in Figure  were increased (all P < 0.05) ( Figure 6A).
Additionally, flow cytometry was employed in order to measure the apoptosis of CSMCs. There were insignificant differences in cell apoptosis among the control, NC and miR-205 mimic +AR overexpression groups (P > 0.05). Compared with the control group and the NC group, the apoptosis rate of CSMCs in the miR-205 mimic group was found to be significantly increased, while decreased rates were observed in the miR-205 inhibitor group and the AR overexpression group (all P < 0.05) ( Figure 6B,C). In comparison with the control group and the NC group, the ex- F I G U R E 6 miR-205 down-regulation or AR overexpression enhances proliferation and suppresses apoptosis of CSMCs in vitro. A, cell viability detected by MTT assay; B, apoptosis rate analysis for CSMCs in response to the treatment of miR-205 mimic, miR-205 inhibitor, AR overexpression, and miR-205 mimic +AR overexpression determined by flow cytometry; C, flow cytometry map demonstrating CSMC apoptosis condition; D, the mRNA levels of Caspase-3, Bax and Bcl-2 in CSMCs treated with miR-205 mimic, miR-205 inhibitor, AR overexpression, and miR-205 mimic +AR overexpression determined by qRT-PCR; E, the protein levels of Caspases3, Bax and Bcl-2 in CSMCs treated with miR-205 mimic, miR-205 inhibitor, AR overexpression, and miR-205 mimic +AR overexpression determined by Western blot analysis; F, the grey value of Caspases3, Bax and Bcl-2 protein bands in CSMCs treated with miR-205 mimic, miR-205 inhibitor, AR overexpression, and miR-205 mimic +AR overexpression determined by Western blot analysis; *, P < 0.05 vs. the control group and the NC group; measurement data were expressed as mean ± SD; data in each group were analysed with repeated measurement or ANOVA; the experiment was repeated three times; erectile dysfunction rats with diabetic mellitus; miR-205, microRNA-205; AR, androgen receptor; NC, negative control; CSMC, cavernous smooth muscle cells; MTT, 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide; qRT-PCR, quantitative real-time polymerase chain reaction.

| D ISCUSS I ON
Erectile dysfunction is a widespread condition affecting diabetic males, with prevalence rates as high as 85%. 23 DM is considered to be a major risk factor for the onset of ED; however, the underlying pathogenesis of DMED remains to be largely unknown. Interestingly, miRs, which are novel and potent mediators of tissue injury, and their correlations with various benign urologic pathologies, specifically ED, remain unexplored. 24 Therefore, owing to various evidence and findings, the current study aimed to explore the role of miR-205 in DMED using multiple modes of testing and experimentation. In the current study, we demonstrated that miR-205 contributed to the pathogenesis of DMED by negatively regulating AR Additionally, it was revealed that AR is a target gene of miR-205, and down-regulation of miR-205 or up-regulation of AR significantly improved the erectile functioning of rats suffering from DMED by promoting proliferation as well as inhibiting apoptosis of CSMCs.
Initially, the current study revealed that compared with normal rats, rats with DMED exhibited significantly down-regulated expressions of AR, and significantly up-regulated miR-205 expressions. Interestingly, low androgen and AR levels in elderly men have been reported to be involved in the incidence and development of DM through interaction with obesity, glucose homeostasis as well as insulin resistance. 17 Next, this study found that miR-205 up-regulation was responsible for a decrease in AR expressions in rats with DMED. Subsequently, AR was identified to be a target gene of miR-205. This particular finding was in accordance with a previous study on PC, in which the expression of miR-205 was found to be negatively correlated with the expression of AR. 17 The most significant effect of miR-205 on cell growth in prostate cells is dependent on androgen. 25 Essentially, the findings of the current study indicate that downregulation of miR-205 or up-regulation of AR can alleviate the erectile function of rats with DMED. According to previous evidence, decreed expressions of AR were strongly associated with ED, leading to inadequate penile arterial blood flow, one of the primary causes of ED. 26 Low androgen levels in the neonatal period changed gene expression of smooth muscle cell differentiation-related biomarkers, which resulted in a decline of CSMCs, and consequently to penile dysfunction. 27 In addition, endothelial progenitor cells (EPC) markers were expressed in the cavernosal sinusoidal endothelial space. 28 It is known that androgens stimulate EPC through an AR-mediated pathway. 29 Several studies have shown that patients suffering from ED exhibit a decreasing number of circulating EPCs. 30,31 These results suggest that high expression of AR may improve the erectile function. 28 Suppression of miR-205 alone was correlated selectively with increased 3 T3-L1 cell proliferation. 32 In a previous study, several miRNAs were confirmed to be aberrantly expressed in the corpus cavernosum of aging rats with ED, including miR-1, miR-200a, miR-203 and miR-206. 33 These aforementioned miRNAs play potent roles via regulation of endothelial nitric oxide (NO) synthase, NO, protein kinase G, the prostaglandin E1 and protein kinase A pathways, and thus lead to the development of aging-induced ED in rats. 33,34 Another finding of the current study is that down-regulated expression of miR-205 or up-regulated expression of AR can prevent tissue fibrosis in the corpus cavernosum. Furthermore, it was also unfounded that overexpression of AR could rescue miR-205 up-regulation mediated DMED. In addition, emerging data suggest that miR-NAs participate in the regulation of fibrosis process. 35 Several recent studies have identified that the miR-200 family, including miR-200a, miR-200b, miR-200c, miR-141, miR-149 and miR-205, function as key regulators of epithelial-mesenchymal transition, from which the myofibroblast, the key cellular mediator of fibrosis, is derived. 36,37 Moreover, lipoxin A4 was also thought to be effective in inhibiting corporal fibrosis, thus improving ED in rats with type I diabetes. 38 Androgen deficiency induces corporal fibrosis through the activation of the Smad and non-Smad pathways, characterized by loss of corpus cavernosum smooth muscles cells, corresponding reduction in SM/C ratio, and accumulation of ECM proteins. 39 Moreover, it was also further reported that androgen deprivation leads to penile tissue atrophy, alterations in dorsal nerve structure, alterations in endothelial morphology, reduction in trabecular smooth muscle cells content, increase in deposition of extracellular matrix and accumulation of fat-containing cells (adipocytes) in the subtunical region of corpus cavernosum. 40 In summary, findings of the current study lead to the conclusion that erectile functioning in rats suffering from DM can be improved by down-regulating the expression of miR-205 or up-regulating the expression of AR in DMED. However, further research is warranted in order to explore the mechanism of AR participation in the downstream pathways of ED.

CO N FLI C T O F I NTE R E S T
The authors have declared no conflicts of interests with respect to the research, authorship and/or publication of this article.