Effects Of MiR-155-5p In Porcine Cumulus Cells On Cumulus Diffusion And Oocyte Maturation

The main purpose of this experiment is to explore the role and regulatory mechanism of miR-155-5p in porcine cumulus cells in cumulus cell proliferation and oocyte maturation during oocyte in vitro maturation,so as to provide data support and theoretical basis for in-depth exploration of porcine oocyte maturation mechanism and search for important genetic molecular markers.Based on the RNA-seq sequencing conducted by the previous research group and the comparison of the transcripts of miRNAand m RNAin cumulus cells around oocytes in germinal vesicular phase(GV)and metaphase of second meiosis(MII),miR-155-5p was selected for research.Targetscan and other online tools were used to predict the target genes.q RT PCR,cell transfection,Western bloting and double luciferase reporter gene detection were used to detect the expression changes of miR-155-5p,CX43,cumulus diffusion and oocyte maturation related genes,c AMPpathway proteins,and to detect the changes of miR-155-5p in cumulus diffusion and oocyte maturation during oocyte in vitro maturation.Theresultsofthis testareas follows;1.The expression of miR-155-5p in MⅡ cumulus cells was significantly higher than that in GV phase,while theexpressionof CX43 wassignificantly lowerthanthatin GVphase(P < 0.05);2.Compared with NC group,the cumulus diffusion area of COCs in miR-155-5p mimics group increased significantly(P < 0.01),while that in miR-155-5p inhibitor group decreased significantly(P < 0.05);In cumulus cells isolated and cultured in vitro,the number of cells and ATP activity in mimics group were significantly increased(P < 0.01),while those in inhibitor group were significantlydecreased(P < 0.05);3.Compared with GV stage,the expression of HAS2,PTGS2,CD44,PTX3 and TNFAIP6 in MⅡ cumulus cells and GDF9,BMP15,Cyclin B1 and CDK1 in MⅡ oocytes increased significantly(P < 0.01),whilethe expressionof CX43 in MⅡ cumulus cellsdecreased significantly(P < 0.05);4.Compared with the control group,the expressions of HAS2,CD44,TNFAIP6 in cumulus cells of the mimics group and GDF9,BMP15,and CDK1 in oocytes of the inhibitor group were significantly increased(P < 0.01),the expressions of PTGS2 in cumulus cells of the mimics group and Cyclin B1 in oocytes of the inhibitor group were significantly increased(P < 0.05),and the expressions of CX43 m RNA and protein in cumulus cells of the mimics group were significantly decreased(P <0.01)and(P <.05),respectively;The expression of HAS2,CD44 and PTX3 in cumulus cells of the inhibitor group decreased significantly(P < 0.05),the expression of PTGS2 and TNFAIP6 in cumulus cells of the inhibitor group and the expression of GDF9,BMP15,cyclin B1 and CDK1 in oocytes of the mimics group decreased significantly(P < 0.01),and the expression of CX43 m RNA and protein in cumuluscellsofthe inhibitorgroupincreased significantly(P < 0.01);5.Compared with the control group,the oocyte maturation rate in mimics group were significantlyhigher,while thoseininhibitorgroup weresignificantlylower(P < 0.05);6.Compared with psicheck2 + NC group,psicheck2-wt + mimics group cou Ld significantly reduce luciferase activity(P < 0.05),and psicheck2-mut + mimics luciferase activity had no significantchange(P > 0.05);7.Compared with the control group,the protein expression of PKR2、Cyclin B1 and p-CDK1(thr14,Tyr15)in the mimics group decreased significantly(P < 0.05),while PKR2,cyclin B1 and p-CDK1 in theinhibitorgroupincreased significantly(P <0.01)and significantly P < 0.05)respectively.The results showed that mimics miR-155-5p promoted the proliferation of porcine cumulus cells,oocyte maturation in vitro;miR-155-5p regulated the expression of CX43 gene and activated the meiosis of porcine oocytes;miR-155-5p CX43-c AMP-MPF pathway.It is speculated that miR-155-5p mayregulatetheinvitro maturation ofporcineoocytesthrough thispathway.