miR-17-92基因簇在胃癌组织中的表达和临床意义

Expression and Clinical Significance of the miR-17-92 Cluster in Gastric Cancer

目的研究miR-17-92基因簇在人胃癌组织中的表达情况和临床意义,并探讨过表达miR-17-92对胃癌细胞生物学特性的影响及潜在机制。方法通过实时荧光定量PCR(qRT-PCR)方法检测miR-17-92基因簇各亚单位在胃癌组织及其对应的癌旁正常组织中的表达差异,并分析其表达水平与胃癌患者临床病理特征的相关性。运用含过表达miR-17-92质粒的慢病毒上清感染MGC-803细胞,并用嘌呤霉素筛选出单克隆细胞,进一步扩增得到稳定过表达miR-17-92的MGC-803胃癌细胞株。采用xCelligence系统实时动态监测MGC-803-miR-17-92胃癌细胞及其对照组细胞的生长情况及侵袭能力。运用Western blot法检测两组细胞中AKT、ERK1/2信号通路相关分子及Integrinβ1蛋白的表达。通过明胶酶谱实验检测两组细胞上清培养液中MMP-2和MMP-9的活性。结果miR-17-92基因簇各亚单位在胃癌组织中的表达均较癌旁正常组织显著升高。miR-17-92基因簇各亚单位的表达水平均与胃癌患者的TNM分期呈正相关,其中miR-17、miR-20的表达水平与胃癌患者的肿瘤分化程度呈正相关,miR-19a、miR-19b、miR-92的表达水平与胃癌患者的肿瘤大小呈正相关。成功构建了稳定过表达miR-17-92的MGC-803胃癌细胞株。MGC-803-miR-17-92细胞的生长速度明显快于MGC-803-control细胞。两组细胞中总的AKT蛋白表达水平差异无统计学意义,然而MGC-803-miR-17-92细胞中AKT蛋白在苏氨酸308位点的磷酸化水平较MGC-803-control细胞显著升高,而其在丝氨酸473位点的磷酸化水平在两组细胞中差异无统计学意义。两组细胞中总的ERK1/2蛋白表达水平差异无统计学意义,然而MGC-803-miR-17-92细胞中磷酸化的ERK1/2蛋白表达显著高于对照组MGC-803-control细胞。MGC-803-miR-17-92细胞的侵袭能力显著强于MGC-803-control细胞。MGC-803-miR-17-92细胞中Integrinβ1的蛋白表达水平及MMP-2的活性显著高于MGC-803-control细胞。结论miR-17-92基因簇在人胃癌组织中的表达水平较癌旁正常组织显著升高,并与胃癌患者的肿瘤大小、分化程度及TNM分期呈正相关。过表达miR-17-92能够显著加快MGC-803细胞的生长,该过程与AKT及ERK1/2信号通路的活化相关。过表达miR-17-92能够显著增强MGC-803细胞的侵袭能力,其机制与Integrinβ1的表达上调及MMP-2的活性增强有关。

Objective To determine the expression pattern of the miR-17-92 cluster in human gastric cancer tissues and observe the relationship between their expressions and its clinicopathological features of gastric cancer patients,and to investigate the biological functions and its mechanisms of the miR-17-92 cluster in human gastric cancer cells.Methods qRT-PCR assay was used to detect the expression of the miR-17-92 cluster in human gastric cancer tissues.Chi-square test was used to analyze the relationship between the miR-17-92 expression and clinicopathological features of gastric cancer patients.Lentivirus transfection was used to establish stably miR-17-92 overexpressing MGC-803 gastric cancer cell lines.Cell growth and invasion abilities of the MGC803-miR-17-92 cells and the MGC-803-control cells were monitored by a real-time xCELLigence system.The protein expression levels of AKT and ERK1/2 pathways between the two established cell lines were measured by Western blot analyses.The expression of Integrinβ1 was detected by Western blot,and the activities of MMP2 and MMP-9 were measured by gelatin zymography experiment.Results The expressions of each subunits of the miR-17-92 cluster in gastric cancer tissues were significantly up-regulated compared with the corresponding adjacent normal tissues.The expression levels of each subunits of the miR-17-92 cluster were positively correlated with TNM stage of gastric cancer patients,while miR-17 and miR-20 were also positively correlated with tumor differentiation,and miR-19a,miR-19b and miR-92 were also positively correlated with tumor size of gastric cancer patients.The MGC-803 gastric cancer cell line stably overexpressing the miR-17-92 cluster was successfully established.miR-17-92 overexpression accelerated cell growth of the MGC-803 cells.Western blotting analyses showed that the expressions of p-AKT(Thr308)and p-ERK1/2 were markedly increased in the MGC-803-miR-17-92 cells compared with those in the MGC-803-control cells,while the total expressions of AKT and ERK1/2 were not affected by the overexpression of miR-17-92.miR-17-92 overexpression significantly promoted cellular invasion abilities of the MGC-803 cells.The protein expression levels of Integrinβ1 and the MMP-2 activities in MGC-803-miR-17-92 cells were significantly increased than those in the MGC-803-control cells.Conclusion The expression of the miR-17-92 cluster in gastric cancer tissues are significantly up-regulated compared with the corresponding adjacent normal tissues,which are positively correlated with tumor size,tumor differentiation and TNM stages of gastric cancer patients.Overexpression of miR-17-92 significantly increases cell growth of the MGC-803 gastric cancer cells,which is associated with the activation of AKT and ERK1/2 signaling pathways.Overexpression of miR-17-92 significantly promotes the invasion abilities of the MGC-803 cells,owing to the up-regulated expression of Integrinβ1 and induced activity of MMP-2.