| MicroRNAs(miRNAs)are a kind of important transcription regulation of non-coding small RNA molecules.Many studies have shown that miRNA has important functions in mammary gland development and lactation of mammalian.Our previous study found that the expression of miR-148 was significantly different in buffalo mammary gland tissues between lactating and non-lactating.And we have verified that the target gene of miR-148a was DNMT1.At present,research of miR-148a mainly focus on cancer cells.However,there has been no report on the proliferation and lactation of buffalo mammary gland cells.This study aimed to observe the effect of regulation of miR-148a gene expression and 5-Aza-dc on mammary epithelial cell proliferation and expression of genes associated with lactation,which laid a foundation for the further understanding the interaction between them on buffalo mammary cell growth regularity and lactation mechanism.The specific research results are as following:1.Primary culture and identification of buffalo mammary epithelial cells.Buffalo mammary epithelial cells were cultured from buffalo mammary gland tissue of lactation and the cells were isolated and purified by trypsin digestion.Then using immunofluorescence,cell karyotype analysis,oil red O staining for its identification,and drawing on the growth curve.The results showed that:mammary epithelial cells were obtained successfully by the method of tissue culture.Purified mammary cells had morphological characteristics of the typical epithelial cells,had a ability of secreting milk fat and was subcultured to 20 generations in vitro.2.The effect of regulation of miR-148a gene expression on mammary epithelial cell proliferation and expression of genes associated with lactation.To screen for the proper concentration of miR-148a on buffalo mammary epithelial cells,miR-148a mimics/inhibitor was respectively transfected into the cells by non-liposomal method.After 48 hours,the cell samples were collected and the expression of target gene DNMT1 was detected by qRT-PCR.The results showed that the expression level of DNMT1 of overexpression group gradually decreased with the increase of miR-148a mimics concentration.Compared with the negative control group,50 nM miR-148a mimics significantly decreased DNMT1 expression(P<0.05);In the miR-148a inhibition group,the expression of DNMT1 gradually increased with the increasIng of the inhibitor concentration.Compared with the negative control group,30 nM miR-148a inhibitor could significantly increase the expression of DNMTl(P<0.05).Then 50 nM miR-148a mimics and 30 nM miR-148a inbihitor were used to treat breast cells for 48 h respectively.The effect of cyclinDl on the cell proliferation and cell cycle and the expression of lactate-related genes such as mTOR,PparG,and Srebp1 were examined.The results showed that compared with the negative control group,overexpression of miR-148a significantly increased the activity of buffalo mammary epithelial cells(P<0.05),increased the proportion of S phase cells and significantly increased the expression of CyclinD1 gene and PparG,Srebp1,and Fabp3 during the lactation(P<0.05);inhibition of miR-148a significantly reduced the activity of buffalo mammary epithelial cells(P<0.05),increased the proportion of G1 phase cells,reduced the proportion of S,G2 phase cells,and significantly reduced the expression of CyclinDl gene and Srebpl,Glutl,and Fabp3 genes during the lactation(P<0.05).3.The effect of 5-Aza-dc treatment on buffalo mammary epithelial cell proliferation and the expression of lactation-related genes.In order to screen the suitable concentration of 5-Aza-dc on buffalo mammary epithelial cells,0.1 μm/L,0.5 μM/L,1 μM/L,5 μM/L 5-Aza-dc was added to medium of buffalo mammary epithelial cell for 72h.Then collection of cell samples,the expression of DNMT1 gene was detected by qRT-PCR.The results showed that compared with the blank group,the expression of DNMT1 gene was significantly decreased in the 0.5 μM/L,1 μM/L,5 μW/L 5-Aza-dc groups(P<0.05),so the low concentration of 0.5 μM/L 5-Aza-dc was selected for follow-up experiments.;After 0.1 μM/L 5-Aza-dc treatment on the cells for 72h,the proliferation of mammary epithelial cells was detected by CCK8 kit.The effects on cell cycle of buffalo mammary epithelial cells was detected by Flow cytometry.The expression of cyclinDl and lactation-related genes such as mTOR,PparG and Srebp1 was detected by qRT-PCR.The results showed that compared with the blank group,addition of 0.5 μM/L 5-Aza-dc significantly increased the viability of buffalo mammary epithelial cells,increased the proportion of S-phase cells,and significantly increased the expression of CyclinDl,miR-148a genes,and lactation-related genes Srebp1,Csnlsl,Fabp3 and Elf5(P<0.05).The above results indicated that miR-148a and DNA methylation were involved in the proliferation of buffalo mammary epithelial cells and regulated the expression of lactation-related genes,both of which were closely related to mammary development and lactation. |
