lncRNA BC002811慢病毒载体构建及对胃癌细胞增殖的影响

Construction of lncRNA BC002811 lentiviral vector and its effect on proliferation of gastric cancer cells

目的构建长链非编码RNA (lncRNA) BC002811慢病毒表达载体,并探讨稳定表达BC002811对胃癌HGC-27细胞增殖的影响。方法将PCR法扩增得到的BC002811全基因序列,与pLVX-EGFP-IRES-neo载体连接,经酶切鉴定和测序验证后,与辅助包装质粒共转染HEK293T细胞,包装成重组慢病毒颗粒,再感染HGC-27细胞,经有限稀释法筛选出稳定表达BC002811的细胞克隆并扩大培养。qPCR法分析BC002811的表达水平,MTS法检测细胞增殖能力。结果双酶切和测序结果表明BC002811重组慢病毒载体构建成功,经HEK293T细胞包装后,测定其病毒滴度为2.2×1011 TU/L。将此病毒感染HGC-27细胞,显示可有效提高BC002811的表达水平(P<0.05)。MTS法检测结果显示,与对照组相比,BC002811组的HGC-27细胞增殖能力增强(P<0.05)。结论成功构建了BC002811重组慢病毒载体,该载体能稳定感染HGC-27细胞,使BC002811过表达并提高细胞的增殖能力。

Objective To construct a lentiviral vector of long non-coding RNA(lncRNA) BC002811 and investigate the effect of stable BC002811 up-regulation on proliferation of gastric cancer HGC-27 cells. Methods BC002811 gene sequence was amplified by PCR, and was ligated with the pLVX-EGFP-IRES-neo vector. After being identified by double enzyme digestion and sequencing analysis, the recombinant vector was co-transfected into HEK293 T cell line with packaging plasmids to produce the lentiviral particles. Lentivirus was used to infect HGC-27 cells, and the cells with stably expressing BC002811 after selection with limiting dilution analysis were amplified and cultured. The expression level of BC002811 was detected by qPCR, and the cell proliferation capacity was determined by MTS. Results The BC002811 recombinant lentivirus plasmid was successfully constructed, which was confirmed by double enzyme digestion and DNA sequencing. The recombinant lentiviral vector was packaged in HEK293 T cells and the titer of concentrated virus was 2.2×1011 TU/L. HGC-27 cells infected with the lentivirus, the expression level of BC002811 was enhanced significantly(P<0.05). MTS assay results showed that the proliferation ability of HGC-27 cells in the BC002811 group was increased as compared with the control group(P<0.05). Conclusion The BC002811 recombinant lentiviral vector was successfully constructed, which could stably transfect HGC-27 cells to yield sustained over-expression of BC002811 and promoted cell proliferation.