摘要
目的 miR-1297在胃癌进展中扮演癌基因的角色,其具体机制尚不明确。本研究旨在探讨miR-1297靶向调控星形细胞上调基因-1(astrocyte elevated gene-1,AEG-1)对胃癌细胞增殖和侵袭能力的影响。方法运用生物信息学软件预测miR-1297的下游靶基因,采用双荧光素酶实验检测miR-1297和AEG-1的靶向关系。将体外培养的MKN-45细胞分为mimics NC组(转染miR-1297 mimics阴性对照)、miR-1297 mimics组、inhibitor NC组(转染inhibitor mimics阴性对照)和miR-1297 inhibitor组,MTT法检测各组细胞增殖活力,Transwell小室实验检测细胞侵袭,RT-qPCR和蛋白质印迹分别检测各组细胞中AEG-1、基质金属蛋白酶9(matrix metallopeptidase 9,MMP-9)和E-钙黏蛋白(E-cadherin)mRNA和蛋白的表达。结果双荧光素酶实验结果显示,在野生型3′UTR中,空白对照组荧光素酶活力均值为0.98±0.01,mimics NC组为0.97±0.02,miR-1297 mimics组为0.56±0.03,组间均值差异有统计学意义,F=836.800,P<0.001;两两多重比较,mimics NC组与miR-1297 mimics组荧光素酶的活力值差异有统计学意义,P<0.001;说明miR-1297可以与AEG-1 3′UTR结合。miR-1297 mimics组细胞的增殖活力和侵袭能力较mimics NC组均降低,均P=0.001;而miR-1297 inhibitor组增殖活力和侵袭能力较inhibitor NC组均升高,均P<0.001。RT-qPCR结果表明,与mimics NC组相比,miR-1297 mimics组AEG-1(P=0.001)和MMP-9(P=0.002)mRNA表达降低,而E-cadherin mRNA表达(P=0.005)升高;与inhibitor NC组相比,miR-1297 inhibitor组AEG-1(P=0.001)和MMP-9(P=0.003)mRNA表达升高,而E-cadherin mRNA表达降低,P=0.012。蛋白质印迹结果表明,AEG-1、MMP-9和E-cadherin蛋白表达与mRNA表达一致。结论 miR-1297通过结合到AEG-1 mRNA的3′UTR促进AEG-1的降解,从而下调MMP-9和上调E-cadherin的活性,抑制胃癌细胞增殖和侵袭。
OBJECTIVE Previous studies had proved miR-1297 could function as oncogene in gastric cancer,but the underlying mechanism is still unclear.This study aimed to investigate the regulation of miR-1297 on AEG-1 and its effects on proliferation and invasion of gastric carcinoma cancer cells.METHODS The target gene of miR-1297 was predicted by bioinformatics tools.The luciferase reporter assay was used to verify the binding effect of miR-1297 on the 3 non-coding AEG-1.MKN-45 cells cultured in vitro were divided into mimics NC group(transfected negative control of miR-1297 mimics),miR-1297 mimics group(transfected miR-1297 mimics),inhibitor NC group(transfected negative control of miR-1297 inhibitor)and miR-1297 inhibitor group(transfected miR-1297 inhibitor).The effect of miR 1297 on cell growth and invasion were detected by MTT and Transwell assay.The expression of AEG-1,MMP-9 and E-cadherin mRNA was detected by RT-qPCR,and the expression of AEG-1,MMP-9 and E-cadherin protein was detected by Western blotting.RESULTS The results of the dual luciferase assay showed that the mean values of the three groups of luciferase in the wild type 3′UTR were 0.98±0.01 in the blank control group,0.97±0.02 in the mimics NC group,and 0.56±0.03 in the miR-1297 mimics group.The mean difference between the groups was statistically significant(F=836.800,P<0.001).There was a statistically significant difference in the activity of luciferase between the two groups of mimics NC group miR-1297 mimics group(P<0.001),indicating miR-1297 can be combined with AEG-1 3′UTR.The proliferation and migration ability of the cells in the miR-1297 mimics group were significantly lower than those in the mimics NC group(both P=0.001),while those in the miR-1297 inhibitor group were significantly higher than those in the inhibitor NC group(both P<0.001).RT-qPCR results showed that compared with the mimics NC group,the mRNA expression of AEG-1(P=0.001)and MMP-9(P=0.002)was decreased in miR-1297 mimics group,while E-cadherin mRNA expression was increased(P=0.005).Compared with the inhibitor NC group,the mRNA expression of AEG-1(P=0.001)and MMP-9(P=0.003)was increased in the miR-1297 inhibitor group,while E-cadherin mRNA expression was decreased(P=0.012).Western blot results showed that AEG-1,MMP-9 and E-cadherin protein expression was consistent with mRNA expression.CONCLUSION miR-1297 decreased the level of AEG-1 through targeting the 3′UTR of the AEG-1 mRNA to down-regulate the activity of MMP-9 and up-regulate the activity of E-cadherin,thus inhibiting the cell growth and invasion of the gastric carcinoma cancer cells.
作者
齐乐
潘英
李素文
吴秀伟
QI Lel;PAN Ying;LI Su-iven;WU Xiu-wei(Department of Gastroenterology,Fourth Affiliated Hospital of Anhui Medical University,Hefei 230000,P.R.China;Department of Gastroenterology,Nanjing Pukou Hospital,Nanjing 210000 R.China;Department of Gastroenterology,First Affiliated Hospital of Anhui Medical University,Hefei 230000,P.R.China;Department of Oncology,Second Affiliated Hospital of Anhui Medical University,Hefei 230000,P.R.China)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2020年第2期93-98,共6页
Chinese Journal of Cancer Prevention and Treatment
