miR-449c通过抑制c-Myc的表达抑制肺腺癌细胞周期进程

miR-449c inhibits the cell cycle progression of lung adenocarcinoma cells by inhibiting the expression of c-Myc

目的:探讨miR-449c对肺腺癌发生发展的影响及可能机制。方法:利用生物信息学分析鉴定高复发风险肺腺癌患者(无病生存期≤6个月)与低复发风险患者(无病生存期>60个月)之间差异表达的microRNA(miRNA)。靶基因预测及通路分析研究可能的机制。RT-PCR检测miR-449c在肺腺癌组织和细胞系中的表达,利用细胞转染实验在肺腺癌细胞系中过表达miR-449c和c-Myc,CCK-8法测定细胞增殖活性,流式细胞术检测细胞周期及凋亡,蛋白质印迹分析检测相关蛋白表达水平。结果:生物信息学分析结果显示,相比低复发风险患者,肺腺癌高复发风险患者的miR-449c表达显著降低。miR-449c表达水平与肺腺癌患者临床分期和淋巴结转移相关,miR-449c低表达患者的预后显著差于高表达患者。通路和功能分析表明,miR-449c可能与细胞周期进程有关。与癌旁组织相比,miR-449c在肺腺癌组织中显著低表达;过表达miR-449c抑制了肺腺癌细胞增殖,导致细胞周期阻滞于G1期,下调了c-Myc及其下游细胞周期蛋白的表达。c-Myc过表达可部分逆转miR-449c对细胞周期蛋白的抑制,miR-449c过表达也可部分逆转c-Myc对细胞周期蛋白的上调。结论:miR-449c低表达的肺腺癌患者预后较差,过表达miR-449c可以通过下调c-Myc调控细胞周期,抑制肺腺癌细胞增殖,提示miR-449c可能作为肺腺癌潜在的治疗靶标之一。

Objective:The purpose of the current study was to explore the effect and possible mechanism of miR-449c on the occurrence and development of lung adenocarcinoma.Methods:Bioinformatics analysis was used to identify the differentially expressed microRNAs between samples with lung adenocarcinoma patients at a high risk of recurrence(disease-free survival≤6 months)and samples with lung adenocarcinoma patients at a low risk of recurrence(disease-free survival>60 months).Target gene prediction and pathway analysis were used to analysis possible mechanisms.RT-PCR was used to detect the expression of miR-449c in lung adenocarcinoma tissues and cell lines.miR-449c and c-Myc were overexpressed in lung adenocarcinoma cell lines by cell transfection experiments.Cell growth activity was determined by CCK-8.Cell cycle and apoptosis were detected by flow cytometry.Western blot analysis was used for relevant protein expression.Results:The results of bioinformatics analysis showed that the expression of miR-449c was significantly decreased in the group of lung adenocarcinoma patients at a high risk of recurrence,compared with that at a low risk of recurrence.The expression of miR-449c was significantly correlated with the clinical stage and lymph node metastasis of patients with lung adenocarcinoma.The prognosis of patients with low miR-449c expression was significantly worse than that of patients with high miR-449c expression.Pathway and functional analysis indicated that miR-449c may be involved in cell cycle progression.Compared with normal tissues,miR-449c was significantly lower expressed in lung adenocarcinoma tissues.Overexpression of miR-449c inhibited lung adenocarcinoma cell proliferation,resulted in cell cycle arrest in G1 phase,and downregulated the expression of c-Myc and its downstream cyclins.Overexpression of c-Myc could partially counteract the inhibition of cyclin by miR-449c.Overexpression of miR-449c could also partially counteract the up-regulation of cyclin expression by c-Myc.Conclusion:Lung adenocarcinoma patients with low expression of miR-449c have a poor prognosis.Overexpression of miR-449c can regulate the lung adenocarcinoma cell cycle by downregulating c-Myc to inhibit the proliferation of lung adenocarcinoma cells.The results suggest that miR-449c may be one of the potential therapeutic targets for lung adenocarcinoma.