摘要
目的探讨长链非编码RNA(lncRNA)肌动蛋白纤维相关蛋白1-反义RNA1(Actin filament-associatedprotein1-antisenseRNA1,AFAP1-AS1)调控结直肠癌(Colorectal cancer,CRC)细胞增殖的分子机制。方法收集2014年7月—2018年6月深圳市第二人民医院消化内科和胃肠外科诊治的38例正常人及38例CRC患者的粪便标本,用Real time-PCR检测lncRNA AFAP1-AS1表达,同时检测人正常结肠上皮细胞株NCM460、人结直肠癌细胞株SW620和HCT116中lncRNA AFAP1-AS1的表达;将靶向siRNA转染到CRC细胞株SW620和HCT116中抑制AFAP1-AS1的表达;通过MTT测定si-AFAP1-AS1转染对CRC细胞增殖的影响;应用免疫印迹检测Cleaved caspase 3、Bcl-2、Bax、p-AKT、total-AKT和PTEN的水平。结果与正常人相比,CRC患者粪便中AFAP1-AS1的表达显著上升(P<0.01);与NCM460细胞相比,SW620和HCT116细胞中AFAP1-AS1的表达也显著升高(P<0.01);si-AFAP1-AS1转染抑制SW620和HCT116的细胞生长(P<0.01)。与NCM460细胞相比,si-AFAP1-AS1干扰上调了SW620和HCT116细胞中Cleaved caspase 3和促凋亡蛋白Bax的表达水平(P<0.05),下调了抗凋亡蛋白Bcl-2的表达水平(P<0.001);此外,si-AFAP1-AS1干扰降低了CRC细胞中p-AKT的蛋白水平,并增加了PTEN的表达(P<0.01)。结论正常人和CRC患者粪便中lncRNA AFAP1-AS1表达的差异有助于CRC的早期诊断,且lncRNA AFAP1-AS1在CRC细胞中表达上调,并通过PTEN/p-AKT信号通路调节CRC细胞的增殖和凋亡。lncRNA AFAP1-AS1有望成为CRC早期诊断的分子靶标。
Objective The aim of this study was to explore the molecular mechanism of long non-coding RNA(LncRNA)AFAP1-AS1 regulating the proliferation of colorectal cancer(CRC)cells.Methods Fecal specimens from 38 normal people and 38 CRC patients diagnosed and treated in the department of digestive medicine and gastrointestinal surgery of Shenzhen Second People′s Hospital from July 2014 to June 2018 were collected to detect the expression of lncRNA AFAP1-AS1 by Real time-PCR.At the same time,the expression of lncRNA AFAP1-AS1 was detected in human normal colon epithelial NCM460 cells,human colorectal cancer SW620 and HCT116 cells.Targeting lncRNA AFAP1-AS1 siRNA(si-AFAP1-AS1)was transfected into SW620 and HCT116 cells to inhibit the expression of AFAP1-AS1.MTT assay was used to examine the effect of si-AFAP1-AS1 on cell proliferation of CRC cells.The levels of cleaved-caspase-3,Bcl-2,Bax,p-AKT,total AKT,and PTEN were detected by Western blot.Results Compared with the normal control group,the expression of AFAP1-AS1 in feces of CRC patients significantly increased(P<0.01);Compared with NCM460 cells,the expression of AFAP1-AS1 in SW620 and HCT116 cells also significantly increased(P<0.01);si-AFAP1-AS1 significantly suppressed the cell viability of SW620 and HCT116 cells(P<0.01).Compared with NCM460 cells,si-AFAP1-AS1 up-regulated the levels of cleaved-caspase-3 and pro-apoptotic protein Bax(P<0.05),and down-regulated the level of anti-apoptotic protein Bcl-2 in SW620 and HCT116 cells(P<0.001).In addition,si-AFAP1-AS1 significantly decreased the level of p-AKT protein and increased the expression of PTEN in CRC cells(P<0.01).Conclusion The difference in the expression of lncRNA AFAP1-AS1 in the stool of normal and CRC patients is beneficial to the early diagnosis for CRC,the expression of lncRNA AFAP1-AS1 is up-regulated in CRC cells regulated by the PTEN/p-AKT signaling pathway.Therefore,lncRNA AFAP1-AS1 is expected to become a molecular target for early diagnosis of colorectal cancer.
作者
曾理
庄树彤
丁世华
陈冲
焦璐
孙大勇
ZENG Li;ZHUANG Shutong;DING Shihua;CHEN Chong;JIAO Lu;SUN Dayong(Department of Gastroenterology,The First Affiliated Hospital of Shenzhen University,The Second People′s Hospital of Shenzhen,Shenzhen,518035,China;Department of Gastrointestinal Surgery,The First Affiliated Hospital of Shenzhen University,The Second People′s Hospital of Shenzhen)
出处
《实用肿瘤学杂志》
CAS
2020年第1期11-16,共6页
Practical Oncology Journal
基金
深圳市卫生计生系统科研项目(编号:201601021
201601024)
