沉默HMGA基因对非小细胞肺癌A549细胞增殖和转移的影响及机制

Effect of HMGA gene silencing on the growth and metastasis of non-small cell lung cancer A549 cells

目的:研究沉默HMGA1和HMGA2基因的表达对非小细胞肺癌细胞A549恶性增殖和转移的影响及其机制。方法:通过MTT实验测定HMGA1和HMGA2基因沉默之后,A549细胞的增殖能力;通过流式细胞术测定HMGA1和HMGA2基因沉默之后,A549细胞凋亡的情况;利用划痕实验测定分别沉默HMGA1和HMGA2基因的表达之后,A549细胞的转移能力;用Q-PCR和Western blot分析HMGA1和HMGA2基因沉默后,抑制A549细胞增殖和转移的分子机制。结果:siRNA-HMGA1和siRNA-HMGA2干扰A549细胞48 h后,A549细胞的生长活力降低(P<0.05)。Q-PCR结果显示,siRNA-HMGA1干扰A549细胞48 h之后,导致细胞凋亡相关基因FOXM1和c-Myc的相对表达量降低(P<0.05),但对细胞侵袭转移相关基因E-Cadherin、N-Cadherin和Vimentin的表达没有影响(P>0.05)。Western blot实验结果进一步证实siRNA-HMGA1干扰A549细胞后,可抑制FOXM1和c-Myc蛋白水平的表达(P<0.05)。Q-PCR结果显示,siRNA-HMGA2不仅可以导致FOXM1和c-Myc的表达量降低(P<0.05),还可促进细胞侵袭转移相关基因E-Cadherin的表达,并抑制N-Cadherin和Vimentin的表达(P<0.05)。Western blot结果进一步证实,沉默HMGA2在A549细胞内的表达后,会导致FOXM1和c-Myc表达量的降低(P<0.05),E-Cadherin表达量的增加(P<0.05),同时引起N-Cadherin和Vimentin表达量的降低(P<0.05)。结论:HMGA1和HMGA2基因沉默后,通过下调FOXM1和c-Myc的表达诱导A549细胞凋亡;HMGA2表达的沉默通过下调N-Cadherin和Vimentin的表达,上调E-Cadherin的表达抑制A549细胞的转移。

Objective:To investigate the effect of HMGA1 and HMGA2 gene silencing on the malignant proliferation and metastasis of human non-small cell lung cancer A549 cells and the corresponding mechanism.Methods:The proliferation capacity of A549 cells was analyzed by MTT assay after silencing HMGA1 and HMGA2 gene.The effect of HMGA1 and HMGA2 gene silencing on the apoptosis of A549 cells were determined by flow cytometry.Scratch assay was used to analyze the migration ability of A549 cells after silencing HMGA1 and HMGA2 genes.Q-PCR and western blot were used to detect the expression of apoptosis and metastasis related factors in mRNA and protein level after HMGA1 and HMGA2 gene silencing.Results:Silencing HMGA1 and HMGA2 genes with siRNA-HMGA1 and siRNA-HMGA2 for 48 h,A549 cell viability decreased(P<0.05).The results of Q-PCR showed that siRNA-HMGA1 inhibited the expression of apoptosis related genes FOXM1 and c-Myc(P<0.05).The effect of siRNA-HMGA1 on the expression of genes related to invasion(E-Cadherin,N-Cadherin and Vimentin)had no statistical difference(P>0.05).The results of western blot confirmed that siRNA-HMGA1 impaired the expression of FOXM1 and c-Myc in A549 cells(P<0.05).Similarly,siRNAHMGA2 inhibited the expression of FOXM1 and c-Myc(P<0.05).Meanwhile,siRNA-HMGA2 caused the expression of E-Cadherin(P<0.05),and inhibited the expression of N-Cadherin and Vimentin(P<0.05).Western blot confirmed the results of Q-PCR,where siRNA-HMGA2 impaired the expression of FOXM1 and c-Myc(P<0.05),increased the expression of E-Cadherin(P<0.05),and blocked the expression of N-Cadherin and Vimentin(P<0.05).Conclusion:Silencing HMGA1 and HMGA2 gene induced the apoptosis of A549 cells through down-regulating the expression of FOXM1 and c-Myc.Silencing HMGA2 gene inhibited the metastasis of A549 cells though down-regulation of N-Cadherin and Vimentin,and upregulating of E-Cadherin.