MiR-185-5p Regulates Angiogenesis By Targeting VEGFA In Polycystic Ovary Syndrome

Objective: Polycystic Ovary Syndrome(PCOS)is a prevalent endocrine disorder that has nearly 7%-9% incident rates in women of reproductive age.PCOS is also one of the leading causes of female infertility.The clinical manifestations of PCOS are heterogeneous.It not only seriously affects the reproductive function of patients,but also increases the incidence of estrogen-dependent tumors.In addition,PCOS also increases the occurrences of metabolic disorders including hyperandrogenism,insulin resistance,type 2 diabetes mellitus and cardiovascular disease.So far,the exact factors that cause PCOS have not been accurately elucidated.The etiology mainly involves the following aspects including genetic factors,environmental factors,high androgen secretion,obesity,inflammation,hyperpubertal development and so on.Previous studies have shown that angiogenesis plays an important role in the pathogenesis and development of PCOS.The prominent features of PCOS are ovarian angiogenesis and abundant blood flow,and the occurrence of PCOS is related to the malregulation of angiogenic factors.Much of literature in this field has highlighted the involvment of vascular endothelial growth factor(VEGF),which is high-expressed in PCOS patients.Di Pietro et al have demonstrated that VEGF levels may affect follicular development and ovarian over-angiogenesis in PCOS rats.Vascular endothelial growth factor A(VEGFA)is the most important vascular endothelial growth factor in VEGF growth factor family.MicroRNA(miRNA)is known as a class of endogenous non-coding RNA of approximately 22-24 nucleotides that impacts complex pathophy-siological processes.However,little is known about the effect of microRNA on follicular development and the course of PCOS.Studies on the regulatory mechanism of microRNA in this field are helpful for in-depth understanding of the pathological mechanism of PCOS and exploration of possible therapeutic targets.MiR-185 includes mi R-185-5p and miR-185-3p.Generally,MiR-185 refers to miR-185-5p.Mi R-185-3p is often degraded and not involved in biological processes.As far as we know,miR-185-5p(miR-185)is firstly identified to control cell growth in human lung cancers.A growing body of studies suggested that miR-185 may also be implicated in a series of diseases,such as tumor,lipid metabolism disorder,neurological disorder,liver fibrosis,dilated cardiomyopathy and idiopathic pulmonary fibrosis.Evidence from Bo Xu et al showed apparent decrease of miR-185-5p expression in the ovaries of PCOS patients,but there was no further specific study.We predicted interactions between VEGFA and miR-185-5p using bioinformatics tools,but the exact function of miR-185-5p in the pathogenesis of PCOS remains to be elucidated.In the current study,we generated classical PCOS animal model and injected them with lentivirus carrying miR-185-5p to investigate its role.Then VEGF-induced human ovarian microvascular endothelial cells(HOMECs)were established to further explore the underlying mechanism of miR-185-5p on angiogenesis.Methods: The dehydroepiandrosterone(DHEA)was subcutaneously injected into rats daily for consecutive 3 weeks.The PCOS rats with HOMA-IR>2.8 were selected for further experiments.Female SD rats were divided into 4 groups(6 in each group):(1)control group(2)PCOS group(3)PCOS + NC group(PCOS rats infected with negative control lentivirus)(4)PCOS + rno-mi R-185-5p group(PCOS rats infected with rno-miR-185-5p overexpressed lentivirus).Lentivirus containing ectopic rno-miR-185-5p or NC was constructed and subcapsularly injected into the ovaries of PCOS rats.After 2 weeks,the fasting rats were given glucose by gavage to assess HOMA-IR and test serum insulin release.Following the above measurements,all animals were sacrificed and the ovaries were harvested immediately for the following examinations.Pathological changes in rat ovarian tissues were detected by HE staining,mRNA expression levels of miR-185-5p and VEGFA in rat ovarian tissues were detected by real-time PCR,and contents of VEGFA,angiopoietin1(ANGPT1),ANGPT2,elelet-derived growth factor B(PDGFB)and PDGFD in ovarian tissues were detected by ELISA.Immunohistochemical analysis was performed to detect the expression of CD34 and ?-SMA in the ovarian tissues of rats in each group.Luciferase reporter assay was conducted to validate the binding activity between miR-185-5p and VEGFA in 293 T cells.Human ovarian microvascular endothelial cells(HOMECs)were purchased and cultured.Construct hsa-mi R-185-5p and its control lentiviral vectors an then infect HOMEC,which were then divided into three groups:(1)control group(2)LV-NC group(3)LV-hsa-miR-185-5p group.After 72 h of infection,Real-time PCR was used to detect the expression of miR-185-5p in the cells of eachgroup.After successful infection,Real-time PCR and Western blot were used to detect the mRNA and protein expression of VEGFA in the cells of each group.After successful infection(72h later),the cells were divided into 6 groups:(1)control group(2)LV-NC group(3)LV-hsa-mi R-185-5p group(4)VEGFA group(5)LV-NC + VEGFA group(6)LV-hsa-miR-185-5p + VEGFA group.VEGFA groups were administrated with recombinant VEGFA 100 ng/ml.HOMECs were applied to undergo MTT assay according to manufacturer's instruction at 0,24,48,72 and 96 hours,respectively.HOMECs were applied to undergo transwell assay at 24,48 hours,respectively.HOMECs were applied to observe tube formation by the phase-contrast microscopy at24 h.Results:The results of Real-time PCR showed that miR-185-5p level was significantly reduced in the ovary of PCOS rats,and expected to be increased by the injection with ectopic lentivirus.To determine the effect of miR-185-5p in PCOS rats,the HOMA-IR was firstly measured,indicating that overexpression of miR-185-5p could restore the high level of HOMA-IR in PCOS rats.Then the serum insulin release at indicated time points following glucose treatment was tested,demonstrating that the remarkable upregulation of insulin release level in PCOS rats were downregualted by miR-185-5p.To observe the ovarian histological changes,HE staining was conducted.It was apparent that ovarian structural integrity,multiple corpora lutea,and ovarian follicles in different stages were presented in the Control rats.However,we saw cystic expansion,granular cell layer reduction and corpora lutea decreasing,but oocyte and corona radiata disappearing in the ovary of PCOS rats.Overexpression of miR-185-5p mitigated the morphological damages of the ovaries and increased the layers of granular cells.PCOS rats showed a significant increase of VEGFA mRNA,but ectopic expression of miR-185-5p lighten this change in PCOS model.Further,the alterations of angiogenesis-associated factors in the ovary were tested using ELISA.Comparing with the control rats,PCOS rats induced remarkable increases of VEGFA and ANGPT1,and decreases of ANGPT2,PDGFB and PDGFD.However,overexpression of mi R-185-5p could reverse the protein changes of angiogenesis-associated factors.In addition,immunohistochemistry staining was performed to detect the expressions of blood vessel endothelial cell markers.The immunopositive materials of CD34 and ?-SMA in theendothelium of ovarian vessel was elevated in PCOS rats,but restored by overexpression of miR-185-5p.Luciferase reporter assay was carried out to explore the associations between miR-185-5p with VEGFA.The results showed that VEGFA was a potential target gene of miR-185-5p.Furthermore,in vitro experiments were designed to better uncover the underlying mechanism of miR-185-5p in PCOS.There was a clear increasing of mi R-185-5p expression in HOMECs by its overexpressing lentivirus.Furthermore,we also demonstrated that the levels of VEGFA mRNA and protein in HOMECs actually suppressed by the overexpression of miR-185-5p.The results of MTT showed that VEGF treatment increased cell viability,and miR-185-5p upregulation could restore cell proliferation to the normal level.The results of Transwell assay indicated that VEGF-induced significant increase of migratory cells was blocked by miR-185-5p overexpression.On the other hand,it was appeared that VEGFA administration in HOMECs caused a large number of formatted tubes,whereas the ectopic expression of miR-185-5p inhibited tube formation.Conclusions:This study shows that miR-185-5p is down-expressed in the ovary of PCOS rats,and its overexpression can attenuate insulin resistance,high serum insulin level and ovarian histological lesion in PCOS.We also found that miR-185-5p could inhibit angiogenesis and maturation of ovarian tissue in PCOS rats.Mechanically,we demonstrates that miR-185-5p directly interacts with VEGFA to inhibit endothelial cell proliferation and migration,as well as tube formation.Taken together,this is the first study to suggest that miR-185-5p acts as a protective effector on PCOS development through inhibiting excessive angiogenesis by targeting VEGFA.