The Mechanism Research Of MiR-21-3p Regulates Cell Proliferation In Bovine Mammary Epithelial Cells

Dairy cow mammary gland undergoes several periodic changes associated with pregnancy cycle,following the proliferation,differentiation and apoptosis of mammary epithelial cells.MicroRNA(miRNA)-mediated gene regulation is important for mammary gland development and lactating process.Our previous lncRNA-seq analysis of gland mammary tissue found that miR-21-3p was potentially associated with multiple distinctly expressed lncRNAs in different stages of cattle lactation.Besides,it was reported that the expression of miR-21 is different in the dry and early lactation period of dairy cow mammary gland,but the molecular mechanisms underlying lactation cycle are not fully understood.Therefore,in this study,bovine mammary epithelial cell line(BMECs)was taken as the research object.First,the effect of miR-21-3p on cell proliferation of BMECs was detected by MTT assay and flow cytometry analysis.After the function of miR-21-3p on BMECs was verified,the target gene of miR-21-3p was predicted by bioinformatics software and further detected and verified by a series of experiments.Furthermore,after the overexpression of miR-21-3p in BMECs,RT-qPCR was used to detect the correlation between lncRNA NONBTAT017009.2 and miR-21-3p,and the regulatory relationship between NONBTAT017009.2 and miR-21-3p with their target gene was further studied by RT-qPCR and dual luciferase assay.In addition,because of the binding sites of STAT3 in the promoter region of miR-21 gene,as well as the possible correlation between STAT3 and IGFBP5-the identified target gene of miR-21-3p,the effect of transcription factors STAT3 on the promoter activity of miR-21 gene was explored by dual luciferase assay,which illuminated the mechanism of miR-21-3p on regulating the proliferation of bovine mammary epithelial cells.The following results were obtained:1.After the transfection of miR-21-3p mimic(or mimic NC)and miR-21-3p inhibitor(inhibitor NC)in bovine mammary epithelial cell line(BMECs),MTT assay showed that the overexpression of miR-21-3p significantly promoted cell viability(P <0.05)and the inhibition of miR-21-3p significantly reduced cell viability(P <0.05)compared with their control groups.Flow cytometry analysis showed that the number of cells in G2 phase was obviously increased in the group of transfection with miR-21-3p mimic,and the percentage in G2+S phase was increased 10.8% compared with the control group.In contrast,thenumber of cells in G2 phase was obviously decreased,and the percentage in G2+S phase was reduced 10.2% compared with the control group,which indicated that miR-21-3p could promote cell proliferation.2.The results of dual luciferase assay showed that comparing with their control groups,the dual luciferase activity ratio(renilla luciferase activity/firefly luciferase activity,R/F)of IGFBP5 3'UTR was significantly decreased(P <0.05)with the overexpression of miR-21-3p,while the dual luciferase activity ratio(R/F)was significantly increased after the inhibition of miR-21-3p(P <0.05).Forthermore,RT-qPCR and western blot results showed that the overexpression of miR-21-3p in BMECs significantly inhibited the expression level of IGFBP5 in mRNA and protein(P <0.05),while the expression of IGFBP5 were significantly up-regulated after the inhibition of miR-21-3p(P <0.05),suggesting that IGFBP5 is a target gene of miR-21-3p.3.RT-qPCR result showed that the expression of NONBTAT017009.2 was significantly inhibited with the overexpression of miR-21-3p.The cells activity was reduced transfecting with NONBTAT017009.2 overexpression vector in BMECs,the expression of miR-21-3p targeted gene IGFBP5 was up-regulated after the overexpression of NONBTAT017009.2 while inhibited the expression of miR-21-3p by RT-qPCR.Further dual luciferase assay result showed that the dual luciferase activity ratio(F/R)of NONBTAT017009.2 was significantly inhibited by the overexpression of miR-21-3p(P<0.05).These results suggested that there was an interaction between NONBTAT017009.2and miR-21-3p,NONBTAT017009.2 could act as a competitive endogenous RNA(ceRNA)involving in the regulation process of miR-21-3p.4.MiR-21 promoter luciferase reporter vector and STAT3 overexpression vector were constructed,and the dual luciferase assay result showed that the overexpression of STAT3 inhibited the luciferase activity of miR-21 promoter(F/R)(P <0.05).This study revealed the influence of miR-21-3p on bovine mammary epithelial cells proliferation.Through constructing a regulatory relationship of NONBTAT017009.2-miR-21-3p-IGFBP5 with the study of related transcription factors to further elucidate the mechanism of miR-21-3p on regulating the proliferation of mammary epithelial cells in dairy cow.It would provide a theory basis for revealing the molecular mechanism of periodic changes in mammary gland development and lactation of dairy cows.