ITGA6基因对非小细胞肺癌脑转移瘤细胞的影响

Effect of ITGA6 gene on non-small cell lung cancer brain metastasis cells

目的深入探究ITGA6基因在非小细胞肺癌(non-small cell lung cancer,NSCLC)脑转移瘤细胞增殖、细胞周期调控、侵袭及迁移过程中的具体作用机制。方法通过挖掘GEO数据库中的NSCLC脑转移瘤相关数据,本研究成功识别出ITGA6基因为与NSCLC脑转移瘤发生和发展相关的重要因子。在此基础上,进一步探究该基因表达水平与NSCLC脑转移瘤患者预后之间的关联性。为验证ITGA6在NSCLC脑转移瘤中的表达模式,本研究进一步采用实时定量逆转录PCR(qRT-PCR)和Western Blot实验技术,在NSCLC脑转移瘤细胞系测定ITGA6表达水平。利用基因转染技术,在体外NSCLC脑转移瘤细胞系H1915-BrM和A549-BrM中分别转染阴性对照(NC组)和ITGA6特异性沉默载体(si-ITGA6组)。通过CCK-8增殖实验评估细胞增殖能力;利用EDU实验进一步验证细胞增殖变化;应用流式细胞技术检测细胞周期分布情况;通过Transwell侵袭实验和划痕愈合实验分析细胞侵袭和迁移能力。利用Western Blot技术检测上皮-间质转化(epithelial-mesenchymal transition,EMT)相关蛋白的表达水平。结果利用GEO数据库系统分析NSCLC脑转移瘤的差异表达基因,并发现ITGA6是其中一个关键的上调基因。临床数据分析表明:ITGA6高表达与NSCLC脑转移瘤患者不良预后显著相关(P<0.05)。进一步通过qRT-PCR和Western Blot技术在实验层面验证ITGA6在肺癌脑转移瘤细胞系中高表达。CCK-8和EDU实验结果表明:si-ITGA6显著抑制NSCLC脑转移瘤细胞H1915-BrM和A549-BrM的增殖(P<0.05)。流式细胞技术揭示:si-ITGA6处理后,细胞在G0/G1期比例增加,并且S期及G2期细胞随之减少,表明细胞周期被阻滞(P<0.05)。Transwell和划痕实验均显示,si-ITGA6显著降低NSCLC脑转移瘤细胞侵袭和迁移能力。Western Blot观察分析:si-ITGA6组中上皮标记物E钙黏蛋白表达显著上升,而间质标记物纤连蛋白、波形蛋白以及基质金属蛋白酶(MMP-2和MMP-9)表达则显著下降,提示EMT过程受到抑制。结论通过特异性沉默ITGA6基因,能够显著抑制NSCLC脑转移瘤细胞增殖、迁移和侵袭能力,同时阻滞细胞周期,并调控EMT相关蛋白的表达。

Objective To deeply investigate the specific mechanisms of ITGA6 gene in the processes of proliferation,cell cycle regulation,invasion,and migration of non-small cell lung cancer(NSCLC)brain metastasis cells.Methods By mining the NSCLC brain metastasis-related data from the GEO database,this study successfully identified ITGA6 as an important factor associated with the occurrence and progression of NSCLC brain metastasis.Based on this,the correlation between the expression level of ITGA6 gene and the prognosis of NSCLC brain metastasis patients was further explored.To verify the expression pattern of ITGA6 in NSCLC brain metastasis,real-time quantitative reverse transcription PCR(qRT-PCR)and Western blot techniques were employed to measure the expression level of ITGA6 in NSCLC brain metastasis cell lines.Using gene transfection technology,negative control(NC group)and ITGA6-specific silencing vector(si-ITGA6 group)were transfected into the NSCLC brain metastasis cell lines H1915-BrM and A549-BrM in vitro.The proliferative ability of the cells was assessed by CCK-8 proliferation assay.The EDU assay was used to further verify the proliferative changes.The flow cytometry was applied to detect the distribution of cell cycle.Transwell invasion assay and wound healing assay were used to analyze the invasion and migration abilities of the cells.Western blotting was used to detect the expression levels of epithelial-mesenchymal transition(EMT)-related proteins.Results Differentially expressed genes in NSCLC brain metastasis were systematically analyzed using the GEO database,revealing ITGA6 as a key upregulated gene.Clinical data analysis indicated that high expression of ITGA6 was significantly associated with poor prognosis in NSCLC brain metastasis patients(P<0.05).Further verification by qRT-PCR and Western blot techniques confirmed the high expression of ITGA6 in lung cancer brain metastasis cell lines.The results of CCK-8 and EDU assays showed that si-ITGA6 significantly inhibited the proliferation of NSCLC brain metastasis cells H1915-BrM and A549-BrM(P<0.05).Flow cytometry revealed that after si-ITGA6 treatment,the proportion of cells in the G0/G1 phase increased,while cells in the S and G2 phases decreased,indicating cell cycle arrest(P<0.05).Both Transwell and wound healing assays showed that si-ITGA6 significantly reduced the invasion and migration abilities of NSCLC brain metastasis cells.Western blot analysis revealed that the expression of the epithelial marker E-cadherin was significantly increased in the si-ITGA6 group,while the expression of mesenchymal markers Vimentin and FN,and matrix metalloproteinases(MMP-2 and MMP-9),was significantly decreased,suggesting inhibition of the EMT process.Conclusions Specific silencing of the ITGA6 gene can significantly inhibit the proliferation,migration,and invasion abilities of NSCLC brain metastasis cells,arrest the cell cycle,and regulate the expression of EMT-related proteins.