Studies On Function Of Pim2 Gene And MiR-135a In Granulosa Cell Growth And Mammalian Ovarian Follicle Development

In mammalian ovaries,follicle development is a complex and precisely regulatory process from the primordial follicle(PF)activation to the ovulation,which is accompanied by follicle atresia and degeneration.Granulosa cells(GCs)provide support for oocyte mature and release,also the morphology and function of GC are continually changed during folliculogenesis.However,dysregulation of apoptosis and proliferation in GCs causes the retardation of follicle development and premature depletion of ovarian follicles,which decreases oocyte quality and shortens the reproductive lifespan of females.In previous studies,transcriptome sequencing of the pre-ovulatory ovarian follicles from Taihu and Large White sows revealed proviral integration site 2(Pim2)and miR-135a was significantly differently expressed.The roles of PIM2 and miR-135a in follicle development were explored.The main results are as follows:1.The role of Pim2 gene in apoptotic resistance of granulosa cell and follicle development(1)The PIM2 kinase inhibited mouse model was generated using C57BL/6J mice treated with SMI-16a(a PIM2 kinase inhibitor).The ratio of ovarian weight to body weight of PIM2 kinase inhibited mice was decreased by 23%,compared with control mice.ELISA assay results showed FSH and LH levels were significantly elevated while serum estradiol level was significantly decreased in PIM2 kinase inhibited mice.H&E staining revealed PIM2 kinase inhibition in mice could decrease the number of primordial follicles,pre-antral follicles and antral follicles,and in particular the number of primordial follicles were decreased by 43%,while secondary follicles were increased by69%,compared with control mice.Ki67 and TUNEL staining results indicated there were more TUNEL-positive GCs and fewer Ki67-positive GCs in PIM2 kinase inhibited mice,compared with control mice.These results suggest that PIM2 kinase inhibition causes the retardation of follicle development and a series of phenotypes analogous to premature ovarian insufficiency(POI)in women.(2)Western blot,fluorescence-activated cell sorting(FACS),CCK-8 and Ki67staining revealed that Pim2 gene could suppress apoptosis and promote proliferation,resulting in maintaining murine granulosa cell(m GC)survival.In addition,q PCR and ELISA demonstrated that Pim2 gene enhanced the expression of steroidogenic and follicle development genes and promoted estradiol synthesis.Subsequently,kinase-inactivated PIM2^K61A was constructed and further abolished the role of PIM2 in Pim2miR-135a apoptotic resistance and estradiol synthesis in m GCs.(3)The global quantitative phosphoproteomic analysis discovered 81 proteins comprising 87 phosphosites with increased phosphorylation levels(fold change>1.3,P<0.05),and 260 proteins comprising 425 phosphosites with decreased phosphorylation levels(fold change<0.77,P<0.05)in Pim2 knockdown(Pim2-KD)m GCs,compared with control m GCs.A subset of downregulated phosphoproteins in Pim2 knockdown m GCs,were enriched in the term of“positive regulation of apoptotic process”(GO:0043065).Notably,phosphoproteins that had increased abundance in Pim2 knockdown m GCs might participate in the steroid biosynthetic process(GO:0006694)and steroid metabolic process(GO:0008202).These results suggest that PIM2-mediated phosphorylation was involved in regulation of the apoptosis and steroid synthesis in m GCs.(4)Combination analysis of downregulated phosphoproteins in Pim2-KD m GCs and PIM2 immunoprecipitates revealed that DAPK3 was a potential PIM2 substrate.Immunoprecipitation assay demonstrated that PIM2 phosphorylated DAPK3 at S306,whereas SMI-16a could abolish this interaction.Confocal analysis and Western blot found that Pim2 overexpression promoted nuclear exclusion of DAPK3 with evidence of dominant existence of DAPK3^WT in the cytoplasm and phosphorylation-null DAPK3^S306Ain the nucleus.Moreover,DAPK3 was able to bind to and phosphorylate p53(p-p53),whereas Pim2 overexpression could abolish the interaction between DAPK3 and p-p53 in m GCs.These data suggested that PIM2 kinase phosphorylated DAPK3,induced its nuclear exclusion and further resulted in suppression of p53-dependent transactivation in m GCs.(5)A series of truncated Pim2 promoters were used to drive luciferase gene expression to determine promoter activity.The luciferase assay showed that the fragment from-247bp to-104bp in the 5'flanking region was crucial for Pim2 promoter activity in m GCs and CHO-K1.The potential transcription factor binding sites including two POU2F1 binding sites(-162bp?-148bp,-127bp?-113bp)were predicted in the Pim2promoter region by BIOBASE software.The site-directed mutagenesis and Ch IP-q PCR assay demonstrated that POU2F1 could specifically bind to the Pim2 promoter region.Moreover,POU2F1 could promote m GC survival via upregulating Pim2 expression.2.The role of miR-135a in suppression of granulosa cell growth during folliculogenesis(1)FACS,Western blot,CCK-8 and immunofluorescence assay revealed that 2021 miR-135a could regulate the progress of G1 phase in m GCs,increase the G1/S ratio and repress m GC proliferation.Meanwhile,miR-135a could also promote m GC apoptosis.The dual luciferase reporter system verified that miR-135a could target the 3'untranslated region(3'UTR)of Tgfbr1 and Ccnd2.RT-q PCR and Western blot demonstrated that miR-135a could suppress Tgfbr1 and Ccnd2 expression.(2)FACS,Western blot,CCK-8 and immunofluorescence assay verified that Tgfbr1gene could promote the progress of G1 phase in m GCs,enhance m GC proliferation activity,and repress m GC apoptosis.miR-135a could suppress the effect of Tgfbr1 and Ccnd2 on m GC proliferation.(3)Confocal immunofluorescence assay found that Tgfbr1 overexpression up-regulated the phosphorylation level of SMAD3 and the localization of SMAD3 in the nucleus;on the contrary,miR-135a overexpression repressed the phosphorylation level of SMAD3 and the localization of SMAD3 in the nucleus.Chromatin immunoprecipitation,RT-q PCR and Western blot demonstrated that Tgfbr1 overexpression enhanced the transcription activity of Ccnd2 and its expression.(4)A series of truncated miR-135a promoters were used to drive luciferase gene expression to determine promoter activity.The luciferase assay showed that the fragment from-250bp to-100bp in the 5'flanking region was crucial for miR-135a promoter activity in m GCs and CHO-K1.The potential transcription factor binding sites including two ESR2 binding sites(-197bp?-183bp,-141bp?-127bp)were predicted in the miR-135a promoter region by BIOBASE software.The site-directed mutagenesis and Ch IP-q PCR assay demonstrated that ESR2 could specifically bind to the miR-135a promoter region.Moreover,Esr2 overexpression or estrogen repressed miR-135a expression in mGCs.