MiR-101靶向调控CREB1抑制肺癌的发展

MiR-101 targets CREB1 to inhibit the development of lung cancer

目的:观察miR-101在肺癌细胞或组织中的表达变化情况,分析miR-101通过靶向调控环磷腺苷反应元件结合蛋白1(cyclic adenosine monophosphate responsive element binding protein 1,CREB1)对肺癌发生发展的影响。方法:实时聚合酶链反应检测肺癌组织,癌旁正常组织,直径≤5 cm,>5 cm,转移,无转移肺癌组织中的miR-101表达量以及各株肺癌细胞中miR-101与mRNA的表达量。MicroRNA靶基因数据库与荧光素酶报告基因检验CREB1为miR-101的潜在靶基因。实时聚合酶链反应和蛋白质印迹法检测miR-101 mimics对CREB1 mRNA及蛋白表达的影响。实时聚合酶链反应检测肿瘤组织与癌旁正常组织CREB1 mRNA的表达量;免疫组织化学检测肿瘤组织和癌旁正常组织中CREB1的表达量。CCK-8检测各转染组的光密度(optical density,OD)值;流式细胞术检测细胞比值。Tranwell迁移实验和细胞划痕实验检测细胞迁移能力。观察21 d裸鼠肿瘤体积及质量;检测miR-101在miR-101 mimics转染组裸鼠肿瘤组织中的表达量;增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)染色检测裸鼠肿瘤细胞增殖情况。结果:与癌旁正常组织相比,肺癌组织中miR-101的表达量显著降低;转移的肺癌组织中miR-101的表达量显著低于无转移肿瘤组织;直径≤5 cm的肿瘤中miR-101的表达量显著高于直径>5 cm的肿瘤;各株肺癌细胞的miR-101表达量均显著低于正常肺细胞,而CREB1 mRNA表达量相反。miR-101可通过结合CREB1的3'-非翻译区(3'-untranslated region,3'-UTR)抑制CREB1的表达。miR-101能够抑制CREB1的mRNA翻译及其蛋白表达。肺癌组织中CREB1 mRNA表达量显著升高。miR-101可抑制肺癌细胞A549的增殖,迁移并使其细胞周期在S期聚集,CREB1补回该抑制作用。miR-101 mimics转染组裸鼠肿瘤体积及质量均显著低于对照组;miR-101过表达能够抑制A549细胞的增殖,抑制肿瘤发展。结论:miR-101通过抑制CREB1表达抑制肺癌的发展,可能成为肺癌诊断及治疗的新靶标。

Objective:To investigate the changes in the expression of miR-101 in lung cancer cells or tissues,and analyze the impact of miR-101 on the development and progression of lung cancer by targeting cyclic adenosine monophosphate responsive element binding protein 1(CREB1).Methods:Real-time PCR was used to detect the expression of miR-101 in lung cancer tumor tissues and adjacent normal tissues,tumors with diameters≤5 or>5 cm,metastatic and non-metastatic lung cancer tumor tissues.The expressions of miR-101 and CREB1 mRNA in each lung cancer cell line were detected.MicroRNA target gene database and luciferase reporter gene assay were used to test the potential targeting relationship between CREB1 and miR-101.The effect of miR-101 mimics on the mRNA and protein expression of CREB1 was detected by real-time PCR and Western blot.The expression of CREB1 in tumor and adjacent normal tissues was detected by immunohistochemical assay;the mRNA expression of CREB1 was detected by real-time PCR in tumor tissues and adjacent normal tissues.Optical density(OD)value of each transfection group was detected by CCK-8 assay.Cell ratio was detected by flow cytometry.Transwell migration assay and cell scratch assay were used to detect cell migration ability.The tumor volume and weight of nude mice at 21 d were observed.The expression level of miR-101 in tumor tissues of nude mice transfected with miR-101 mimics was detected.Proliferating cell nuclear antigen(PCNA)staining results were used to detect tumor cell proliferation in nude mice.Results:The expression level of miR-101 in lung cancer tissues was significantly lower than that in the adjacent normal tissues.The expression level of miR-101 in metastatic lung cancer tissues was significantly lower than that in non-metastatic tumor tissues.The expression level of miR-101 in tumors with diameter≤5 cm was significantly higher than that in tumors with diameter>5 cm.The expression level of miR-101 in all lung cancer cells was significantly lower than that in normal lung cells,while the expression level of CREB1 mRNA was opposite.miR-101 inhibited the expression of CREB1 via binding to the 3'-untranslated region(3'-UTR)of CREB1.miR-101 can inhibit the mRNA translation and protein expression of CREB1.The mRNA expression level of CREB1 was significantly increased in lung cancer tumor tissues.miR-101 inhibited the proliferation and migration of A549 cells,and caused the cell cycle to aggregate in S phase,CREB1 compensated for this inhibitory effect could antagonize this inhibitory effect.The tumor volume and weight of nude mice transfected with miR-101 mimics were significantly lower than those of the control group.Overexpression of miR-101 inhibited the proliferation of A549 cells and tumor development.Conclusion:MiR-101 may inhibit the development of lung cancer via downregulating the expression of CREB1 and may be a new target for the diagnosis and treatment of lung cancer.