The Regulation Mechanism Of Lnc-GULP1-2:1/COL3A1 Involved In Granulosa Gell Proliferation And Apoptosis

BackgroundLong non-coding RNA is a kind of RNA molecule with a transcript length of more than 200 nt and lacking protein coding ability.It should be noted that lncRNA is a generic term that encompasses different classes of RNA transcripts,including enhancer RNA,snoRNA hosts,intergenic transcripts,and sense or antisense transcripts that overlap other transcripts.Unlike miRNAs or proteins,lncRNA functions are currently incapable of inferring from sequences or structures.The role of lncRNA is mainly achieved from the aspects of chromatin modification,transcriptional regulation and post-transcriptional regulation.LncRNAs are invoLVed in a variety of biological functions and pathological processes,including proliferation,differentiation,development,apoptosis and carcinogenesis.For example,forced expression of lncRNA-Amhr2 in mouse granulosa cells results in decreased mRNA levels of Amhr2,and activation of lncRNA-Amhr2 increases Amhr2 promoter activity.Although the field continues to develop,there is still a lack of studies on the expression profile of lncRNA in human ovarian granulosa cells.Granulosa cells play important roles in follicular development,oocyte maturation and follicular atresia.They proliferate,differentiate and apoptosis under the action of various hormones and cytokines formed at different developmental stages of follicles,which have an irreplaceable regulatory effect on follicular development.Granulosa cells support oocyte growth and development,regulate meiosis,and regulate global transcriptional activity in the oocyte genome.In turn,oocytes promote granulosa cell proliferation and differentiation.In many ovarian-related diseases,abnormal states of granulosa cells were observed.In the related studies of primary ovarian insufficiency(POI),apoptosis of ovarian granulosa cells was observed at different levels,such as molecular level,tissue level and gene level.POI refers to the loss of function of the ovary before age 40,accompanied by amenorrhea,hypergonadotropism and hypoestrogenism.The causes of POF mainly include genetic disorders,autoimmune diseases,tuberculosis of the genital tract,smoking,ovarian surgery,radiation and/or chemotherapy.A report from the St.Jude Lifetime Cohort reported that the prevalence of POI in childhood cancer survivors to be 10.9%.In our previous study,next generation sequencing suggested that lncGULP1-2:1 and col3a1 were significantly down-regulated in ovarian cortical tissue in patients with premature ovarian failure than normal ovarian cortical tissue.Co-expression network analysis showed that COL3A1 was the cis-predicted transcript of lnc-GULP1-2:1.ObjectiveTo explore the role of lnc-GULP1-2:1 in ovarian granulosa cells;to examine the effect of lnc-GULP1-2:1 on granulosa cell proliferation;to study the interaction of lnc-GULP1-2:1 with COL3A1;to explore the lncGULP1-2: 1/COL3A1 Regulatory mechanism during the development of cisplatin-induced POI.MethodsIn this study,RT-PCR was used to detect the relative expression of lnc-GULP1-2:1/COL3A1 in granulosa cells from patients with different ovarian function reserve.The localization and expression of lnc-GULP1-2:1 in ovarian granulosa cells were detected by FISH.Lentivirus was used to construct a stable KGN stably overexpressing lnc-GULP1-2:1,and then CCK8 was used to detect the effect of overexpression of lnc-GULP1-2:1 on proliferation and apoptosis of KGN.The effect of cisplatin on the proliferation of granulosa cells was analyzed by CCK8.The effect of cisplatin on the expression of lnc-GULP1-2:1/COL3A1 on KGN was analyzed by real-time PCR and western blot.Results1.The expression level of lnc-GULP1-2:1 in granulosa cells was significantly higher in the PCOS group than NC group,and decreased significantly in the DOR group.The expression of lnc-GULP1-2:1 was mainly concentrated in the cytoplasm,but there is also a small amount of expression in the nucleus.2.Overexpression of lnc-GULP1-2:1 can inhibit the proliferation of KGN cells,up-regulate the apoptosis regulator Bax,and the expression of CCND2,whose activity is required for cell cycle G1/S transition,was significantly decreased,while the expression of cyclin-dependent kinase inhibitor P16 was up-regulated.3.The expression level of COL3A1 in granulosa cells was significantly higher in the PCOS group than NC group,and decreased significantly in the DOR group.Overexpression of lnc-GULP1-2:1 upregulate the expression of COL3A1 mRNA and protein.The overexpression of lnc-GULP1-2:1 significantly changed the subcellular localization of COL3A1 protein and increased its expression in the nucleus.4.The down-regulation of COL3A1 expression by siRNA in KGN significantly inhibited the proliferation of KGN but had no significant effect on the expression of lnc-GULP1-2:1.Up-regulation of lnc-GULP1-2:1 expression and down-regulation of COL3A1 can synergistically inhibit KGN proliferation and alter the expression of proliferation-and apoptosisrelated genes.5.The chemotherapeutic drug cisplatin can significantly inhibit the proliferation of KGN cells,and promote the expression of Bax gene,and inhibit the expression of Bcl-2 and Bcl-XL genes.Meanwhile,the expression of CCND2,whose activity is required for cell cycle G1/S transition,was significantly decreased,while the expression of cyclindependent kinase inhibitor P16 was up-regulated.Cisplatin can upregulate the expression of lnc-GULP1-2:1 and inhibit the expression of COL3A1 in KGN cells.Overexpression of lnc-GULP1-2:1 enhanced the inhibitory effect of cisplatin on KGN cell proliferation.ConclusionOverexpression of Lnc-GULP1-2:1 can change the distribution of COL3A1 protein in granulosa cells,increase the expression of COL3A1 protein in the nucleus,affect the expression of cell cycle-related proteins,and inhibit the proliferation of granulosa cells.Cisplatin can inhibit granulosa cell proliferation by up-regulating the expression of lnc-GULP1-2:1,affecting the intracellular localization of COL3A1 protein.Cisplatin can also directly down-regulate the expression of COL3A1 mRNA and protein in cells,affect the expression of cell cycleassociated proteins,and inhibit the proliferation of granulosa cells.