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 A549 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-H1 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 A549-99 cells detected by Western blot. Downregulation of nm23-H1 gene expression enhanced the invasiveness of the NL9980-99 and A549-99 cells compared with the controls.
Conclusions: 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-H1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的正常表达；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。