RhoA、RhoC串联RNA干扰载体的构建及在结直肠癌细胞中的表达

Construction and identification of recombinant RhoA and RhoC shRNAs Tandem expression vector and its expression in human colorectal carcinoma cells

目的探讨RhoA、RhoC串联RNA干扰载体的构建及在结直肠癌细胞中的表达。方法设计合成4对靶向RhoA与RhoC基因的各两对寡核苷酸序列，退火后分别克隆人shRNA表达载体；通过一系列的酶切与连接，将该4段干扰片段串联起来，构建一个同时表达4个shRNA的重组质粒。经酶切和测序鉴定重组质粒。脂质体法转染HCTll6，荧光定量聚合酶链反应（FQ—PCR）检测各细胞中RhoA、RhoCmRNA的表达，噻唑蓝（MTT）比色法检测细胞的增殖活性。结果经酶切和测序证实重组质粒pGenesil2．7-A1＋A2＋C1＋C2构建成功。转染该重组质粒后细胞中RhoA、RhoCmRNA的表达水平分别为（0．78±0．11）和（0．90±0．21），明显低于空白对照组（0±0．15、0±0．09）和阴性对照组（一0．05±0．12、0．11±0．10，P〈0．05），且细胞的生长增殖率（63．8±12．5）％也显著低于对照组和质粒对照组（101．6±13．7）％（P〈0．05）。结论成功构建4个shRNA串联的重组表达载体，该载体可在体外高效表达。

Objective To construct and identify recombinant RhoA and RhoC shRNAs Tandem expression vector pGenesl/2. 7-A1 ＋ A2 ＋ C1 ＋ C2 and observe its expression in human colorecta/carcinoma cells HCT116. Methods Four pairs of hairpin-like oligonucleotide sequences special for human RhoA or RhoC gene were designed and synthesized. The annealed oligonucleotide fragments were subcloned into four plasmids each with a different promotor. Then the four oligonucleotide fragments were series connected to construct an efficient multiple shRNAs expression system. The tandem array multiple shRNAs expression vector was confirmed by enzyme digestion and DNA sequencing and then was transfected into HCT116 using LipofectamneTM 2000. The expression of RhoA or RhoC mRNA was detected by fluorescence quantitative polymerase chain reaction （FQ-PCR）. Cellular proliferation inhibitory activity was determined by methyl thiazolyl tetrazolium （MTF） assay, Results Enzyme digestion and DNA sequencing showed that the oli- gonucleotide fragments were correctly inserted into pGenesil2. 7-A1 ＋ A2 ＋ C1 ＋ C2 plasmid. After transfec- tion, the expression of RhoA or RhoC mRNA was （0. 78 ＋- 0. 11 ） or （0. 90 ＋ 0. 21 ） , significantly lower than that in control group （0 ±0. 15,0 ＋0. 09） and plasmid control group （ -0.05 -＋0. 12,0. 11 ±0. 10） （P 〈0. 05 ） , and the proliferation rate （63.8± 12. 5 ）% was also lower as compared with control group and plasmid control group （ 101.6 ± 13.7 ） % （ P 〈 0. 05 ）. Conclusion The recombinant tandem array multiple shRNAs expressing vectors can effectively silence the expression of RhoA or RhoC in human colorectal carcinoma cells.