长链非编码MEG3靶向miR-144表达对结肠癌细胞增殖转移潜能影响

Effect of long-chain non-coding MEG3 targeting miR-144 on the proliferation and metastasis of colon cancer cells

目的母系表达基因3(maternal expression gene 3,MEG3)在多种恶性肿瘤中表达异常,本研究评估MEG3和miR-144在结肠癌细胞中的表达状态,并进一步探讨MEG3与miR-144的相互作用,研究其与下游相关通路对结肠癌增殖和转移过程中的作用及其机制。方法收集2016-01-10-2017-01-10宁波市鄞州区第二医院手术切除并且经病理检查确诊为结肠癌组织80例,同时取配对癌旁正常结肠组织80例。人结肠癌细胞株HT-29、SW480、SW620及RKO细胞株购于上海细胞研究所。qPCR检测MEG3和miR-144在不同结肠癌细胞株中的表达情况以及MEG3在结肠癌组织和癌旁正常组织中的表达;分析MEG3和结肠癌患者的临床病理资料之间的关联;双荧光素酶报告基因检测MEG3与miR-144之间的相互作用;MTT增殖实验和Transwell侵袭实验检测抑制MEG3后结肠癌细胞的增殖和侵袭能力的变化情况,及抑制miR-144表达后对结肠癌细胞增殖和侵袭能力的恢复情况;蛋白质印迹法检测抑制MEG3后PTEN/AKT信号通路蛋白的表达情况。结果与其他结肠癌细胞株相比,SW620细胞中MEG3表达最低(0.52±0.08),t=9.968,P<0.001,miR-144的表达水平最高(1.25±0.11),t=9.648,P<0.05;结肠癌组织中MEG3的表达水平相比癌旁正常组织明显降低;MEG3的表达水平与结肠癌的病理分期相关以及淋巴结转移情况有关,随着分期越高,MEG3在结肠癌组织中表达越高,淋巴结转移的患者中MEG3的表达也相对较高;双荧光素酶实验证实MEG3能与miR-144的3′UTR特异性结合,可以调控miR-144的表达与活性;抑制MEG3的表达后可以促进结肠癌细胞的增殖能力(3.52±0.59),t=3.289,P<0.05和侵袭能力(321.52±23.24),t=15.900,P<0.001,差异有统计学意义;同时抑制miR-144的表达水平过后,结肠癌细胞的增殖(3.63±0.48,t=4.303,P<0.05)和侵袭能力(89.52±8.19,t=15.327,P<0.001)得到一定程度的恢复;抑制MEG3的表达后,PTEN/AKT信号通路被相应的激活,PTEN(1.19±0.15)%,t=10.954,P<0.001;AKT(1.29±0.18)%,t=8.881,P<0.001。结论 MEG3可以调控miR-144的表达通过PTEN/AKT信号通路影响结肠癌细胞的增殖和侵袭能力,为临床结肠癌诊治分子靶标研究提供一定的理论支持。

OBJECTIVE In this study,the expression of MEG3 and miR-144 in colon cancer cells was evaluated,and the interaction between MEG3 and miR-144 was further explored.The relationship between MEG3 and miR-144,the role of downstream related pathways in the proliferation and metastasis of colon cancer and its mechanism were studied.METHODS Eighty case pathologically confirmed colon cancer tissues and 80 case adjacent normal tissues were collected during operation in Anhui Medical University First Affiliated Hospital from 2016-01-10 to 2017-01-10.The expressions of MEG3 in colon cancer tissue and adjacent normal tissue were detected by qPCR.The association between clinical and pathological data of MEG3 and colon cancer patients were analyzed.Double luciferase reporter gene was used to detect the interaction between MEG3 and miR-144.MTT proliferation assay and Transwell invasion assay were used to detect the changes of proliferation and invasion ability of colon cancer cells after MEG3,and the inhibition of the colon cancer cell proliferation and invasion of the recovery after the inhibition of miR-144 expression.Western blotting was used to detect the expression of PTEN/AKT signaling pathway after MEG3.RESULTS Compared with other colon cancer cell lines,the expression of MEG3[（0.52±0.08）,t=9.968,P〈0.001]was the lowest and the expression level of miR-144 was the highest in SW620 cells[（1.25±0.11）,t=9.648,P〈0.05].The expression of MEG3 was related to the pathological stage of colon cancer and the lymph node metastasis.The higher the staging,the higher the expression of MEG3 in colon cancer.Double luciferase assay confirmed that MEG3 could specifically bind to the 3′UTR of miR-144 and regulate the expression and activity of miR-144.Inhibition of MEG3 expression can promote colon cancer cell proliferation[（3.52±0.59）,t=3.289,P〈0.05]and invasion[（321.52±23.24）,t=15.900,P〈0.001]ability.While the inhibition of miR-144 expression level,colon cancer cell proliferation[（3.63±0.48）,t=4.303,P〈0.05]and invasion ability [（89.52±8.19）,t=15.327,P〈0.001]had a certain degree of recovery;after inhibition of MEG3 expression,the PTEN/AKT signaling pathway was activated accordingly[PTEN（1.19±0.15）% ,t=10.954,P〈0.001;AKT（1.29±0.18）% ,t=8.881,P〈0.001].CONCLUSION MEG3 can regulate the expression of miR-144 and influence the proliferation and invasion of colon cancer cells through PTEN/AKT signaling pathway.