The Regulatory Effect Of MiR-21-5p On The Apoptosis And Lactation Of Dairy Goat Mammary Epithelial Cells

miR-21 is one of the earliest detected miRNAs in mammals.miR-21-5p,as a member of the miR-21 family,was found to be highly expressed in epithelial ovarian cancer,breast cancer,thymic epithelial tumors,and gastric cancer.It plays a role in inhibiting apoptosis.At present,the research on miR-21-5p is mostly limited to tumors,and there are few reports on breast development and lactation.In our previous study,through high-throughput sequencing and bioinformatics research,it was found that mir-21-5p was differentially expressed in the breast tissue of dairy goats during the pre-lactation period and peak lactation period.Studies have shown that miRNA regulates the growth,development and lactation function of breast epithelial cells by regulating the expression of target genes.The bioinformatics website predicted that PDCD4 may be its target gene,and speculated that miR-21-5p may regulate milk goat mammary gland development and lactation through PDCD4.For this reason,In this research,dairy goat mammary epithelial cells(GMECs)were used as the research object,using RT-PCR,Western blot,CCK-8,EDU,flow cytometry,ELISA and other technologies,by the method of constructing an overexpression vector and synthesizing interfering RNA study the mechanism of miR-21-5p targeting PDCD4 to regulate the proliferation,apoptosis and lactation of mammary epithelial cells in dairy goats.This study may provide a scientific basis for studying the development and lactation function of dairy goats.The main findings are as follows:1.The regulation of miR-21-5p on the proliferation,apoptosis and lactation function in mammary epithelial cells of dairy goat.The results of EdU and CCK-8 showed that miR-21-5p promoted the proliferation of GMECs(P<0.05);flow cytometry detection of cell apoptosis showed that miR-21-5p inhibited the apoptosis of GMECs(P<0.01).ELISA results showed that miR-21-5p promoted the secretion of triglycerides and β-casein from GMECs(P<0.01).Western blot was used to detect the expression of related proteins in the PI3K/m TOR and ERK signaling pathways.The results showed that miR-21-5p can significantly up-regulate the phosphorylation levels of PI3 K,m TOR,and S6K(P<0.01),and activate the PI3K/m TOR signaling pathway At the same time,it significantly increased the phosphorylation level of ERK and the expression of Bcl-2(P<0.01),down-regulated the expression of Bax(P<0.01),and activated the ERK pathway.The above results indicate that miR-21-5p can inhibit the apoptosis of GMECs throughthe ERK pathway,and promote the secretion of triglycerides and β-casein by GMECs through the PI3K/m TOR signaling pathway.2.The regulation of miR-21-5p to PDCD4 gene in mammary epithelial cells of dairy goat.The bioinformatics prediction software found that the 3’UTR region of PDCD4 contains the targeted binding site of miR-21-5p.The results of dual luciferase activity test showed that miR-21-5p can reduce the luciferase activity of the wild-type vector(P<0.05),but has no significant effect on the mutant vector.RT-q PCR and Western blot results showed that miR-21-5p can significantly reduce PDCD4 m RNA and protein expression(P<0.01).The above studies indicate that PDCD4 is the target gene of miR-21-5p,and miR-21-5p negatively regulates the expression of PDCD4.3.The regulation of overexpression and interference with PDCD4 on the prolifera tion,apo-ptosis and lactation function in mammary epithelial cells of dairy goat.The results of EdU and CCK-8 showed that overexpression of PDCD4 can inhibit cell proliferation(P<0.01),and interference with PDCD4 can promote the proliferation of GMECs(P<0.01);the results of cell apoptosis detection by flow cytometry showed that overexpression of PDCD4 can promote Cell apoptosis(P<0.01),interference with PDCD4 can inhibit the apoptosis of GMECs(P<0.01).ELISA results showed that interference with PDCD4 could promote the secretion of triglycerides and β-casein by GMECs(P<0.01).Western blot was used to detect the expression of related proteins in the PI3K/m TOR and ERK signaling pathways.The results showed that interference with PDCD4 can significantly up-regulate the phosphorylation levels of PI3 K,m TOR,and S6K(P<0.01),activate the PI3K/m TOR signaling pathway,and promote GMECs secrete β-casein and triglycerides;at the same time,it significantly up-regulates the phosphorylation level of ERK and the expression of Bcl-2(P<0.01),and down-regulates the expression of Bax(P<0.01),which activates the ERK pathway,thereby promoting GMECs proliferate and inhibit their apoptosis.The above results indicate that PDCD4 can regulate the proliferation and apoptosis of dairy goat mammary epithelial cells and the secretion of triglycerides and β-casein through the ERK and PI3K/m TOR signaling pathways.In summary,miR-21-5p can activate PI3K/m TORand ERK signaling pathways by negatively regulating PDCD4,thereby regulating proliferation,apoptosis and the secretion of triglycerides and β-casein of GMECs.