Study On The Mechanism Of MiR-195/497/106b To Regulate The Buffalo Oocytes Maturation By Targeting HAS2

Cumulus cells provide material transportation and nutritional support for oocytes during maturation of oocytes and its physiological state is closely related to the growth and maturation of oocytes.The maturation process of oocytes is usually accompanied by the expansion of cumulus cells,but there are few reports on the regulation of cumulus cell expansion,especially the mechanism of miRNA in regulating cumulus cell expansion is not yet clear.To this end,the cumulus cells derived from buffalo GⅤ and MⅡ oocytes were selected for miRNA high-throughput sequencing.Bioinformatics analysis was used to screen out the differentially expressed miRNAs that had a predicted targeting relationship with the cumulus expansion gene HAS2,and then the dual luciferase test was used to verify the targeting relationship.Subsequently,the role of miRNAs in the process of buffalo oocyte maturation in vitro and its molecular mechanism of regulating oocyte maturation were explored,which would provide a theoretical basis for improving the efficiency of buffalo oocyte maturation in vitro.The main results of this study are as follows:1.Construction and analysis of miRNA expression profiles of cumulus cells derived from buffalo GⅤ/MⅡ oocytesHigh throughput sequencing was used to construct the miRNA expression profile of cumulus cells derived from buffalo GⅤ/MⅡ oocytes.Furthermore,the differential expression analysis of miRNAs in the two periods was performed,and 69 significantly different miRNAs were obtained,including 24 up-regulated miRNAs and 45 down-regulated miRNAs in the cumulus cells derived from MⅡoocytes.A total of 7956 predicted target genes were predicted from these 69 differentially expressed miRNAs.After KEGG enrichment analysis,a total of 276 significantly enriched signal pathways were obtained,including VEGF,glycolysis/gluconeogenesis,carbon metabolism,nitrogen metabolism and MAPK signal pathways.7 miRNAs were randomly selected for RT-qPCR and found to be consistent with the sequencing results.A total of 12 significantly different miRNAs had a predicted targeting relationship with HAS2 and 4 miRNAs such as miR-195,miR-497,miR-106b and miR-27a-3p were screened to verify the targeting relationship with HAS2.2.Verification of miR-195/497/106b-HAS2 targeting relationshipRT-qPCR showed that miR-195,miR-497,miR-106b and miR-27a-3p had opposite expression relationships with HAS2 in cumulus cells derived from GV/MII oocytes.The addition of miR-195,miR-497 and miR-106b mimic to cumulus cells respectively could reduce the expression level of HAS2 mRNA and protein,while the addition of miR-27a-3p mimic reduced the mRNA level of HAS2,but had no effect on its protein expression level.Adding miR-195 inhibitor to cumulus cells increased the protein expression level of HAS2,but had no effect on its mRNA expression level.Adding miR-497 inhibitor to cumulus cells increased the expression level of HAS2 mRNA and protein.Adding miR-106b inhibitor to cumulus cells increased the mRNA level of HAS2,but had no effect on its protein expression level.The addition of miR-27a-3p inhibitor to cumulus cells increased the mRNA expression level of HAS2,but reduced its protein expression level.Through the dual luciferase test,it was found that miR-195,miR-497 and miR-106b had a targeting relationship with HAS2,while miR-27a-3p had no targeting relationship with HAS2.3.Effect of miR-195/497/106b and HAS2 on in vitro maturation of buffalo oocytesThe addition of miR-195/497/106b mimic to the buffalo oocyte maturation medium respectively reduced the expansion area of buffalo cumulus cells,the maturation efficiency of oocytes,and the subsequent cleavage rate and blastocyst rate of parthenogenetic embryos.However,when miR-195/497/106b inhibitor was added separately,the opposite results were obtained.Using adenovirus as a carrier to overexpress HAS2 during the buffalo oocyte maaturation could significantly increase the cumulus cells expansion area,oocyte maturation efficiency,and subsequent cleavage rate and blastocyst rate of parthenogenetic embryos(P<0.05),while interference with HAS2 expression resulted in the opposite results.4.The mechanism of miR-195/497/106b targeting HAS2 to regulate the biological process of buffalo cumulus cellsThe addition of miR-195/497/106b mimic to the buffalo cumulus cells respectively could all reduce the proliferation of cumulus cells,increase the level of apoptosis(P<0.05),and increase the proportion of G1 phase cells.When miR-195/497/106b inhibitor was added separately,the ratio of cumulus cells in G1 phase was reduced but had no significant effect on cell proliferation.Inhibiting the expression of miR-195/miR-497 respectively decreased the level of apoptosis,while inhibition of miR-106b expression had no effect on the level of apoptosis(P>0.05).After the cumulus cells were treated with miR-195/497/106b mimic at 100 nM respectively,Western blot was used to detect the proteins expression levels related with PI3K/AKT signaling pathway which affects cell biological process,found that the proteins expression levels of PI3K P110,PI3K P85,p-AKT/AKT,p-ERK1/2/ERK1/2,PCNA,cyclinD1 and CDK6 were significantly lower than the corresponding miR-195/497/106b mimic NC group(P<0.05),while P53,BAX and caspase3 expression levels were significantly increased(P<0.05).However,after treating cumulus cells with miR-195/497/106b inhibitor at 100 nM respectively,the proteins expression levels of p-AKT/AKT,p-ERK1/2/ERK1/2 and cyclinDl were significantly higher than the corresponding miR-195/497/106b inhibitor NC group(P<0.05),the expression levels of BAX and caspase3 were significantly reduced(P<0.05).Overexpression of HAS2 in cumulus cells could improve the cell proliferation,reduce the cell apoptosis(P<0.05),reduce the proportion of G1 phase cells(P<0.05),increase the proteins expression levels of HAS2,PI3K P110,PI3K P85,p-AKT/AKT,p-ERK1/2/ERK1/2,BCL2,PCNA,cyclinD1,CDK6 and CDK2(P<0.05),reduce the protein expression level of BAX(P<0.05),while after interfering with the expression of HAS2,the results were reversed.Compared with the miR-195 mimic+Ad-control group,the miR-195 mimic+Ad-HAS2 group increased the cumulus cell proliferation,decreased the cell apoptosis(P<0.05)and the proportion of cells in G1 phase(P<0.05),and increased the proteins expression levels of HAS2,PI3K P85,p-AKT/AKT,cyclinD1 and CDK6(P<0.05),the same results were obtained when miR-497 mimic+Ad-HAS2 and miR-106b mimic+Ad-HAS2 were used to treat cumulus cells respectively.Compared with the miR-195 inhibitor+NC siRNA group,the miR-195 inhibitor+HAS2 siRNA group decreased the cumulus cell proliferation(P<0.05),increased the cell apoptosis(P<0.05)and the proportion of cells in G1 phase(P<0.05),decreased the proteins expression levels of HAS2,PI3K P110,p-AKT/AKT,p-ERK1/2/ERK1/2,BCL2,PCNA,cyclinD1,CDK6 and CDK2(P<0.05),the same results were obtained when miR-497 inhibitor+HAS2 siRNA and miR-106b inhibitor+HAS2 siRNA were used to treat cumulus cells respectively.The above results indicate:(1)There are 69 significantly differentially expressed miRNAs in the cumulus cells derived from buffalo GV/MII oocytes,of which 24 are up-regulated and 45 are down-regulated in cumulus cells of MII oocytes.(2)HAS2 is the target gene of miR-195,miR-497 and miR-106b.(3)miR-195/497/106b regulates PI3K/AKT signaling pathway in cumulus cells by targeting HAS2 to affect cumulus cell proliferation,apoptosis and cycle,and then regulates the buffalo oocyte maturation in vitro.