Screening And Verifying Ovary-specific LncRNA Related To Hippo Signaling Pathway And Preliminary Investigation On Ovarian Aging In Mice

Objection:This study aimed to observe the changes of lncRNA expression profile during the physiological aging process of the ovary,screen and verify ovary-specific lncRNAs related to Hippo signaling pathway,and explore their expressions in the ovarian tissues at different stages.The results of this study will provide new information for further enriching molecular network of ovarian development regulation and provide reference for finding effective biomarkers of early ovarian aging.Methods:1.The expression profiles of lncRNA in ovarian tissues of different functional states were observed by High throughput sequencing to detect the expression of lncRNA in ovary tissues of young mice(3 months),ovarian dysfunction group(11 months)and ovarian failure group(17 months).2.Bioinformatics(GO and KEGG enrichment analysis)was used to annotate the target genes of lncRNA differentially expressed in the sequencing results,and the corresponding target genes were enriched by signal pathways.Ten lncRNAs were randomly selected and verify the reliability of the sequencing results by q-PCR.3.Screening ovary-specific lncRNA related to Hippo signaling pathway from three groups: young group(3 months),ovary dysfunction group(11 months)and ovary failure group(17 months),young group(3 months)and ovarian failure group(17 months),respectively to construct a co-expression network of lncRNA-RNA.4.The relative expression levels of ovary-specific lncRNA related to Hippo signaling pathway in 10 tissues of heart,liver,spleen,lung,kidney,thymus,ovary,muscle,stomach and brain were detected by q-PCR and RT-PCR respectively.Three ovary-specific lncRNA of Hippo signaling pathway were obtained.5.Differential expression of three lncRNAs in ovary tissues under different functional states was detected by q-PCR and RT-PCR techniques.6.Further bioinformatics analysis of the three lncRNAs was carried out,and the Sanger sequence of the PCR products was sequenced by RT-PCR technology to detect whether the PCR products consist with the complete sequence reported in the database.Results:1.The results of high throughput sequencing showed that there were 545 differentially expressed lncRNA(including 361 up-regulated and 184 down-regulated)in the ovary dysfunction group compared with the younger group.There were 300 known lncRNAs(156 up-regulated and 144 down-regulated),245 new predicted lncRNAs(205 up-regulated and 40 down-regulated).There were 664 differentially expressed lncRNAs(394 up-regulated and 270 down-regulated)in ovary failure group,of which 453(283 up-regulated and 170 down-regulated)were known,and 211(111 up-regulated and 100 down-regulated)were newly predicted.There were 598 differentially expressed lncRNAs(including 255 up-regulated and 43 down-regulated)in the ovary failure group compared with the ovary dysfunction group,of which 301(including 185 up-regulated and 116 down-regulated)were known,297(including 70 up-regulated and 227 down-regulated)were newly predicted.2.Bioinformatics analysis results:(1)GO analysis showed that lncRNA was significantly enriched in protein localization,cell-cell junction maintenance,protein binding during ovarian dysfunction.(2)KEGG pathway analysis showed that differentially expressed lncRNA target genes participated in Hippo signaling pathway,MAPK signaling pathway,Wnt signaling pathway,Ras signaling pathway,B cell receptor signaling pathway,RNA degradation,RNA transport and other pathways.3.The random selection of lncRNA was verified by q-PCR,and the results showed that the expression trend was consistent with the sequencing results.4.The results of q-PCR and RT-PCR showed that there are three ovary-specific lncRNA related to Hippo signaling pathway: ENSMUST00000210794,ENSMUST00000156781,ENSMUST00000144657.Bioinformatics analysis showed that ENSMUST00000210794 is mainly target to the LATS2 molecule of Hippo pathway,and also participates in Notch signaling pathway and MAPK signaling pathway.ENSMUST00000156781 is mainly target to TEAD1 and TEAD2 molecules of Hippo pathway.It also participates in FoxO signaling pathway,TGF-beta signaling pathway,PI3K-Akt signaling pathway,AMPK signaling pathway,TNF signaling pathway and oocyte meiosis pathway.ENSMUST00000144657 mainly targets the TEAD1 molecule of Hippo pathway,and also participates in the NF-kappa B and MAPK signaling pathways.5.The results of q-PCR and RT-PCR showed that the expression of ENSMUST00000156781 decreased with aging,while the expression of ENSMUST 00000144657 increased with aging.6.RT-PCR results showed that the amplified products of ENSMUST00000210794 were consistented with the sequence alignment in the database.Conclusions:1.There are a lot of abnormal expression of lncRNA in the process of ovarian physiological aging,which may play an important role in the occurrence and development of ovarian aging.2.Three lncRNAs related to Hippo signaling pathway have ovarian tissue specificity and are significantly associated with ovarian aging.