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

Abstract:

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.

Key words: Pleckstrin-2, Tumor microenvironment, Lung cancer cells, Cancer-associated fibroblasts