普列克底物蛋白2/miR-196a信号轴介导肿瘤微环境中肺癌细胞的通讯机制研究

A study on communication mechanism of lung cancer cells in tumor microenvironment mediated by pleckstrin-2/miR-196a signal axis

背景与目的:阐明介导肿瘤相关成纤维细胞(cancer-associated fibroblasts,CAFs)与肿瘤细胞之间通讯的信号分子仍然是一个巨大的挑战,这些信号分子对癌症转移至关重要。本研究旨在探讨普列克底物蛋白2(pleckstrin-2,PLEK2)/miR-196a信号轴介导肿瘤微环境中肺癌细胞的通讯机制。方法:选择人肺腺癌细胞系H1299和人胚胎肺细胞MRC-5作为研究对象。用表达PLEK2的慢病毒(PLEK2)和载体对照(Vector)转染H1299细胞,并在转染24 h后分离外泌体(Vector_exo、PLEK2_exo)。采用miR-196a模拟物或抑制剂转染MRC-5细胞。通过蛋白质印迹法(Western blot)分析PLEK2和上皮-间充质转化(epithelial-mesenchymal transition,EMT)相关蛋白水平,采用聚合酶链反应(polymerase chain reaction,PCR)分析miR-196a表达,采用transwell实验测定细胞转移和侵袭能力。将6只雌性BALB/c-nu小鼠随机分为Vector组和PLEK2组,每组3只。通过尾静脉向各组小鼠注射转染Vector或PLEK2的H1299细胞。4周后,取出肺组织进行H-E染色和免疫组织化学染色分析α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)表达。所有动物实验均经长沙市第一医院(中南大学湘雅医学院附属长沙医院)伦理委员会批准(伦理编号为EI-2021-103)。结果:与Vector组相比,PLEK2组小鼠肺结节数和转移灶中α-SMA表达显著增加(P<0.001)。与Vector组相比,PLEK2组H1229细胞中miR-196a表达水平显著增加(P<0.05),并且PLEK2_exo中miR-196a表达水平显著高于Vector_exo(P<0.05)。与Vector_exo组相比,PLEK2_exo组MRC-5细胞中miR-196a、α-SMA和成纤维细胞活化蛋白(fibroblast activation protein,FAP)表达水平显著增加(P<0.05)。与阴性对照(negative control,NC)相比,miR-196a转染的MRC-5细胞中α-SMA和FAP表达水平显著增加(P<0.05)。相反,通过miR-196a抑制剂(si-miR-196a#1、si-miR-196a#)转染,α-SMA和FAP表达水平被显著抑制(P<0.05)。与NC-CM组相比,miR-196a-CM组H1299细胞的转移、侵袭细胞数和波形蛋白(vimentin)表达均显著增加(P<0.001),E-钙黏蛋白(E-cadherin)表达显著降低(P<0.001)。此外,与Vector_exo-CM组相比,PLEK2_exo-CM组H1299细胞的转移、侵袭细胞数和vimentin表达均显著增加(P<0.01),E-cadherin表达显著降低(P<0.001)。结论:PLEK2上调能够增强肺癌细胞来源的外泌体miR-196a水平,从而促进CAFs激活。激活的CAFs能够进一步增强肺癌细胞的侵袭能力。

Background and purpose:It is still a great challenge to clarify the signal molecules that mediate the communication between cancer-associated fibroblasts(CAFs)and tumor cells.These signal molecules are very important for cancer metastasis.The purpose of this study was to explore the communication mechanism of pleckstrin-2/miR-196a signal axis mediated by lung cancer cells in tumor microenvironment.Methods:Human lung adenocarcinoma cell line H1299 and human embryonic lung cell MRC-5 were selected as the research objects.H1299 cells were transfected with lentivirus(PLEK2)expressing PLEK2 and Vector control,and exosomes(Vector_exo,PLEK2_exo)were isolated after 24 h of transfection.MRC-5 cells were transfected with miR-196a mimetic or inhibitor.The expressions of PLEK2 and epithelial-mesenchymal transition(EMT)-related proteins were analyzed by Western blot.The expression of miR-196a was analyzed by polymerase chain reaction(PCR),and the metastasis and invasion ability of cells were determined by transwell assay.Six female BALB/c-nu mice were randomly divided into Vector group and PLEK2 group,with 3 mice in each group.Mice in each group were injected with H1299 cells transfected with Vector or PLEK2 through the tail vein.After 4 weeks,lung tissue was taken out for H-E staining and immunohistochemical staining to analyze the expression ofα-smooth muscle actin(α-SMA).All animal experiments were approved by the ethics committee of First Hospital of Changsha City(Changsha Hospital,Xiangya School of Medicine,Central South University)(ethics number:EI-2021-103).Results:Compared with the Vector group,the number of pulmonary metastatic nodules and the expression ofα-SMA in metastatic cancer in PLEK2 group increased significantly(P<0.001).Compared with Vector group,the expression level of miR-196a in H1229 cells in PLEK2 group increased significantly(P<0.05),and the expression level of miR-196a was significantly higher in PLEK2_exo than in Vector_exo(P<0.05).Compared with Vector_exo group,the expression levels of miR-196a,α-SMA and fibroblast activation protein(FAP)in MRC-5 cells in PLEK2_exo group increased significantly(P<0.05).Compared with the negative control(NC),the expression levels ofα-SMA and FAP in MRC-5 cells transfected with miR-196a increased significantly(P<0.05).On the contrary,by transfection with miR-196a inhibitors(si-miR-196a#1 and si-miR-196a#),the expression levels ofα-SMA and FAP were significantly inhibited(P<0.05).Compared with NC-CM group,the number of metastatic cells,invasive cells and the expression of vimentin in miR-196a-CM group increased significantly(P<0.001),and the expression of E-cadherin decreased significantly(P<0.001).In addition,compared with Vector_exo-CM group,PLEK2_exo-CM group had significant increase in number of metastatic and invasive cells and the expression of vimentin(P<0.01),and significant decrease in the expression of E-cadherin(P<0.001).Conclusion:Upregulation of PLEK2 can enhance the level of exosomes miR-196a derived from lung cancer cells,thereby promoting the activation of CAFs.The activated CAFs can further enhance the invasive ability of lung cancer cells.