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 of α-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 of α-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 of α-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 of α-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.
关键词
普列克底物蛋白2/肿瘤微环境/肺癌细胞/肿瘤相关成纤维细胞
Key words
Pleckstrin-2/Tumor microenvironment/Lung cancer cells/Cancer-associated fibroblasts
维普
万方数据