The Role And Mechanism Of Ovarian Aging Related LncRNA-lnc81 In The Functional Regulation Of Ovarian Granulosa Cells And Female Germ Stem Cells Mediated By The Hippo Signaling Pathway

Further study on the mechanism of ovarian aging is crucial for the prevention and treatment of premature ovarian failure and lengthening female reproductive life.The function and lifespan of ovary are determined by the quantity and quality of follicles.It is well known that granulosa cells(GCs)activity is important for maintaining ovarian morphology and function.Recently,newly found female germ stem cells(FGSCs)have become a potential source to maintain the ovarian structure and function.Previous study from others and ours demonstrated that the Hippo signaling pathway,which can control the size,growth and regeneration of tissues and organs by sensing and responding to mechanical signals,played pivotal roles in regulating the proliferation of GCs and FGSCs,and probably affected the process of ovarian aging.However,the mechanisms have not been fully elucidated,and it is not clear whether there is any ‘crosstalk’ between GCs and FGSCs.Therefore,to investigate lncRNAs which may play an important role in ovarian function regulation and related diseases by function as transcriptional or posttranscriptional regulatory molecules,this study screened,verified and identified the lncRNAs closely related to the Hippo signaling pathway,and observe the function and mechanism of lncRNAs closely related to Hippo-TEAD2 in GCs and FGSCs.To our knowledge,this study was the first time that revealed lnc81,a new lncRNA closely related to ovarian aging.Lnc81 was shown to regulate the proliferation and apoptosis of GCs and FGSCs through the Hippo-TEAD2-CCN1/2 pathway.All of these results added new materials for the mechanism research of maintaining ovarian physiological function in mammals,and provided important theoretical basis for treatment or delay of female ovarian aging.Part Ⅰ: Identification,expression and localization of lncRNAs related toovarian aging and targeting to Hippo-TEAD2In order to study the role of lncRNAs in the ovarian function regulation,we usedbioinformatic analysis technology to screen lncRNAs related to ovarian aging,and especially focused on the lncRNAs related to Hippo-TEAD2.Using PCR,Western blot,RIP,RACE and other experimental techniques,we identified the existence of lnc81 and verified the combination of lnc81 and TEAD2.Then we observed the expression changes of lnc81 during the ovarian aging process and the subcellular localization of lnc81 in GCs and FGSCs.Simultaneously,the relative expression of CCN1 and CCN2,the target genes of TEAD2,were detected in different periods of ovarian tissue.The results showed that:(1)Lnc81 was a lncRNA closely related to ovarian aging.The full length of lnc81 was 1877 bp,and it localized on chromosome4.(2)lnc81 was highly presented in mice ovarian tissue.With the increase of mice age,the expression level of lnc81 increased significantly(p< 0.05).(3)Lnc81 was presented both in ovarian GCs and FGSCs,and mainly located in the nucleus.(4)Lnc81 can bind to TEAD2,a main component of the Hippo signaling pathway.(5)The mRNA and protein expression of CCN1 decreased significantly with age(p<0.05,compared to the 3 d group).In addition,the mRNA and protein expression of CCN2 increased significantly in 3 m group(p< 0.05,compared to the 3 d group),but decreased significantly at 11 m and 17 m groups(p< 0.05,compared to the 3 d group).These results indicated that the lncRNA-lnc81 in mice ovary is related to the ovarian aging,and it may be related to Hippo-TEAD2 and target genes CCN1 and CCN2.Part Ⅱ: Biological function and mechanism of lnc81 in ovarian granulosa cellsTo clarify the biological role of lnc81 in ovarian granulosa cells,siRNA was used to downregulate the expression of lnc81 in GCs,the proliferation and apoptosis of GCs were observed,and the expression changes of TEAD2,CCN1 and CCN2,the downstream genes of the Hippo signaling pathway,were analyzed.In addition,we further studied the role and mechanism of CCN1 and CCN2 in the function regulation of human granulosa cells.The results showed that:(1)Downregulation of lnc81 can inhibit the proliferation of granulosa cells.(2)The apoptosis of granulosa cells can be promoted by the downregulating the expression of lnc81.(3)The expression of TEAD2,CCN1 and CCN2 increased significantly after downregulating lnc81(p<0.05).(4)YAP mediated the sphingosine-1-phosphate(S1P)-induced upregulation ofCCN1,CCN2,and COX2 expression.All of these results indicated that the Hippo signaling pathway play an important role in the regulation of ovarian granulosa cells function,and lnc81 can regulate the function of granulosa cells through the Hippo signaling pathway.Part Ⅲ: Biological function and regulatory mechanism of lnc81 in female germ stem cells(FGSCs)and the regulatory process of FGSCs by GCsTo further verify the physiological function of lnc81 in FGSCs,we observed the effect of lnc81 on the proliferation and apoptosis of FGSCs by using CCK analysis,EDU staining,Tunel staining and so on.In addition,the proliferation of FGSCs was observed after co-culturing with GCs,and the possible mechanism of FGSC proliferation was explored by PCR and Western blot.The results showed that:(1)The proliferation of FGSCs can be inhibited by the downregulation of lnc81.(2)The apoptosis of FGSCs can be promoted by the downregulation of lnc81.(3)After downregulating lnc81,the expression of TEAD2,CCN1 and CCN2 increased significantly(p< 0.05),the mRNA and protein expression of MVH decreased significantly(p< 0.05),while there was on change of OCT4 expression.(4)Compared with the group that FGSCs were cultured alone,the proliferation rate of FGSCs decreased significantly when they were cultured with GCs(p< 0.05),and the proliferation level was lower when the density of GCs was higher(p< 0.05).After co-culturing with GCs,the expression of lnc81 in FGSCs decreased,and the decline level increased with the increase of the GCs numbers.There was no change of the mRNA and protein expression of CCN1 in FGSCs after co-culturing with GCs.Compared to the control group,the mRNA expression of PCNA decreased significantly(p< 0.05).While the rate of mRNA and protein expression level of BAX/BCL-2 increased compared to the control group and the low density group(p<0.05).All of these results verified the role of lnc81 in regulation of FGSCs proliferation and apotosis,and showed that lnc81 mediated the regulation of FGSCs proliferation by GCs,indicating the role of lnc81 in the ovarian microenvironment communication.