CAJ | 학술논문

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,退火后分别克隆入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HCT116,荧光定量聚合酶链反应(FQ-PCR)检测各细胞中RhoA、RhoC mRNA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、RhoC mRNA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(-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.
목적 탐토RhoA、RhoC천련RNA간우재체적구건급재결직장암세포중적표체.방법 설계합성4대파향RhoA여RhoC기인적각량대과핵감산서렬,퇴화후분별극륭입shRNA표체재체;통과일계렬적매절여련접,장해4단간우편단천련기래,구건일개동시표체4개shRNA적중조질립.경매절화측서감정중조질립.지질체법전염HCT116,형광정량취합매련반응(FQ-PCR)검측각세포중RhoA、RhoC mRNA적표체,새서람(MTT)비색법검측세포적증식활성.결과　경매절화측서증실중조질립pGenesil2.7-A1+A2+C1+C2구건성공.전염해중조질립후세포중RhoA、RhoC mRNA적표체수평분별위(0.78±0.11)화(0.90±0.21),명현저우공백대조조(0±0.15、0±0.09)화음성대조조(-0.05±0.12、0.11±0.10,P＜0.05),차세포적생장증식솔(63.8±12.5)%야현저저우대조조화질립대조조(101.6±13.7)%(P＜0.05).결론　성공구건4개shRNA천련적중조표체재체,해재체가재체외고효표체.
Objective To construct and identify recombinant RhoA and RhoC shRNAs Tandem expression vector pGenesil2. 7-A1 + A2 + C1 + C2 and observe its expression in human colorectal 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 HCT 116 usingLipofectamneTM 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 (MTT) assay. Results Enzyme digestion and DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pGenesil2. 7-A1 + A2 + C1 + C2 plasmid. After transfection, 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.