| Micro RNAs(mi RNAs)are small non-coding RNAs and act as post-transcription regulation RNA molecules,numerous mi RNAs play a key role in development and specific biological processes of mammary gland development and lactation.It had been discovered that the inhibition of mi RNA-221 could probably promote the proliferation of mouse mammary epithelial cellsand up regulated growth hormone receptor(GHR).However,the function and mechanism of mi R-221 remained unclear in bovine mammary gland development.Therefore,this experiment was to investigate the function and regulatory mechanism of mi R-221 in bovine mammary gland epithelial cells by MTT,flow cytometry,dual luciferase report analysis and q RT-PCR afer overexpression and inhibition of mi R-221.The results were as follows:1.The mi R-221 mimic,inhibitor and their controls were transfected into bovine mammary epithelial cells.After 24 h,the cell vitality was analyzed by MTT method.The result showed that mi R-221 mimic could extremely significantly inhibit the cell activity and mi R-221 inhibitor could significantly promote the cell vitality(P < 0.001);Further the flow cytometry was used to analyze the cell cycle.Compared with the control group,the number of cells in G2 phase significantly decreased after the overexpression of mi R-221,and the number of cells in G2 + S phases decreased 13.12 %.The inhibition of mi R-221 caused the number of cells in G2 phase increased obviously,and the number of cells in G2 phase + S phase increased 10.57 %.The results showed that mi R-221 could inhibit the cell vitality and cell proliferation of bovine mammary epithelial cells.2.In order to study the molecular mechanism of mi R-221 that inhibited cell proliferation,the target genes of mi R-221 were screened and identified.the potential target genes of mi R-221 were revealed in JAK2-STAT5 and PI3K-AKT/ m TOR signal pathways.The gene of m TOR,IRS1,PIK3R1,IGF1,STAT5 a,PRLR and AKT3 were identified as the potential target genes of mi R-221 through bioinformatic softwares.The recombinant vectors containing 3'UTR of target genes were constructed by dual fluorescence reporter vector psi CHECK?-2.The Recombinant plasmid was cotransfected into HEK293 T cells with 50 nmol/ ?L mi R-221 mimic and 100 nmol/ ?L inhibitor and their controls.The results showed that the ratio of synthetic renilla luciferase gene / synthetic firefly luciferase gene(R/ F)of m TOR,IRS1,IGF1,STAT5 a and PRLR genes significantly reduced after the overexpression of mi R-221,while the ratio of R/ F of IRS1,STAT5 a and AKT3 genes significantly increased by inhibiting mi R-221 expression,which indicated that mi R-221 could interact with the 3'UTR regions of IRS1 and STAT5 a genes.The bovine mammary epithelial cells were further transfected with mi R-221 mimic,mimic-NC,inhibitor and inhibitor-NC,and then the target genes of IRS1,STAT5 a were validated by q RT-PCR.The results showed that the overexpression of mi R-221 could significantly inhibit the m RNA expression of STAT5 a and IRS1 genes(P < 0.05,P < 0.001),and inhibiting mi R-221 could significantly promote the m RNA expression of STAT5 a and IRS1 genes(P < 0.01,P < 0.001).Then,the result of Western Blot was coodinant with q RT-PCR result,which indicated that mi R-221 could regulate the expression of IRS1 and STAT5 a at m RNA and protein levels,STAT5 a and IRS1 were the target genes of mi R-221.3.Furthermore,the effect of mi R-221 on the expression of downstream genes in JAK2-STAT5 and PI3K-AKT/ m TOR signal pathways was investigated by q RT-PCR method,the results showed that the overexpression of mi R-221 could significantly inhibit m RNA expression of PIK3R1,SOCS3,AKT3 and m TOR(P < 0.05);while the inhibition of mi R-221 could significantly increase the expression levels of SOCS3 and m TOR genes(P < 0.05).In summary,mi R-221 had a certain regulation effect on the proliferarion of bovine mammary gland epithelial cells,it could inhibit the cell proliferation by tatgeting IRS1 and STAT5 a genes.And mi R-221 also could affecte the m RNA expression of downstream genes of SOCS3 and m TOR in JAK2-STAT5 and PI3K-AKT/ m TOR signaling pathways,and this study would provide a theoretical basis to reveal the function and regulation mechanism of mi R-221 in the development of bovine mammary gland... |
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