shRNA抑制非小细胞肺癌H460SM细胞锚定非依赖生长和侵袭

目的:探讨慢病毒介导α6整合素(integrinα6,ITGA6)shRNA对人非小细胞肺癌(non-small cell lung cancer,NSCLC)H460SM细胞生长和侵袭的影响。方法:实时定量PCR和Western blotting检测ITGA6在9种NSCLC细胞中的表达。慢病毒介导ITGA6 shRNA感染H460SM细胞后,实时定量PCR和Wesern blotting检测H460SM中ITGA6的表达,显微镜下观察H460SM细胞形态的变化,MTS法检测细胞增殖,软琼脂实验检测H460SM细胞锚定非依赖性生长,流式细胞术检测细胞凋亡和细胞周期,迁移和侵袭实验检测H460SM细胞体外迁移和侵袭能力。结果:ITGA6在7种人NSCLC细胞中均有表达,H460SM细胞中ITGA6表达水平明显高于亲本H460细胞(8.75±0.09)vs(5.78±0.26),P<0.01]。慢病毒介导ITGA6shRNA稳定感染H460SM细胞后的H460SM-75、H460SM-76细胞中,ITGA6表达水平明显低于阴性对照组H460SM-NS细胞(0.11±0.04)、(0.22±0.04)vs(1.00±0.01),P<0.01]。H460SM-75、H460SM-76细胞增殖活性与H460SM-NS细胞无差异(P>0.05),且凋亡率、细胞增殖指数、G0/G1期细胞的比例也无差异(P>0.05)。H460SM-75、H460SM-76细胞的克隆形成率明显低于H460SM-NS细胞(18.87±1.47)%、(18.85±1.11)%vs(20.81±1.38)%,P<0.05],迁移率(43.92±0.41)%、(24.10±0.33)%vs(100.00±0.50)%,P<0.01]和侵袭率(7.04±2.96)%、(4.68±0.27)%vs(100.00±6.74)%,P<0.01]也明显低于H460SM-NS细胞。结论:抑制ITGA6的表达可以抑制NSCLC细胞锚定非依赖性生长、迁移和侵袭,但不影响NSCLC细胞增殖和凋亡。

shRNA inhibits anchorage independent growth and invasion of human non-small cell lung cancer cell line H460SM

Objective:To investigate the effect of lentivirus-mediated integrin α6(ITGA6) shRNA on growth and invasion of human non-small cell lung cancer(NSCLC) H460SM cells.Methods: The expression levels of ITGA6 in 9 human NSCLC cell lines were evaluated by quantitative-PCR(Q-PCR) and Western blotting.Lentiviral ITGA6 shRNA were transfected into H460SM cells and ITGA6 expression was detected by Q-PCR and Western blotting;the cellular morphology change was observed under a microscope;cell proliferation was detected by MTS assay;anchorage-independent growth was determined by colony formation assay in soft agar;apoptosis and cell cycle were analyzed by flow cytometry;and cell invasion and migration were determined by invasion and migration assay.Results: ITGA6 was expressed in 7 NSCLC cell lines.A significantly higher level of ITGA6 expression was seen in H460SM cells compared with the parental H460 cells(8.75±0.09] vs 5.78±0.26],P<0.01).After H460SM cells were stably transfected with lentiviral ITGA6 shRNA(H460SM-75,H460SM-76 cells),a significant down-regulation of ITGA6 expression was observed in H460SM-75 and H460SM-76 cells compared with negative control H460SM-NS cells(1.00±0.01] vs 0.11±0.04],0.22±0.04],P<0.01).The cell viability of H460SM-75 and H460SM-76 cells had no difference with H460SM-NS cells(P>0.05),as well as the apoptotic rate,cell proliferative index and proportion of G0/G1 phase cells as compared with the negative control(P>0.05).The colony formation rate of H460SM-75 and H460SM-76 cells was significantly decreased as compared with H460SM-NS cells(20.81±1.38]% vs 18.87±1.47]%,18.85±1.11]%,P<0.05),and the migration rate(100.00±0.50]% vs 43.92±0.41]%,24.10±0.33]%,P<0.01) and invasion rate(100.00±6.74]% vs 7.04±2.96]%,4.68±0.27]%,P<0.01) were also significantly decreased.Conclusion: Knockdown of ITGA6 expression can inhibit anchorage-independent cell growth,migration and invasion of NSCLC cells,but have no effect on cell proliferation and apoptosis.