The Effect Of WNT11 On The Proliferation Of Goat Granulosa Cells Based On CeRNA

The proliferation of granulosa cells in mammals is important for promoting ovarian development and improving ovulation rate.Noncoding RNAs are widely involved in follicle development,granulosa cell proliferation,and oocyte maturation in female animals,in which miRNAs and lnc RNAs can jointly participate in regulation in the form of endogenous competing RNAs（ce RNAs）.However,the ce RNA interaction network that regulates the growth and development of goat granulosa cells is rarely studied.In this study,the results of whole transcriptome sequencing of black goat ovary tissue in the early stage of our laboratory showed that an important candidate gene WNT11 was significantly associated with goat prolificacy traits.Integrated whole transcriptome analysis found that chi-miR-1343 and lnc＿000466 might be important regulatory elements regulating the expression of WNT11.Based on the above research results,this study intends to overexpress/interfere candidate target genes and their regulatory elements by culturing goat granulosa cells in vitro to analyze the molecular mechanism of lnc＿000466 mediated chi-miR-1343 targeting WNT11 in regulating goat granulosa cell proliferation.The main findings are as follows:1.The results of WNT11 regulating the proliferation of goat granulosa cells showed that the expression of WNT11 in the ovary of Yunshang black goat in the high-yield group was significantly higher than that in the low-yield group by using RT-q PCR and Western Blot（P<0.05）.The m RNA and protein expressions of cell proliferation factors（CDK4,CCND1,CCND2）were significantly increased（P<0.05）,and the expressions of apoptosis factors（Caspase3,Caspase9,Beclin1）were significantly decreased（P<0.05）.cell counting kit-8（CCK-8）and 5-ethynyl-2-deoxyuridine（Ed U）assays showed that overexpression of WNT11 promoted the proliferation of granulosa cells（P<0.05）.ELISA test showed that the secretion of estradiol（E₂）and Progesterone（P₄）in granulosa cells also increased significantly after overexpression of WNT11（P<0.05）;after interference with WNT11,these results showed the opposite trend.2.The regulation of WNT11 expression by chi-miR-1343 showed that the expression pattern of chi-miR-1343 was opposite to that of WNT11 in high/low-yield Yunshang black goat ovaries（P<0.05）.Overexpression of chi-miR-1343 in goat granulosa cells inhibited the m RNA and protein expression levels of WNT11（P<0.05）.Dual-luciferase reporter group results indicated that WNT11 was the target gene of chi-miR-1343;After transfection of chi-miR-1343 mimics and inhibitors in granulosa cells,it was found that chi-miR-1343 inhibited granulosa cell proliferation and promoted granulosa cell apoptosis by targeting WNT11 gene in granulosa cells.ELISA results showed that overexpression of chi-miR-1343 significantly decreased the content of E₂ and P₄ in granulosa cells（P<0.05）,while inhibition of chi-miR-1343 showed the opposite result.3.In the study on the regulatory pathway of lnc＿000466-chi-miR-1343-WNT11,dual luciferase reporter group indicated that lnc＿000466 could competitively bind to chi-miR-1343.RT-q PCR detection showed that the expression pattern of lnc＿000466 in the ovaries of Yunshang black goat in high and low yield group was opposite to that of chi-miR-1343.Overexpression of lnc＿000466 resulted in a significant decrease in the expression of chi-miR-1343（P<0.05）,whereas interference with lnc＿000466 resulted in the opposite;After overexpression of lnc＿000466 in granulosa cells,Ed U and CCK-8detection showed that lnc＿000466 promoted the proliferation of granulosa cells and inhibited the apoptosis of granulosa cells,while interfering with lnc＿000466 inhibited the proliferation of granulosa cells and promoted apoptosis;After overexpression of lnc＿000466,the expression of WNT11 protein increased,and RIP results showed that there was an RNA-proteins complex between lnc＿000466 and WNT11.In conclusion,WNT11 gene plays an important role in promoting granulosa cell proliferation and inhibiting granulosa cell apoptosis,and promote its secretion of E₂ and P4,while chi-miR-1343 inhibits granulosa cell proliferation and secretes E₂ and P4 by targeting WNT11,which promoted granulosa cell apoptosis.Lnc＿000466 is a ce RNA of chi-miR-1343,which can promote granulosa cell proliferation and inhibit its apoptosis.This study preliminarily revealed the role of the ce RNA regulatory network of chi-miR-1343-lnc＿000466-WNT11 on granulosa cell proliferation,with a view to provide reference for the development of goat granulosa cells and the molecular mechanism of goat multiple-ovulation.