Effect Of LncPHLPP1-miR-338-y-CCND1 Axis Regulation PI3K/AKT Pathway On Proliferation And Differentiation Of Yak Follicular Somatic Cells

The yak is an important economic livestock species in the highlands,but its seasonal heat and low fertility severely restrict the development of the pastoral economy.Follicular development in yaks is a very complex physiological process involving the combined action of several tissues and organs and regulatory factors.GCs(Granulosa cells,GCs)and CCs(Cumulus cells,CCs)play a crucial role in follicle and oocyte development,with various changes in the morphology and function of GCs occurring throughout the follicular development cycle.GCs secrete steroid hormones that are important in regulating the ovulatory cycle and maintaining pregnancy status in yaks.Research has now confirmed that nc RNA(non-coding RNA,nc RNA)play an important role in mammalian follicle development,but their role in regulating functions related to yak GCs and CCs has rarely been reported.The whole transcriptomic analysis of GCs and CCs was carried out in this experiment,in which Lnc RNA(Long non-coding RNA,Lnc RNA)and mi RNA(micro RNAs,mi RNAs)had a regulatory role in the proliferation and differentiation of GCs and CCs.The aim of this study was to investigate the effects of Lnc RNA and mi RNA on the proliferation and differentiation of GCs and CCs in yaks and their mechanisms of action,and to provide a theoretical foundation for further improving the follicular development rate in yaks.Therefore,the following experiments were conducted in this study and the corresponding results were obtained:1.In this experiment,in order to clarify the important roles of Lnc RNA,mi RNA and m RNA,whole transcriptomics sequencing analysis was performed and the selected RNA sequences were validated using q RT-PCR.The results of the trial showed that a total of 6223 m RNAs(2197 up-regulated and 4026 down-regulated),643 Lnc RNAs(204 up-regulated and 439 down-regulated)and 559 mi RNAs(311 up-regulated and248 down-regulated)were significantly altered between the two groups.The selection of 12 differential Lnc RNAs,12 differential m RNAs and 8 differential mi RNAs,which are involved in G0 processes such as cell adhesion,cell differentiation,regulation of developmental processes,cell proliferation,embryonic development,signal transduction,apoptosis,and aromatic compounds,was validated.KEGG pathways such as ECM-receptor interaction,MAPK signaling,Hippo signaling,PI3K/AKT signaling,cell cycle,cell adhesion,leukocyte transendothelial migration and actin cytoskeleton regulation was significantly enriched and screened for significant relationships related to follicle development for Lnc PHLPP1-mi R-338-y-CCND1.2.The new gene Lnc PHLPP1 was obtained by whole transcriptomic sequencing,and the expression of genes and proteins related to proliferation and differentiation,cell cycle,and cell cycle inhibitors in the PI3K/AKT pathway were examined using FISH,q RT-PCR,Western-blotting,ELISA,Scratching and Dual luciferase assays,respectively.Hormone secretion levels and cell migration rates were measured,and mi RNA-Lnc RNA targeting relationships were verified.The results showed that Lnc PHLPP1 was localized in the cytoplasm of GCs and CCs.The si RNA-Lnc PHLPP1 vector was then constructed and transfected into GCs and CCs,which significantly regulated proliferation and differentiation-related gene expression and protein activity,and significantly inhibited the proliferation and migration ability of GCs and CCs.The results suggest that Lnc PHLPP1 is involved in the regulation of cell proliferation and differentiation,and promotes the proliferation and migration of GCs and CCs.The secretion of E2 and P4 in yak GCs and CCs is facilitated by Lnc PHLPP1.mi R-338-y and Lnc PHLPP1 are targeted and bind specifically to each other.3.This assay detects the expression of genes and proteins related to proliferation and differentiation,cell cycle and cell cycle inhibitors in the PI3K/AKT pathway by FISH,q RT-PCR,Western-blotting,ELISA,Scratching and Dual luciferase assays,respectively.Hormone secretion levels,cell migration rates and validation of mi RNAm RNA targeting relationships were also examined.The results showed that mi R-338-y was localized to the cytoplasm of GCs and CCs as verified by FISH in yaks.The mi R-338-y mimics and inhibitor vectors were constructed and transfected in GCs and CCs,respectively.While mimics inhibit the migratory capacity of GCs and CCs,inhibitors promote the migratory capacity of GCs and CCs,and both significantly regulate cell proliferation-related gene expression and protein activity in the PI3K/AKT pathway.The mimics and inhibitor of mi R-338-y regulated the secretion of E2 and P4 in yak GCs and CCs,respectively.Lnc PHLPP1 was inversely regulated by mi R-338-y,and there was a targeting relationship and specific binding between mi R-338-y and CCND1.In summary,in vitro culture of yak GCs and CCs,with altered levels of Lnc PHLPP1 and mi R-338-y,respectively,affected cell cycle,proliferation and differentiation-related gene and protein expression,and regulated cell migration capacity as well as E2 and P4 secretion levels.A targeting relationship exists between Lnc PHLPP1-mi R-338-y-CCND1.Downregulation of Lnc PHLPP1 increased mi R-338-y expression and decreased CCND1 expression,while inhibition of mi R-338-y reversed the downregulation of CCND1 by Lnc PHLPP1.The above results provide a theoretical basis to further explore the role of non-coding RNAs in the proliferation and differentiation of animal reproduction-related cells and their mechanisms of action on follicle development and oocyte maturation.