调控SUV39H1/2对水牛乳腺细胞增殖及STAT5a表达的影响

Effects of Regulating SUV39H1/2 Expression on Proliferation of Mammary Epithelial Cells and the Expression of STAT5a Gene in Buffalo

为了解SUV39H1/2基因对水牛STAT5a基因表达的调控作用,首先克隆不同长度水牛STAT5a启动子区,采用双荧光素酶方法检测启动子活性.利用SUV39H1/2-shRNA慢病毒感染水牛乳腺上皮细胞,检测细胞增殖及相关基因表达变化,并分析DNA甲基化水平.结果显示:克隆获得的2190 bp,355 bp,417 bp STAT5a启动子片段均具有活性,2190 bp片段活性显著高于其他两个片段(p<0.05),2190 bp启动子区存在sp1,p300,p53,NF-kB等转录结合位点.下调SUV39H1/2基因表达后,乳腺上皮细胞周期阻滞在S期,G0/G1期细胞比例无显著差异,G2期比例显著减少(p<0.05).qRT-PCR结果显示,抑制SUV39H1/2基因表达使乳腺上皮细胞分化及乳蛋白合成相关基因STAT5a,mTOR,且乳脂合成相关基因srebp1,fabp3表达水平均显著降低(p<0.05);STAT5a启动子区甲基化程度为31.5%,高于对照组15.5%.结果表明:抑制SUV39H1/2基因表达能够促进水牛乳腺上皮细胞增殖;SUV39H1/2基因可能通过调控STAT5a启动子甲基化水平,进而调控乳腺上皮细胞增殖分化、乳汁和乳蛋白合成相关基因表达.

To understand the regulatory effect of SUV39H1/2 gene on the expression of STAT5a gene in buffalo,the different lengths of buffalo STAT5a promoter region were cloned.The double luciferase method was used to detect the activity of promoter.Buffalo mammary epithelial cells infected with SUV39H1/2-shRNA lentivirus were used to detect cell proliferation and related gene expression,and analyze DNA methylation levels.The results showed that the cloned 2,190 bp,355 bp and 417 bp of STAT5a promoter fragments were all active,and the activity of the 2,190 bp fragment was significantly higher than that of the other two fragments(p<0.05).There were some transcriptional binding sites such as sp1,p300,p53,NF-kB in the 2,190 bp promoter region.After downregulating the expression of SUV39H1/2 gene,the breast epithelial cell cycle was blocked in the S phase,there was no significant difference in the proportion of cells in the G0/G1 phase,and the proportion in the G2 phase decreased significantly(p<0.05).qRT-PCR results showed that the expression of SUV39H1/2 gene was inhibited,while the expression levels of breast epithelial cell differentiation,milk protein synthesis-related genes STAT5a and mTOR,milk lipid synthesis-related genes srebp1 and fabp3 were significantly reduced(p<0.05).The degree of methylation of STAT5a promoter region was 31.5%,which was higher than that of the control group by 15.5%.The above results showed that inhibiting the expression of SUV39H1/2 gene could promote the proliferation of buffalo mammary epithelial cells.The SUV39H1/2 gene may regulate the proliferation and differentiation of breast epithelial cells and the expression of genes related to milk and milk protein synthesis by regulating the level of methylation of STAT5a promoter.