LncRNA miR143HG调控肺鳞癌H520细胞生物学行为的机制研究

Mechanism Research of lncRNA miR143HG on Regulating the Biological Behavior of Lung Squamous Cell Carcinoma H520 Cells

背景与目的肺鳞癌(lung squamous cell carcinoma,LUSC)具有高发病率和高致死率,并且其临床预后较差。然而,目前对于LUSC尚无有效的靶向治疗方案。LncRNA miR143HG作为一种长链非编码RNA(long noncoding RNA,lncRNA),已被证实在多种肿瘤的发生发展中发挥着重要作用。然而lncRNA miR143HG在LUSC细胞中所扮演的生物学角色尚不清楚。因此,本研究旨在探讨lncRNA miR143HG调控LUSC H520细胞生物学行为的作用机制。方法基于肿瘤基因组图谱(The Cancer Genome Atlas,TCGA)数据库,对lncRNA miR143HG进行泛癌分析和差异表达分析。使用受试者工作特征(receiver operating characteristic,ROC)曲线和timeROC曲线评估lncRNA miR143HG对LUSC诊断和预后的预测效果。确定各通路对于lncRNA miR143HG的富集程度。利用实时定量聚合酶链反应(quantitative real-time polymerase chain reaction,qRT-PCR)检测BEAS-2B和H520细胞中lncRNA miR143HG以及miR-155的表达。将H520细胞随机分为空白对照组(不做任何处理)、阴性对照组(转染lncRNA-negative control,即lncRNANC)、lncRNA miR143HG组(转染lncRNA miR143HG)和lncRNA miR143HG+miR-155组(共转染lncRNA miR143HG和miR-155)。采用CCK-8法检测细胞增殖能力;划痕实验检测细胞迁移能力;Transwell实验检测细胞侵袭能力;流式细胞术检测细胞凋亡率;qRT-PCR和蛋白免疫印迹实验检测Wnt/β-Catenin通路相关基因和蛋白的表达情况。结果泛癌分析和差异分析结果共同显示,除肾透明细胞癌外,其他癌组织中lncRNA miR143HG的表达均高于健康组织,且在LUSC中的差异具有统计学意义。ROC曲线和timeROC曲线的评估结果提示lncRNA miR143HG对于LUSC的诊断及预后的预测具有重要意义。富集于lncRNA miR143HG高表达的通路主要包括黏着斑、血管平滑肌收缩和钙信号通路等;而富集在lncRNA miR143HG低表达的通路主要包括氧化磷酸化、细胞周期、基础转录因子等。qRT-PCR结果显示,与BEAS-2B细胞相比,lncRNA miR143HG在H520细胞中低表达,而miR-155在H520细胞中高表达(P<0.05)。与阴性对照组比较,lncRNA miR143HG组细胞中lncRNA miR143HG基因、Wnt的基因和蛋白以及β-Catenin的基因和蛋白表达水平显著增加,而miR-155基因表达显著降低,且细胞增殖能力、细胞迁移能力和细胞侵袭能力也明显降低,但细胞凋亡率明显升高(P<0.05)。另外,与lncRNA miR143HG组比较,过表达miR-155能够逆转lncRNA miR143HG介导的生物学行为,差异具有统计学意义(P<0.05)。结论LncRNA miR143HG对于H520细胞的生物学行为具有重要意义。LncRNA miR143HG通过下调miR-155表达抑制H520细胞的增殖、迁移和侵袭能力并促进H520细胞的凋亡,其分子机制可能与Wnt/β-Catenin通路相关。

Background and objective There is a high morbidity,mortality,and poor clinical prognosis of lung squamous cell carcinoma(LUSC).However,there is currently no effective targeted treatment plan for LUSC.As a long noncoding RNA(lncRNA),lncRNA miR143HG has been proven to play an important role in the occurrence and development of various tumors.However,the biological role played by lncRNA miR143HG in LUSC cells is still unclear.Therefore,this study aimed to investigate the mechanism of lncRNA miR143HG on regulating the biological behavior of LUSC H520 cells.Methods Pan-cancer analysis and differential expression analysis of lncRNA miR143HG were performed based on The Cancer Genome Atlas(TCGA)database.The predictive effect of lncRNA miR143HG on the diagnosis and prognosis of LUSC was evaluated by adopting the receiver operating characteristic(ROC)curve and timeROC curve.The enrichment degree of each pathway to lncRNA miR143HG was determined.The expression of lncRNA miR143HG and miR-155 in BEAS-2B cells and H520 cells was detected using quantitative real-time polymerase chain reaction(qRT-PCR).H520 cells were randomly divided into blank control group(without any treatment),negative control group(transfected with lncRNA-NC),lncRNA miR143HG group(transfected with lncRNA miR143HG),and lncRNA miR143HG+miR-155 group(co-transfected with lncRNA miR143HG and miR-155).The approaches of CCK-8,wound healing test,Transwell assay,flow cytometry,qRT-PCR,and Western blot were respectively employed to detect the cell proliferation ability,cell migration ability,cell invasion ability,cell apoptosis rate,and expression level of related genes and proteins of the Wnt/β-Catenin pathway.Results The results of pan-cancer analysis and differential analysis collectively showed that except for renal clear cell carcinoma,the expression of lncRNA miR143HG in other cancer tissues was higher than that in healthy tissues,and the differences were significant in LUSC.The evaluation results of the ROC curve and timeROC curve suggested that lncRNA miR143HG was of great significance in the prediction of diagnosis and prognosis of LUSC.The pathways enriched in high expression of lncRNA miR143HG mainly included focal adhesion,vascular smooth muscle contraction,calcium signaling pathways,and so on;the pathways enriched in the low expression of lncRNA miR143HG embraced oxidative phosphorylation,cell cycle,basic transcription factors,etc.The qRT-PCR results showed that lncRNA miR143HG was low expressed but miR-155 was highly expressed in H520 cells when compared to BEAS-2B cells(P<0.05).Compared with the negative control group,the expression levels of the gene of lncRNA miR143HG,the gene and protein of Wnt,as well as the gene and protein ofβ-Catenin were significantly increased,while the gene expression of miR-155,the ability of cell proliferation,cell migration,and cell invasion were significantly reduced,but the cell apoptosis rate was dominantly elevated in cells of lncRNA miR143HG group(P<0.05).In addition,compared with the lncRNA miR143HG group,overexpression of miR-155 could reverse the biological behavior mediated by lncRNA miR143HG,and the difference was statistically significant(P<0.05).Conclusion LncRNA miR143HG was of great significance for the biological behavior of H520 cells.LncRNA miR143HG inhibited the ability of proliferation,migration,and invasion,as well as enhanced the apoptosis of H520 cells by downregulating miR-155 expression,which may be related to the Wnt/β-Catenin pathway.