稳定靶向干扰长链非编码RNA BC002811胃癌SGC-7901细胞株的构建

Establishment of gastric cancer SGC-7901 cell line with stable lncRNA BC002811 silencing

目的 BC002811在胃癌中的生物学功能及其作用机制尚不明确。文中旨在构建长链非编码RNA (lncRNA BC002811的短发夹结构RNA (shRNA)重组慢病毒载体,建立稳定干扰BC002811表达的胃癌SGC-7901细胞株。方法实验根据不同BC002811 siRNA转染人胃癌SGC-7901细胞分为:siRNA-1组(siRNA-1正义链及反义链)、siRNA-2组(siRNA-2正义链及反义链)、siRNA-3组(siRNA-3正义链及反义链)、对照组(siRNA-NC正义链及反义链)。取SGC-7901细胞内对BC002811表达抑制效果最明显的siRNA序列构建shRNA慢病毒载体,定义为shRNA组。包装重组慢病毒及测定滴度,建立稳定靶向干扰BC002811表达的SGC-7901胃癌细胞株,MTS法检测细胞增殖能力。结果 siRNA-1组SGC-7901细胞内BC002811的RNA干扰效率较其他组升高。因此,后续实验选择siRNA-1设计BC002811 shRNA。对照组、shRNA组的病毒滴度分别为4.5×108、3.7×10~8TU/mL,提示病毒已包装成功。shRNA组BC002811表达水平[(10%±1%)]较对照组明显降低[(100%±4%)],差异有统计学意义(P<0.01),提示稳定靶向干扰BC002811表达的SGC-7901胃癌细胞株构建成功。与对照组比较,shRNA组的SGC-7901细胞生长速度明显减慢(P<0.05),提示下调BC002811表达可抑制SGC-7901细胞增殖。结论成功建立了稳定靶向干扰BC002811表达的SGC-7901胃癌细胞株,为BC002811在胃癌中的功能及其作用机制研究奠定了基础。

Objective The aim of this study was to construct a recombinant lentivirus-mediated short hairpin RNA(shRNA)expression vector targeting the long non-coding RNA(lncRNA)BC002811 and establish a gastric cancer SGC-7901 cell line with stable BC002811 down-regulation.Methods Three small interfering RNAs(siRNA)were designed and synthesized.The best sequence for RNA interference was selected by real-time quantitative PCR(qPCR)and inserted into the lentiviral vector pLVX-shRNA2.After identification by DNA sequencing,the lentiviral vectors carrying BC002811 shRNA were packaged in HEK293T cells.The lentiviral particles were collected to infect human gastric cancer SGC-7901 cells.After screened by limiting dilution analysis,the SGC-7901 cell line with stable BC002811 down-regulation was established,the expression level of BC002811 detected by qPCR,and the effect of BC002811 on the proliferation of the cells analyzed by MTS.Results The results of qPCR showed that BC002811 siRNA-1 was the most effective siRNA sequence,with a knockdown efficiency of 87%.The recombinant lentiviral vector was packaged in the HEK293T cells with a viral titer of 3.7×10^8 TU/mL in the shRNA-1 group as compared with 4.5×10^8 TU/mL in the control.The expression of BC002811 in the shRNA-1 group was only 10%of that in the control group(P<0.01),which indicated the successful establishment of the gastric cancer SGC-7901 cell line with stable BC002811 down-regulation.BC002811 knockdown significantly inhibited the proliferation of the SGC-7901 cells in the shRNA-1 group as compared with the control.Conclusion A recombinant lentiviral vector expressing BC002811 shRNA was successfully constructed and the gastric cancer cell line SGC-7901 with stable BC002811 silencing was established.