摘要
目的探讨miR-641在肺腺癌中的表达情况及其分子生物学作用机制。方法收集延安大学附属医院胸外科2018-01-01-2019-03-31行手术切除的30例肺腺癌患者癌组织及对应周围正常肺组织标本,采用qPCR法检测肺腺癌组织和肺腺癌细胞系A549、H1299和H157中miR-641表达水平,分析miR-641表达与肺腺癌患者临床特征关系。MTT法检测miR-641对肺腺癌A549细胞增殖能力的影响;Transwell侵袭及迁移实验检测miR-641对肺腺癌A549细胞迁移及侵袭能力的影响;靶基因双荧光素酶实验检测miR-641与CYR61的相互作用;PCR和蛋白质印迹法检测A549细胞中CYR61表达。结果 qPCR法检测miR-641在肺腺癌组织、肺组织、A549、H1299、H157和WI-38细胞中表达值分别为0.14±0.02、1.01±0.08、0.35±0.04、0.47±0.05、0.48±0.04、0.98±0.08,F=775.537,P<0.001;肺腺癌组织和肺组织miR-641表达差异有统计学意义,t=57.786,P<0.001。miR-641表达与TNM分期(P=0.004)和淋巴结转移(P=0.013)有关联。NC组和miR-641 mimics组A549细胞增殖活力分别为0.98±0.07和8.23±0.53,t=23.489,P<0.001。miR-641mimics转染A549细胞24、48和72h后,细胞增殖活力分别为1.23±0.19、1.86±0.21和3.05±0.29,F=333.197,P<0.001。Transwell侵袭及迁移实验结果显示,与NC组(101.42±10.14)%和(97.24±7.63)%相比,miR-641mimics组迁移及侵袭力分别为(71.43±6.12)%和(63.47±7.12)%,t值分别为13.620和12.430,均P<0.001。Target Sacn生物信息网站预测miR-641基因序列与CYR61基因序列存在结合片段,靶基因双荧光素酶实验显示,野生型CYR61 3′-UTR相对双荧光素酶活性低于突变型CYR61 3′-UTR,F=49.832,P<0.001。NC组和miR-641mimics组CYR61蛋白表达水平分别为1.00±0.09和0.61±0.10,t=5.024,P=0.004。MTT增殖实验显示,与miR-641mimics相比较,miR-641mimics+pSUPER-CYR61转染A549细胞24、48和72h后细胞增殖活性增加,分别为1.61±0.15、2.29±0.23和3.46±0.34,F=356.301,P<0.001。Transwell迁移实验表明,NC组、miR-641mimics组和miR-641mimics+pSUPER-CYR61组A549细胞迁移力分别为(100.00±7.23)%、(45.61±5.67)%和(97.12±3.17)%,F=53.994,P<0.001;Transwell侵袭实验表明,NC组、miR-641mimics组和miR-641mimics+pSUPER-CYR61组A549细胞侵袭力分别为(100.00±7.82)%、(39.42±6.07)%和(85.62±5.37)%,F=34.893,P<0.001。结论 miR-641在肺腺癌组织和细胞中处于低表达状态,可抑制肺腺癌细胞增殖、迁移和侵袭,其作用机制可能与靶向调节CYR61有关,可成为肺癌基因治疗的潜在靶点。
Objective To explore the expression of miR-641 in lung adenocarcinoma and its molecular biological mechanism.Methods A total of 30 cases of resected lung adenocarcinoma and corresponding normal lung tissues between January 1,2018 and March 31,2019 were involved in this study.The expressions of miR-641 in lung adenocarcinoma tissues,normal lung tissues as well as A549,H1299 and H157 cell lines were detected by qPCR.The relationship between the expression of miR-641 and the clinical features of lung adenocarcinoma was investigated.MTT assay was used to detect the effect of miR-641 on the proliferation of A549 cells.Transwell invasion and migration assay was used to detect the effect of miR-641 on the migration and invasion of A549 cells.Dual luciferase reporter assay was used to detect the interaction between miR-641 and CYR61.PCR and western blot were used to detect the expression of CYR61 in A549 cells.Results The expressions of miR-641 in lung adenocarcinoma,lung tissue,A549,H1299,h157 and WI-38 cells were0.14±0.02,1.01±0.08,0.35±0.04,0.47±0.05,0.48±0.04 and 0.98±0.08,respectively(F=775.537,P<0.001).There was significant difference in the mean expression of miR-641 between lung adenocarcinoma and lung tissue(t=57.786,P<0.001).The miR-641 expression was correlated with TNM stage(P=0.004)and lymph node metastasis(P=0.013).The proliferation activity of A549 cells in NC group and miR-641 mimics group were 0.98 ±0.07 and8.23±0.53(t=23.489,P<0.001).After transfection of A549 cells with miR-641 mimics for 24,48 and 72 h,the cell proliferation activities were 1.23±0.19,1.86±0.21 and 3.05±0.29,respectively(F=333.197,P<0.001).The results of Transwell invasion and migration experiment showed that compared with NC group(101.42±10.14)% and(97.24±7.63)%,the migration and invasion of miR-641 mimics group were(71.43±6.12)% and(63.47±7.12)%,respectively(t values were 13.620 and 12.430,all P<0.001).Target sacn bioinformatics website predicted the binding fragment between miR-641 gene sequence and CYR61 gene sequence.The target gene double luciferase experiment showed that after lung adenocarcinoma A549 cells were transfected with wild-type CYR61 3′-UTR and mutant CYR613′-UTR vector respectively,the relative double luciferase activity of wild-type CYR61 3′-UTR was lower than that of mutant CYR61 3′-UTR,F=49.832,P<0.001.Western blot showed that transfection of miR-641 mimics(1.00±0.09)into lung adenocarcinoma cells could reduce the expression of CYR61 protein(0.61±0.10),t=5.024,P=0.004.MTT proliferation assay showed that compared with mir-641 mimics,the proliferation activity of A549 cells transfected with miR-641 mimics+ psuper-cyr61 increased at 24,48 and 72 hours,which were 1.61±0.15,2.29±0.23 and 3.46±0.34(F=356.301,P<0.001),respectively.Transwell migration experiment showed that the migration ability of A549 cells in NC group,miR-641 mimics and miR-641 mimics+psuper-cyr61 transfected group were(100.00±7.23)%,(45.61±5.67)% and(97.12±3.17)%(F= 53.994,P<0.001),respectively;Transwell invasion experiment showed that the invasiveness of A549 cells in NC group,miR-641 mimics group and miR-641 mimics+ psuper-cyr61 group were(100.00±7.82)%,(39.42±6.07)% and(85.62±5.37)%(F=34.893,P<0.001),respectively.Conclusion miR-641 is lowexpressed in lung adenocarcinoma tissues and cells,it can inhibit the proliferation,migration and invasion of lung adenocarcinoma cells,and its mechanism of action may be related to the targeted regulation of CYR61,which may become a potential target for lung cancer gene therapy.
作者
杨扬
王凯
任清泉
许瑞彬
郭建峰
YANG Yang;WANG Kai;REN Qing-quan;XU Rui-bin;GUO Jian-feng(Department of Thoracic Surgery,Affiliated Hospital of Yan’an University,Yan'an 716000,China)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2021年第21期1611-1617,共7页
Chinese Journal of Cancer Prevention and Treatment
