慢病毒介导小干扰RNA沉默人结肠癌HT-29细胞芳香烃受体核转位蛋白基因的研究

Lentivirus-mediated small interfering RNA silencing aryl hydrocarbon receptor nuclear translocatorgene in human colon cancer cells HT29

目的获取有效沉默人结肠癌HT29细胞的芳香烃受体核转位蛋白基因（ARNT）的短发卡RNA（shRNA）重组慢病毒。方法构建4组表达ARNT—shRNA的慢病毒载体和1组无效干扰慢病毒载体并测序鉴定。用合成的5组重组慢病毒转染HT29细胞，通过克隆稀释扩大培养获取5组稳定转染细胞株，实时荧光定量聚合酶链反应（FQ—PCR）和Westernblot检测沉默效率。结果成功构建4组表达ARNT．shRNA的重组慢病毒，其中1组（LV—ARNT—RNAi一3）转染HT29细胞后能有效沉默ARNT基因，mRNA相对表达下降70％（P〈0．05），蛋白水平表达下降60％（P〈0．05）。结论重组慢病毒LV—ARNT—RNAi一3可以下调人结肠癌HT29细胞ARNT基因的表达。

Objective To obtain the recombinant lentivirus vector with expressing aryl hydrocar- bon receptor nuclear translocator （ARNT）-short hairpin RNA （shRNA） that could suppress the expression of ARNT gene in human colorectal cancer HT29 cells effectively. Methods Construct four lentiviral vector with expressing ARNT-shRNA and a negative control lentiviral vector and have the sequencing appraisal. After the five recombinant lentivirus vector were transfected into the HT29 cells, gain five stable transfection cell lines through enlarging cultivation of cloning dilution, the ARNT mRNA and protein expression were ex- amined by real-time quantitative polymerase chain reaction and Western blotting, respectively. Results Four groups of recombinant lentivirus vector that could express ARNT-shRNA were successfully construc- ted. One of the groups （ LV-ARNT-RNAi-3 ） could significantly suppress the expression of ARNT gene in HT29 cells, the mRNA expression fell by 70% （ P 〈 0. 05 ） and the protein expression fell by 60% （ P 〈 0. 05 ） ,respectively, compared with the control group. Conclusion LV-ARNT-RNAi-3 can efficiently in- hibit ARNT expression in HT29 cells.