miR-451对肺癌细胞的增殖抑制及其靶基因的初步鉴定

Inhibitory effect of microRNA-451 on proliferation in lung cancer A549 cells and primary identification for its target genes

目的探讨微小RNA(microRNA,miRNA)对非小细胞肺癌细胞增殖的作用及调控机制。方法 Lipofectamine 2000转染已构建成功的miR-451真核表达载体至非小细胞肺癌细胞A549,通过MTT检测肺癌细胞增殖状况,流式细胞术检测细胞周期和细胞凋亡情况,运用生物信息学技术对miR-451预测靶基因进行GO分析和KEGG信号途径分析,对炎症相关的预测靶基因PSMB8进行进一步的验证。构建含有PSMB8 3'UTR野生型和突变型质粒,通过双荧光素酶检测A549细胞PSMB8表达水平,Western blot检测PSMB8和NOS2的表达情况。结果 MTT结果显示过表达miR-451能显著抑制A549细胞增殖。流式细胞术检测发现miR-451组细胞阻滞在G2期,而细胞凋亡未见明显差异。GO分析发现miR-451预测靶基因涉及PSMB8、CAB39、YWHAZ等17个基因,PSMB8主要参与细胞代谢调控,细胞周期与细胞增殖;KEGG信号途径分析结果显示靶基因主要涉及microRNA在细胞周期、PI3KAKT、m TOR等信号通路。17个miR-451预测靶基因进行生物信息学研究发现PSMB8含有miR-451的结合位点,且在多个物种中呈高度保守。荧光素酶活性检测发现,过表达miR-451可抑制PSMB8荧光素酶活性表达。Western blot结果发现过表达miR-451的A549细胞PSMB8呈下调,而低表达miR-451的正常支气管上皮16HEB细胞PSMB8呈上调。Western blot和细胞荧光免疫化学检测发现炎症因子NOS2在肺癌中表达也降低。结论 miR-451可能通过靶向炎症相关基因PSMB8/NOS2调控非小细胞肺癌细胞增殖,并参与肺癌的发生与发展。

Objective To determine the role of microRNA-451（ miR-451） in the proliferation in non-small cell lung cancer cells and investigate the underlying regulative mechanism. Methods The eukaryotic expression vector of miR-451（ p Genesil-miR-451） was transfected into A549 cells using Lipofectamine 2000,and then the cell proliferation was tested by MTT assay,and cell apoptosis and cell cycle were examined by flow cytometry. Bioinformatics analysis was used for miR-451 target prediction. The target gene functional analysis was carried on by GO and KEGG pathway analysis. Among the candidate genes,PSMB8 was chosen. The luciferase gene expression vectors containing PSMB8 3’ UTR widetype and PSMB8 3’UTR mutant were constructed. And the luciferase activity was detected by dual-luciferase reporter assay.Moreover,the expression of PSMB8 and NOS2 was determined by Western blotting. Results MTT assay showed that the cell proliferation was inhibited by over-expression of miR-451 in A549 cells. Flow cytometry indicated that the cells were arrested at G2 phase in the A549 cells after transfection of p Genesil-miR-451,however,no significant difference was seen in the apoptotic rates among the transfected cells,control cells and A549 cells（ P 〉 0. 05）. By bioinformatics analysis,there were 17 potential target genes found for miR-451.Meanwhile,PSMB8 was found to contain a phylogenetically conserved binding site with miR-451. Evidence showed that miR-451 negatively regulated the expression of PSMB8 through PSMB8 3’ UTR. Moreover,the expression of PSMB8 was declined in miR-451 over-expressed A549 cells,while the expression of PSMB8 was enhanced in miR-451 down-expressed normal 16 HEB cells by Western blotting. Additionally,the expression of NOS2 was also decreased in miR-451 A549 cells. Conclusion MiR-451 may regulate the cell proliferation of non-small cell lung cancer cells by targeting inflammatory factors PSMB8 / NOS2,and participate in the regulation of the occurrence and development of lung cancer.