lncRNA RPPH1通过调控miR-326/TCF4信号通路影响胃癌细胞增殖和迁移的机制研究

Mechanism of lncRNA RPPH1 influencing proliferation and migration of gastric cancer cells through regulation of miR-326/TCF4 signaling pathway

目的探究长链非编码RNA(lncRNA)RPPH1(下文简称RPPH1)对胃癌细胞增殖和迁移的影响及作用机制。方法采用实时荧光定量PCR(qRTPCR)法检测人胃癌细胞BGC-823、MGC-803、AGS、SGC-7901、MKN-45,以及人正常胃上皮细胞GES-1中RPPH1的表达水平。收集2019年6月至2021年8月在三亚市人民医院接受胃癌切除术的30例胃癌患者的肿瘤组织和癌旁正常组织标本,采用qRT-PCR法检测组织中RPPH1和miR-326的表达水平,采用蛋白质印迹法检测组织中TCF4的表达水平。采用双荧光素酶报告基因实验检测RPPH1与miR-326、miR-326与TCF4的靶向关系。应用GraphPad Prism 7.0软件分析RPPH1、miR-326和TCF4表达水平的相关性。将MGC-803细胞分为Control组、si-RPPH1组、si-RPPH1+miR-326 inhibitor组和si-RPPH1+miR-326 inhibitor+siTCF4组,采用CCK-8法检测各组细胞的增殖能力,采用Transwell小室法检测各组细胞的迁移和侵袭能力。结果RPPH1在人胃癌组织及人胃癌细胞BGC-823、MGC-803、AGS、SGC-7901和MKN-45中均呈高表达。双荧光素酶报告基因实验检测结果显示,miR-326是RPPH1的靶基因,TCF4是miR-326的靶基因。胃癌组织中RPPH1与miR-326的表达水平呈负相关(r=-0.504,P<0.001),RPPH1与TCF4的表达水平呈正相关(r=0.672,P<0.001),而miR-326与TCF4的表达水平呈负相关(r=-0.499,P<0.001)。与Control组比较,si-RPPH1组的细胞增殖能力、迁移能力和侵袭能力均显著减弱,TCF4的表达水平显著降低,差异均有统计学意义(P均<0.05);与si-RPPH1组比较,si-RPPH1+miR-326 inhibitor组的细胞增殖能力、迁移能力和侵袭能力均显著增强,TCF4的表达水平显著升高,差异均有统计学意义(P均<0.05);与si-RPPH1+miR-326 inhibitor组比较,si-RPPH1+miR-326 inhibitor+siTCF4组的细胞增殖能力、迁移能力和侵袭能力均显著减弱,TCF4的表达水平显著降低,差异均有统计学意义(P均<0.05)。结论RPPH1在胃癌组织和胃癌细胞中均呈高表达,并可能通过调控miR-326/TCF4信号通路影响胃癌细胞的增殖和迁移。

Objective This paper intends to investigate the effect and mechanism of lncRNA RPPH1 on the proliferation and migration of gastric cancer cells.Methods qRT-PCR was used to detect the expression level of RPPH1 in human gastric cancer cells BGC-823,MGC-803,AGS,SGC-7901,MKN-45,and normal gastric epithelial cells GES-1.The tumor tissue and paracancerous tissue samples of 30 patients with gastric cancer who underwent gastrectomy in Sanya People's Hospital from June 2019 to August 2021 were collected.The expression levels of RPPH1 and miR-326 were detected by qRT-PCR,and the expression level of TCF4 was detected by Western blotting.The targeting relationship between RPPH1 and miR-326,and between miR-326 and TCF4 were detected by double luciferase reporter gene assay.The correlation between the expression levels of RPPH1,miR-326,and TCF4 was analyzed by utilizing GraphPad Prism 7.0 software.MGC-803 cells were divided into the Control group,the si-RPPH1 group,the si-RPPH1+miR-326 inhibitor group,and the si-RPPH1+miR-326 inhibitor+siTCF4 group.The proliferation ability of each group of cells was detected by CCK-8,while the migration and invasion ability of each group of cells was detected by transwell.Results RPPH1 is highly expressed in human gastric cancer tissue and human gastric cancer cells BGC-823,MGC-803,AGS,SGC-7901,and MKN-45.The double luciferase reporter gene test results show that miR-326 is the target gene of RPPH1,and TCF4 is the target gene of miR-326.The expressions of RPPH1 and miR-326 in gastric cancer tissue are negatively correlated(r=-0.504 and P<0.001),RPPH1 is positively correlated with TCF4(r=0.672 and P<0.001),and miR-326 is negatively correlated with TCF4(r=-0.499 and P<0.001).Compared with the Control group,the cell proliferation,migration,and invasion abilities of the si-RPPH1 group are reduced,and the expression level of TCF4 is decreased,with statistically significant differences(P<0.05).Compared with the si-RPPH1 group,the cell proliferation,migration,and invasion abilities of the si-RPPH1+miR-326 inhibitor group are enhanced,and the expression level of TCF4 is increased,with statistically significant differences(P<0.05).Compared with the si-RPPH1+miR-326 inhibitor group,the cell proliferation,migration,and invasion abilities of the si-RPPH1+miR-326 inhibitor+siTCF4 group are reduced,and the expression level of TCF4 is decreased,with statistically significant differences(P<0.05).Conclusion RPPH1 is highly expressed in both gastric cancer tissues and gastric cancer cells and may affect the proliferation and migration of gastric cancer cells through regulation of miR-326/TCF4 signaling pathway.