| Background: Lung adenocarcinoma often has extensive metastases at the time of consultation.Although significant progress has been made in the treatment of lung adenocarcinoma,metastasis is still the main cause of death.Currently,the prognosis of lung adenocarcinoma patients is still poor.PFN1(profilin 1,PFN1)is an actin-binding protein.Studies have shown that PFN1 is involved in the regulation of tumor metastasis.However,PFN1 has different roles in different tumors and can promote or inhibit tumor metastasis.The roles of PFN1 in lung adenocarcinoma metastasis is not yet clear,and the mechanism by which PFN1 promotes or inhibits metastasis is not the same in different tumors.Studies have confirmed that PFN1 is an important regulatory protein for cell membrane transport.Microvesicles(MVs)are a type of extracellular vesicles that are produced by directly budding of plasma,and their production and secretion are closely related to cell membrane transporting.Numerous studies have confirmed that MV involved in the regulation of tumor metastasis.In this study,we explored the roles of PFN1 and MV in lung adenocarcinoma metastasis,and deeply studied the mechanism of PFN1 in promoting lung adenocarcinoma metastasis.Methods: The expression of PFN1 in lung adenocarcinoma was detected by tissue microarray immunohistochemistry.We established PFN1 overexpression lung adenocarcinoma cell lines by lentiviral transfection.Knocking down of the expression of PFN1 was achieved by transfection with PFN1 si RNA.Wound healing assay and Transwell migration assay were used to evaluate the changes of cell migration abilities.Proteomic analysis was used to analyze the difference of protein expression between PFN1 overexpressing cells and EV(empty vector)cells.MVs were extracted from sera and cell supernatants using sequential differential centrifugation,and the extracted MVs were qualitatively and quantitatively analyzed using western blotting,electron microscopy and flow cytometry.PKH67 was used to label MVs to detect its uptake by cells.Protein expression was determined by western blot and m RNA expression was determined by real-time quantitative PCR(RTq PCR).Protein interaction was detected by protein coimmunoprecipitation.Rho kinase(ROCK)activity was evaluated by ROCK kinase kit according to the manufacturer’s instructions.ROCK inhibitor Y27632 was used reverse effects of PFN1 on lung adenocarcinoma metastasis and secretion of MVs.In vivo metastatic tumor model was established by intracardiac injection of H1299 cells.And intraperitoneal injection of Y27632 reversed the pro-metastatic effect of PFN1 in vivo.Results: PFN1 is highly expressed in lung adenocarcinoma tissues.In vitro experiments,the cell migration ability was enhanced after PFN1 overexpression and impaired after PFN1 knockdown.The same results were obtained in vivo experiments.Analysis of proteomic data found that the differential proteins were mainly involved in cell membrane transport,vesicle transport and signal transduction.Further research found that the number of MVs in the sera of patients with metastatic lung adenocarcinoma was significantly higher than that of MVs isolated from the sera of patients with non-metastatic lung adenocarcinoma.It was found that the amount of MVs secreted by cells increased after PFN1 overexpression,and the expression of PFN1 and the key protein of MV secretion-phosphorylated myosin light chain(p-MLC),was significantly positively correlated.Cell experiments found that PFN1 overexpressed cell lines derived MVs significantly promoted the migration of lung adenocarcinoma cells,and the same results were obtained in the in vivo experiments.Further mechanism studies found that PFN1 could interact with ROCK1.ROCK kinase assay found that PFN1 could enhance the kinase activity of ROCK1,which ultimately led to the enhanced phosphorylation of MLC,thereby promoting the secretion of MV and the migration of lung adenocarcinoma.Both in vivo and in vitro experiments revealed that inhibition of ROCK1 activity by Y27632 partially reversed the metastatic enhancement induced by PFN1.Conclusions: Our study revealed that PFN1 acts as a metastasispromoting protein by enhancing MV secretion.These new insights suggest that PFN1 is a potential therapeutic target for lung adenocarcinoma metastasis. |
