shRNA靶向P AX6对乳腺癌MCF-7细胞增殖、凋亡及上皮间质转化的影响

摘要

目的：探讨短发夹RNA（ shRNA）靶向沉默人类配对盒基因（ PAX6）对乳腺癌MCF⁃7细胞增殖、凋亡及上皮间质转化（ EMT）的影响。方法根据GenBank数据库中PAX6序列，设计、合成互补并特异性编码其 shRNA序列的寡核苷酸，克隆至线性化 pSUPER． puro 质粒载体中，将 pSuper． puro PAX6 shRNA 质粒（转染组）和阴性对照质粒 pSuper． puro luciferase shRNA（阴性对照组）分别转染人MCF⁃7细胞，建立稳定低表达PAX6的MCF⁃7细胞株，同时设未行转染的MCF⁃7细胞为空白对照组。经实时定量PCR验证后，噻唑蓝（ MTT）法检测各组转染24、48、72、96 h后的增殖抑制率，流式细胞术检测各组转染后的细胞凋亡和细胞周期情况，采用Western blotting检测各组转染96 h后的EMT相关标记分子（ E⁃钙粘蛋白、纤连蛋白和波形蛋白）水平。结果转染组的PAX6水平均低于阴性对照组和空白对照组（ P＜0�05）；与其余两组相比，转染组的增殖抑制率、凋亡率及促凋亡基因Bax、Bad mRNA水平均升高，而抑制凋亡基因Bcl⁃2 mRNA水平降低，且细胞周期的G0／G1期细胞比例升高，但S期和G2／M期细胞比例均降低，以上差异均有统计学意义（ P＜0�05）；转染组的钙粘蛋白水平升高，而纤连蛋白和波形蛋白水平均降低，与其余两组的差异均有统计学意义（ P＜0�05）。结论通过shRNA抑制PAX6基因表达可抑制乳腺癌细胞增殖并诱导凋亡和细胞周期阻滞，对EMT过程有一定抑制效果，为乳腺癌的基因沉默靶向治疗提供了依据。%Objective To investigate the effect of short hairpin RNA( shRNA) targeting gene paired⁃box 6 ( PAX6) on the proliferation, apoptosis and epithelial mesenchymal transition ( EMT) of breast cancer MCF⁃7 cells. Methods According to the Gen⁃Bank sequence, the PAX6 sequence was designed and the shRNA sequence of PAX6 was synthesized, and then cloned into the vector of Puro pSUPER. plasmid. The MCF⁃7 cells were randomly transfected with pSuper. puro PAX6 shRNA plasmid ( transfection group) or negative control plasmid pSuper. puro luciferase shRNA ( negative control group) as to establish the MCF⁃7 cell line with stable low expression of PAX6. The MCF⁃7 cells without transfection were used as blank control group. After the verification by qPCR, thiazolyl blue( MTT) method was used to detect the proliferation inhibition rate with the plasmid for 24, 48, 72 and 96 h. The flow cytometry was employed to evaluate the apoptosis and cell cycle. Western blotting was used to detect the levels of EMT related markers( E⁃cadher⁃in, Fibronectin and Vimentin) after transfection for 96 h. Results The level of PAX6 in the transfection group was lower than that in the negative control group and blank control group ( P<0�05) . Compared with other two groups, the proliferation inhibition rate, apop⁃tosis rate and mRNA level of Bax and Bad were increased, while the Bcl⁃2 level was decreased in transfection group with statistical sig⁃nificant difference ( P<0�05) . The transfection with pSuper. puro PAX6 shRNA plasmid increased the proportion of G0/G1 phase but decreased the proportion of S phase and G2/M phase (P<0�05). The levels of E⁃cadherin in the transfection group were increased, while the levels of Fibronectin and Vimentin were decreased with significant differences ( P<0�05) . Conclusion The shRNA targeting PAX6 inhibits proliferation, induces apoptosis and cell cycle arrest and has a certain inhibition effect on EMT process, thus providing a basis for the gene silencing target of breast cancer.