摘要
目的MiR-106a-5p表达异常与多种癌症的增殖、迁移和侵袭功能密切相关,如肾细胞癌、结肠癌和骨肉瘤等。本研究探讨miR-106a-5p重组人转化生长因子B受体-2(recombinant transforming growth factor beta receptorⅡ,TGFBR2)对肺腺癌增殖、迁移和侵袭的影响和其分子机制。方法starBase 3.0平台调取并分析TCGA数据库中miR-106a-5p在肺腺癌组织中的表达谱;选取肺腺癌细胞系PC-9和H1299,实时荧光定量PCR检测miR-106a-5p在肺腺癌细胞系中的表达。设置miR-NC组和miR-106a-5p inhibitor组,CCK-8实验、Transwell实验检测miR-106a-5p表达下调对肺腺癌细胞增殖、迁移和侵袭的影响。荧光素酶报告基因检测miR-106a-5p靶向TGFBR2。qRT-PCR、蛋白质印迹等检测miR-106a-5p对TGFBR2的mRNA和蛋白表达的影响。分析TGFBR2表达与肺腺癌患者生存预后之间的相关性。结果miR-106a-5p在肺腺癌组织和细胞中呈现高表达,均P<0.05;CCK-8检测细胞增殖结果显示,在第3、4和5天时,PC-9细胞的miR-106a-5p inhibitor组细胞增殖活性低于miR-NC组细胞增殖活性,P3d=0.001,P4d=0.001,P5d=0.001,且H1299细胞的miR-106a-5p inhibitor组细胞增殖活性低于miR-NC组细胞增殖活性,P3d=0.001,P4d=0.001,P5d=0.001。Transwell迁移实验结果提示,肺腺癌PC-9细胞中的迁移数目miR-106a-5p inhibitor组为21.32±2.68,对照miR-NC组为108.74±3.89,差异有统计学意义t=23.72,P<0.001;肺腺癌H1299细胞中的迁移数目miR-106a-5p inhibitor组为23.19±3.07,NC组为123.52±6.44,差异有统计学意义,t=42.46,P<0.001。Transwell侵袭实验结果显示,肺腺癌PC-9细胞中的侵袭数目miR-106a-5p inhibitor组为17.38±1.25,miR-NC组为62.38±3.06,差异有统计学意义,t=23.73,P=0.001;肺腺癌H1299细胞中的侵袭数目miR-106a-5p inhibitor组为16.48±1.36,miR-NC组为57.22±4.01,差异有统计学意义,t=17.72,P=0.007。荧光素酶报告基因显示TGFBR2是miR-106a-5p的靶基因,miR-106a-5p负向调控TGFBR2的表达。CCK-8方法检测TGFBR
OBJECTIVE It is reported that the abnormal expression of miR-106a-5p is associated with proliferation,migration and invasion of multiple tumors such as renal cell carcinoma,colon cancer,osteosarcoma and so on.The study was to explore the effects and mechanism of miR-106a-5p overexpression on lung adenocarcinoma cells proliferation,migration and invasion.METHODS The expression profile of miR-106a-5p in lung adenocarcinoma tissues was analyzed from starBase v 3.0integrated from TCGA database.The expression of miR-106a-5p in lung adenocarcinoma cell lines PC-9and H1299were examined by quantitative Real time PCR(qRT-PCR).Cells were transfected with miR-106a-5p inhibitors.CCK-8assay was used to measure the proliferation ability of lung adenocarcinoma cells in miR-NC group and miR-106a-5p inhibitor group.Transwell assay was used to detect the migration and invasion of lung adenocarcinoma cells in miR-NC group and miR-106a-5p inhibitor group.TGFBR2was determined as the direct target of miR-106a-5p by luciferase reporter assay.TGFBR2expression regulated by miR-106a-5p in lung adenocarcinoma cell lines was detected by qRT-PCR and western blot.The relationship between TGFBR2expression and prognosis of lung adenocarcinoma patients was analyzed.RESULTS MicroRNA-106a-5p was up-regulated in lung adenocarcinoma tissues and cell lines,all P<0.05.CCK-8assay showed that the proliferation activity of miR-106a-5p inhibitor group in PC-9cells was lower than that of control group at 3rd,4th and 5th day(P3d=0.001,P4d=0.001,P5d=0.001),and the proliferation activity of miR-106a-5p inhibitor group in H1299cells was lower than that of control group at 3rd,4th and 5th day(P3d=0.001,P4d=0.001,P5d=0.001);Transwell migration assay indicated that the number of cell migration of the inhibitor group in PC-9cells was 21.32±2.68,significantly less than that in miR-NC group(108.74±3.89),t=23.72,P<0.001;the number of cell migration of the inhibitor group in H1299cells were 23.19±3.07,significantly less than that in miR-NC group(123.52±6.44),t=42.
作者
张成伟
毕昕
闫睿
ZHANG Cheng-wei;BI Xin;YAN Rui(Department of Thoracic Surgery,Third Medical Center of Chinese PLA General Hospital,Beijing100039,P.R.China)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2020年第16期1284-1291,1297,共9页
Chinese Journal of Cancer Prevention and Treatment
