lncRNA MALAT1通过miR-211靶向调控PI3K/Akt信号通路对食管癌细胞凋亡及侵袭的影响

Study on the Effect of lncRNA MALAT1 Targeting PI3K/Akt Signal Pathway on Apoptosis and Invasion of Esophageal Cancer Cells Through miR-211 Targeting

[目的]探讨肺腺癌转移相关转录物1(metastasis-associated lung adenocaroma transcript 1,MALAT1)通过miR-211靶向调控磷脂酰肌醇-3-激酶(phosphoinositide 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)信号通路对食管癌(esophagus cancer,EC)细胞凋亡及侵袭的影响。[方法]选取2018年10月至2020年8月在焦作市人民医院肿瘤外科行EC根治术治疗的25例EC患者的癌组织和癌旁组织(癌周3 cm以上)。使用定量聚合酶链反应(quantitative polymerase chain reaction,q PCR)检测EC患者癌灶、相对癌旁组织和人食管癌细胞KYSE30、KYSE150、KYSE450和Ec109细胞中MALAT1的表达。依据转染不同将细胞分为NC组(阴性对照sh-NC)、sh-MALAT1组(转染shMALAT1)、miR-211 mimics组(转染miR-211 mimics)、sh-MALAT1+miR-211 inhibitor组(转染shMALAT1+miR-211 inhibitor)。使用EDU实验、Transwell、AnneinⅤ-FITC/PI双染流式细胞术检测各组细胞的增殖、侵袭和凋亡能力;使用荧光素酶报告分析荧光素酶活性;通过Western blot分析各组细胞中lncRNA MALAT1、miR-211、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)、基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)、血管内皮生长因子-A(vascular endothelial growth factor-A,VEGF-A)、PI3K/Akt的表达情况。[结果]与癌旁组织相比,lncRNA MALAT1在EC组织中明显高表达,且Ec109细胞系中lncRNA MALAT1的表达明显高于其他细胞系,差异具有统计学意义(P<0.05)。双荧光素酶报告分析表明,miR-211是lncRNA MALAT1潜在的靶向microRNAs。与NC组相比,sh-MALAT1组和miR-211 mimics组EDU阳性细胞率和侵袭细胞数均明显降低,凋亡细胞数明显增加;与sh-MALAT1组相比,sh-MALAT1+miR-211 inhibitor组EDU阳性细胞率和侵袭细胞数均明显升高,凋亡细胞数明显增加,差异有统计学意义(P<0.05)。与NC组相比,sh-MALAT1组和miR-211 mimics组的MMP-9、MMP-2、VEGF-A、PI3K和p-Akt表达明显降低;与sh-MALAT1组相比,sh-MALAT1+miR-211 inhibitor组的MMP-9、MMP-2、VEGF-A、PI3K和p-Akt表达明显增加,差异均有统计学意义(P<0.05)。[结论]lncRNA MALAT1可通过靶向miR-211,激活PI3K/Akt信号通路,促进MMP-9、MMP-2和VEGF-A的表达,从而促进细胞增殖和侵袭,抑制细胞凋亡。

[Objective]To explore the effect of lncRNA MALAT1 targeting PI3K/Akt signal pathway on apoptosis and invasion of esophageal cancer cells through miR-211 targeting.[Methods]Cancerous tissues and paracancerous tissues of 25 patients with EC who underwent EC radical resection in Tumor Surgery Department,Jiaozuo People’s Hospital from October 2018 to August 2020 were selected.qPCR was used to detect the expression of lncRNA MALAT1 in cancer foci and paracancerous tissues of EC patients,human esophageal cancer cells KYSE30,KYSE150,KYSE450 and Ec109 cells.According to different transfection,the cells were divided into NC group(negative control sh-NC),shMALAT1 group(transfection sh-MALAT1),miR-211 mimics group(transfection miR-211 mimics)and sh-MALAT1+miR-211 inhibitor group(transfection sh-MALAT1+miR-211 inhibitor).The proliferation,invasion and apoptosis of cells in each group were detected by EDU test,Transwell and AnneinⅤ-FITC/PI double staining flow cytometry,and luciferase activity was analyzed by luciferase report.The expressions of lncRNA MALAT1,miR-211,MMP-9,MMP-2,VEGF-A and PI3K/Akt were analyzed by Western blot.[Results]The expression of lncRNA MALAT1 in EC was significantly higher than that in paracancerous tissues,and the expression of lncRNA MALAT1 in Ec109 cell line was significantly higher than that in other cell lines.Double luciferase report analysis showed that miR-211 was a potential targeting microRNAs for lncRNA MALAT1.Compared with NC group,the rate of EDU positive cells and the number of invasive cells in sh-MALAT1 group and miR-211 mimics group were significantly lower and the number of apoptotic cells was significantly higher than that in sh-MALAT1 group.Compared with sh-MALAT1 group,the rate of EDU positive cells and invasive cells and the number of apoptotic cells in sh-MALAT1+miR-211 inhibitor group were significantly higher than those in sh-MALAT1+miR-211 inhibitor group.Compared with NC group,the expression of MMP-9,MMP-2,VEGF-A,PI3K and p-Akt in sh-MALAT1 group and miR-211 mimics group decreased significantly,while the expression of MMP-9,MMP-2,VEGF-A,PI3K and p-Akt in sh-MALAT1+miR-211 inhibitor group increased significantly compared with sh-MALAT1 group.[Conclusion]lncRNA MALAT1 can target miR-211,activate PI3K/Akt signal pathway and promote the expression of MMP-9,MMP-2 and VEGF-A,so as to promote cell proliferation,invasion and inhibit apoptosis.