慢病毒介导的稳定沉默nm23-H1基因的肺癌细胞株的建立及生物学行为改变

摘要

Background and objective The nm23-H1 gene is an important tumor metastatic suppressor gene. Our previous study showed that the downregulation of nm23-H1 gene expression using small interfering RNA (siRNA) in NL9980 lung cancer cells greatly enhanced their invasiveness. To further explore the molecular mechanisms after nm23-H1 gene knockdown, we established transgene NL9980 and A549 lung cancer cell lines with stable nm23-H1 gene silencing through the lentivirus-mediated short hairpin RNA (shRNA) method. Methods The human large cell lung cancer NL9980 and human lung adenocarcinoma AS49 cells were transfected with shRNA lentiviral particles specific for the nm23-H1 gene, and were then selected through puromycin. Puromycin-resistant clones were generated and screened using reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time polymerase chain reaction (qPCR), and Western blot analysis. shRNA rescue experiments were performed to restore the nm23-H1 gene expression in the shRNA-expressing cells. Invasiveness was determined through a Boyden chamber assay. Results The puromycin-resistant clones (NL9980-99 and A549-99) showed very low levels of nm23-Hl mRNA and protein expression under RT-PCR, qPCR, and Western blot analysis. Meanwhile, the shRNA rescue experiment restored the nm23-H1 expression in the NL9980-99 and AS49-99 cells detected by Western blot. Down-regulation of nm23-H1 gene expression enhanced the invasiveness of the NL9980-99 and AS49-99 cells compared with the controls. Conclusion The lung cancer cell lines NL9980-99 and A549-99 with stable nm23-H1 gene silencing were successfully established and their invasiveness was greatly increased after nm23-H1 gene knockdown.%背景与目的 nm23-H1基因是重要的肿瘤转移抑制基因.前期研究发现利用化学合成的小干扰RNA(small interfering RNA,siRNA)抑制nm23-H1基因的表达可明显增强肺癌细胞的侵袭力.为了进一步研究nm23-H1基因沉默后的分子生物学机制,本研究利用慢病毒介导的短发夹RNA(short hairpin RNA,shRNA)建立nm23-H1基因稳定沉默的肺癌细胞株.方法 将表达特异性抑制nm23-H1基因shRNA的慢病毒转染人大细胞肺癌细胞株NL9980和肺腺癌细胞株A549,通过嘌呤霉素筛选出稳定转染细胞株.逆转录PCR、定量PCR及Western blot法检测nm23-Hl基因表达,并通过shRNA抵抗的nm23-H1基因重组质粒转染拯救实验验证,侵袭小室实验检测侵袭力改变.结果 逆 转录PCR、定量PCR和Western blot法检测稳定转染细胞株NL9980-99和A549-99中nm23-H1基因在mRNA和蛋白水平表达均明显降低；shRNA抵抗的nm23-H1基因重组质粒转染拯救实验重现nm23-H1正常表达；侵袭小室实验显示NL9980-99和A549-99细胞侵袭力明显增强.结论 成功建立nm23-H1基因稳定沉默的人大细胞肺癌细胞株NL9980-99和人肺腺癌细胞株A549-99,nm23-H1基因沉默后使NL9980-99和A549-99细胞的侵袭力明显增强.