PGC-1β基因干扰载体的构建及其对乳腺肿瘤细胞增殖的影响

Construction of Interference Vector of PGC-1β and Its Effect on Proliferation of Breast Cancer Cells

目的构建过氧化物酶体增殖物激活受体辅助活化因子1β(peroxisome proliferative actives receptorγcoactivator-1β,PGC-1β)基因干扰载体,并将其转入人乳腺肿瘤细胞MCF-7中,验证其干扰效应及对肿瘤细胞增殖的影响。方法应用PCR技术扩增得到人PGC-1β的全长序列,克隆入p GEM-T easy载体中,筛选阳性克隆,经酶切鉴定、测序鉴定证实克隆成功。构建人PGC-1β基因干扰载体,命名为p Genesil/sh PGC-1β。将干扰载体利用Lip3000转至乳腺肿瘤细胞系MCF-7中,通过Real-time PCR、Western blot技术检测外源基因PGC-1β的表达。同时利用MTT、平板克隆形成实验检测转染干扰载体对MCF-7细胞增殖能力的影响。结果经双酶切鉴定证实构建的PGC-1β干扰载体的酶切片段与预期大小一致;Real-time PCR与Western blot结果显示与MCF-7细胞组及转染空载体组相比,转染干扰载体组MCF-7细胞中PGC-1βm RNA与PGC-1β蛋白的表达水平均下降(P<0.05)。平板克隆形成和MTT实验结果表明,转染干扰载体组与空载体组及MCF-7细胞组相比,细胞的增殖能力减弱(P<0.01)。结论成功构建PGC-1β基因干扰载体(p Genesil/sh PGC-1β),转染MCF-7细胞可抑制其增殖能力,为后续深入研究PGC-1β对肿瘤细胞的作用机制奠定了基础。

Objective To construct gene interference vector of peroxisome proliferative actives receptorγcoactivator-1β（PGC-1β）and to verify its interference effect and the proliferation of tumor cells after transfecting into breast cancer cell MCF-7. Methods The full length gene sequence of human PGC-1β was amplified by polymerise chain reaction（PCR）technique. The fragment was cloned into easy p GEM-T vector and screening positive clones were confirmed by restriction enzyme digestion.Interference vector of human PGC-1βgene was constructed and was named as p Genesil/sh PGC-1β. The interference vector was transferred into the breast tumor cell line MCF-7 by Lip3000 and the expression of exogenous gene PGC-1β was detected by Real-time PCR and Western blotting technology. Meanwhile,the effect of transfection interference vector on the proliferation of MCF-7 cells was detected by MTT and plate cloning assays. Results Restriction enzyme digestion results confirmed that PGC-1β was successfully constructed and the enzyme fragment was in agreement with the expected size. Real-time PCR and Western blot results showed that compared with the MCF-7 cell group and the empty vector group,the expression of PGC-1β m RNA and PGC-1β protein were significantly decreased when transfected with interference vector in MCF-7 cells（P0.05）. The results of plate cloning and MTT assays showed that the proliferation ability of the transfected interference vector group was significantly lower than that of the empty vector group and the MCF-7 cell group（P0.01）. Conclusion The PGC-1β vector was successful constructed andits inhibitory effect on the proliferation of MCF-7 cells it was verified,which would provide the foundation for the biologic function study of PGC-1β on tumor cell.