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目的通过构建靶向四个半LIM结构域蛋白1(FHL1)基因的慢病毒介导的短发夹RNA(shRNA),观察FHL1表达降低对He La细胞和Hep G2细胞生长的影响。方法构建FHL1基因的shRNA干扰病毒载体,将重组质粒转染人胚肾293T细胞,实时定量PCR(qRT-PCR)和Western blot法检测转染p Lenti-H1 FHL1 shRNA对FHL1表达的抑制效果。慢病毒感染宫颈癌He La细胞和Hep G2肝癌细胞,经2~3周嘌呤霉素筛选后得到FHL1稳定低表达的细胞,利用生长曲线实验和克隆形成实验检测FHL1基因表达降低后对细胞生长的影响,利用软琼脂实验检测对肿瘤细胞非锚定依赖性生长的影响。结果 qRT-PCR和Western blot结果显示,构建的shRNA病毒载体能够明显抑制FHL1的表达;生长曲线实验和克隆形成实验显示,慢病毒介导的FHL1 shRNA能够明显促进肿瘤细胞的生长;软琼脂实验结果表明,FHL1低表达能够促进肿瘤细胞的非锚定依赖性生长。结论构建的p Lenti-H1 FHL1 shRNA慢病毒载体可有效地抑制FHL1表达,明显促进He La细胞和Hep G2细胞的增殖。
OBJECTIVE: To investigate the effect of FHL1 expression on the growth of HeLa cells and Hep G2 cells by constructing a lentivirus-mediated short hairpin RNA (shRNA) targeting four and a half LIM-domain protein 1 (FHL1) genes. Methods The recombinant plasmid was transfected into human embryonic kidney 293T cells by shRNA interference virus vector of FHL1 gene. The inhibition of FHL1 expression by p Lenti-H1 FHL1 shRNA was detected by real-time quantitative PCR (qRT-PCR) and Western blot. Lentiviral infection of cervical cancer He La cells and Hep G2 liver cancer cells, after 2 to 3 weeks of puromycin screening obtained FHL1 stably low expression of cells, the use of growth curve experiments and colony formation assay FHL1 gene expression decreased after the cell growth Effect on the non-anchorage-dependent growth of tumor cells using soft agar assay. Results The results of qRT-PCR and Western blot showed that the constructed shRNA vector could significantly inhibit the expression of FHL1. The growth curve and clone formation assay showed that lentivirus-mediated FHL1 shRNA can significantly promote the growth of tumor cells. The result of soft agar It is indicated that low expression of FHL1 can promote the non-anchorage-dependent growth of tumor cells. Conclusion The p Lenti-H1 FHL1 shRNA lentiviral vector can effectively inhibit the expression of FHL1 and promote the proliferation of He La cells and Hep G2 cells.