let-7g对小鼠乳腺发育和泌乳相关功能基因Tgfbr1的作用及其机理

运用生物信息学方法对let-7g进行靶基因预测,构建含有与其结合位点互补序列的荧光素酶报告质粒将其与phRL-TK及let-7gmimics或negative control共转染小鼠乳腺上皮细胞,双荧光素酶报告系统检测试剂盒测定荧光素酶的表达;用let-7ginhibitor和let-7g mimics或negative control分别转染小鼠乳腺上皮细胞,并应用细胞活力分析技术、qRT—PCR技术、Western blotting、HPLC分析let-7g的表达变化及其对小鼠乳腺上皮细胞的影响。结果显示：经酶切及测序证实荧光素酶报告质粒构建成功;将荧光素酶报告基因、phRL—TK与let-7g mimics共转染小鼠乳腺上皮细胞,荧光素酶活性与对照组（即共转染荧光素酶报告基因、phRL-TK与Negative Control的小鼠乳腺上皮细胞组）相比显著降低（P〈0．05）;与阴性对照组和空白对照组相比较,let-7g inhibitor转染后,TGFβ3RI蛋白表达量显著增加（P〈0．01）,细胞活性显著增强（P〈0．05）,β-酪蛋白表达量增加（P〉0．05）,而let-7g mimics转染后,TGFβRI蛋白表达量显著减少（P〈0．01）,细胞活性显著降低（P〈0．05）,β-酪蛋白表达量显著减少（P〈0．05）。结果表明,在小鼠乳腺上皮细胞中,let-7g能够靶向结合Tgfbr1,且负调控其表达;let-7g可通过抑制靶蛋白TGFβRI的表达,进而调控小鼠乳腺上皮细胞的增殖及β-酪蛋白的分泌。

The role and mechanism of let-7g on tgfbrl related to development and lactation of mouse mammary gland

Computational approaches were used for let-7g target prediction. A luciferase gene expression vector containing let-7g binding site complementary sequences was constructed. The Firefly luciferase expression plasmids were named pMIR-report-let-7gT. The Firefly luciferase expression plasmid pMIR-report-let-7gT and the Renilla luciferase construct phRL-TK with either let-7g mimics or negative control were cotransfected into mouse mammary epithelial cells. The Firefly and Renilla luciferase activity as detected by using the dual glo luciferase assay system after cotransfection. After transfection of let-7g inhibitor and let-7g mimics or negative control in mouse mammary epithelial cells,expression of let-7g and the effect of let-7g on mouse mammary epithelial cells were determined by CASY-technology,qRT-PCR,Western blotting and HPLC. The recombinant vector was verified by restriction enzyme digestion and sequencing. Luciferase activity was significantly decreased in mouse mammary epithelial cells cotransfected with recombinant vector,phRL-TK and let-7g mimics,compared with cells transfected with recombinant vector,phRL-TK and negative control （P〈0.05）. Western blotting detection demonstrated a significant increase of TGFβR I protein level （P〈0.01） and cytoactive （P〈0.05） in cells transfected with let-7g inhibitor,and expression of β-casein was significantly increased （P〉0.05）; but not in cells transfected with let-7g mimics, TGFβR I protein level （P〈0.01） and cytoactive （P〈0.05） was significantly decreased, and expression of β-casein was decreased （P 〈0.05） ,compared to control group cells. These data indicated that let-7g could negatively regulate the expression of target Tgfbr1 by complementary combination in mouse mammary epithelial cells;let-7g could regulate the proliferation of mouse mammary epithelial cells and expression of β-casein by suppressing the expression of TGFβR I .