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目的探讨TPX2基因沉默对人肺腺癌A549细胞增殖的影响。方法构建3个靶向TPX2基因的短发夹RNA(shRNA)表达质粒,转染A549细胞,RT-PCR检测TPX2基因mRNA的转录水平;筛选干扰效果最佳的质粒转染的细胞,RT-PCR检测增殖相关基因CDK4和CyclingD1 mRNA的转录水平,Western blot检测TPX2蛋白的表达水平,显微镜下观察细胞的生长情况,MTT法检测细胞的增殖活力。结果 TPX2 shRNA-3质粒干扰效果最佳,有效沉默TPX2基因后,A549细胞中TPX2基因mRNA的转录水平及蛋白的表达水平均受到明显抑制(P<0.01),与空白对照组比较,抑制率分别为73.8%和82.8%;CDK4和CyclingD1基因mRNA的转录水平也明显降低(P<0.01或<0.05),与空白对照组比较,抑制率分别为76.7%和67.3%;A549细胞的生长和增殖也明显受到抑制,转染后72 h,增殖抑制率可达61.55%(P<0.05)。结论成功构建了靶向TPX2基因的shRNA表达质粒,其在mRNA和蛋白水平有效抑制A549细胞TPX2基因的表达后,细胞生长受到抑制,增殖能力减弱。
Objective To investigate the effect of TPX2 gene silencing on the proliferation of human lung adenocarcinoma A549 cells. Methods Three short hairpin RNA (shRNA) expression plasmids targeting TPX2 gene were constructed and transfected into A549 cells. The transcription level of TPX2 mRNA was detected by RT-PCR. The transfected cells were screened by RT-PCR. The transcription level of CDK4 and CyclingD1 mRNA were detected. The expression of TPX2 protein was detected by Western blot. The growth of the cells was observed under the microscope. The proliferation activity of the cells was detected by MTT assay. Results The interference effect of TPX2 shRNA-3 plasmid was the best. After TPX2 gene was effectively silenced, the transcription level of TPX2 mRNA and protein expression of A549 cells were significantly inhibited (P <0.01). Compared with the blank control group, the inhibition rates were Were 73.8% and 82.8%, respectively. The mRNA transcription levels of CDK4 and CyclingD1 were also significantly decreased (P <0.01 or <0.05), compared with the control group, the inhibition rates were 76.7% and 67.3% Was significantly inhibited, 72 h after transfection, proliferation inhibition rate up to 61.55% (P <0.05). Conclusion The shRNA expression plasmid targeting TPX2 gene was successfully constructed. After TPX2 gene was effectively inhibited at mRNA and protein level, the cell growth was inhibited and the proliferation ability was weakened.