自噬基因Beclin 1 shRNA表达质粒的构建及其对宫颈癌细胞HeLa生长的作用

目的 构建自噬基因Beclin 1小发夹RNA(short hairpin RNA,shRNA)表达质粒,探讨Beclin 1抑制后对宫颈癌HeLa细胞生长的作用.方法 根据人Beclin 1 mRNA编码序列,设计RNA干扰靶点,构建Beclin 1 shRNA表达质粒,脂质体法转染人宫颈癌HeLa细胞,通过荧光定量PCR(RT-PCR)和Western blot检测其对HeLa细胞自噬基因Beclin 1 mRNA及蛋白表达的影响.MTT法分析其对细胞增殖、流式细胞仪检测其对细胞周期和细胞凋亡的影响.结果 构建的shRNA表达载体可以使HeLa细胞中自噬基因Beclin 1的mRNA及其蛋白含量降低,转染质粒的细胞生长增殖速度加快,凋亡率降低.结论 成功构建了针对自噬基因Beclin 1的shRNA表达载体,通过转染HeLa细胞,可有效抑制细胞中Beclin 1的表达,并促进细胞生长,抑制凋亡.

Construction of shRNA Expression Vectors for Autophagy Gene Beclin 1 and Effect of the vectors on Transfected HeLa Cells in Vitro

Objective Autophagy gene Beclin 1 plays an important role in several types of human cancer. In this study, RNA interference (RNAi) technique was employed to determine the effect of inhibiting Beclin 1 on the growth of tumor cells. Methods According to the encoding sequence of mRNA of Beclin 1, the target site for the RNAi technique was designed and the vector for shRNA (short hairpin RNA) expression in tumor cells was constructed. The HeLa cell line was transfected with the sfRNA to inhibit the expression of Beclin 1. Results The constructed vector significantly inhibited the expression of the mRNA and protein of Beclin 1 in the HeLa cells. The growth of the transfected cells was promoted, and less apoptosis cells were identified in these cells. Conclusions The shRNA expression vector can effectively inhibit the expression of Beclin 1 in the HeLa cells, and promote the growth of HeLa cells.