MiR-188-5p From CAFs-derived Exosomes Promotes OSCC Cell Migration And Invasion Through PTEN

ObjectiveOral squamous cell carcinoma(OSCC),with high malignant degree and low 5-year survival rate,is one of the most common types of malignant tumors in Head and Neck.Metastasis is one of the main reasons of poor prognosis of OSCC.Therefore,the prevention and treatment of OSCC metastasis are urgent issues to be solved in clinical and basic research.Previous studies on the mechanism of OSCC metastasis mainly focuses on the migration and invasion of OSCC cells.Studies have shown that not only the genetic changes in tumor cells,but also the local tumor microenvironment is associated with the occurrence,development and metastasis of tumors.Cancer-associated fibroblast(CAF)is one of the most important interstitial cell types in the tumor microenvironment.CAFs are closely responsible for the development and metastasis of tumors.PTEN is an important tumor suppressor gene,which is involved in the proliferation,apoptosis,migration and invasion of cancer cells.This study aims to investigate the mechanism of CAFs regulating PTEN expression in OSCC cells and to illustrate the role of CAFs in migration and invasion of OSCC cells.Methods1.Revealing the role of CAFs in CAL-27 cell migration and invasionA-Smooth muscle actin(a-SMA)was used to identify the CAFs in OSCC.CAL-27 cells were co-cultured with CAFs or fibroblasts islated from normal adjacent tissues(NFs),and transwell assay was used to evaluate the cell migration and invasion capacity of CAL-27 cells.2.Demonstrating the mechanism of CAFs regulating CAL-27 cell migration and invasionExosomes were extracted from CAFs and NFs,and these two exosomes were added to the culturing medium of CAL-27 cells.Then transwell assay was used to evaluate the cell migration and invasion capacity of CAL-27 cells.The expression of miR-382-5p in exosomes was detected with quantatitive real-time PCR(qRT-PCR).CAL-27 cells were co-cultured with CAFs with or without miR-382-5p knockdown,and cell migration and invasion were measured using transwell assay.MiR-382-5p were labeled with FITC and transfected into CAFs;and CAFs were co-cultured with CAL-27 cells to verify whether miR-382-5p can be transferred from CAFs to CAL-27 cells.3.Illustrating the mechanism of miR-382-5p regulating CAL-27 cell migration and invasionMiR-382-5p inhibitors were transfected into CAL-27 cells and PTEN expression was measured using Western blot analysis and qRT-PCR.MiR-382-5p inhibitors were transfected into CAL-27 cells with or without PTEN knockdown,and cell migration and invasion were measured using transwell assay.PTEN 3’-UTR activity reportor(pmir-PTEN)was constructed to verify whether miR-382-5p regulating PTEN through the putative binding site in PTEN 3’-UTR.ResultsFibroblasts were isolated from tumor tissue of OSCC and the corresponding adjacent normal tissue,and CAFs were identified according to the positive a-SMA expression.CAL-27 cells co-cultured with CAFs show high capacity of migration and invasion compared with the cells co-cultured with NFs.The exosomes separated from CAFs and NFs were used to culture with CAL-27 cells respectively.Transwell experiment shows that the CAL-27 cells cultured with exosomes from CAFs process stronger migration and invasion ability than those cultured with exosomes from NFs.RNA was extracted from CAFs and NFs-derived exosomes.QRT-PCR assay shows that the expression of miR-382-5p in CAFs-exosomes was significantly higher than that in NFs-exosomes.CAL-27 cells were co-cultured with CAFs with or without miR-382-5p knockdown,and transwell experiment shows that the migration and invasion ability of CAL-27 cells decreased significantly after miR-382-5p knockdown.MiR-382-5p labeled with FITC fluorescence was transfected into CAFs and CAFs were co-cultured with CAL-27 cells.MicroRNA-382-5p could be transferred into CAL-27 cells,however,this transferring can be blocked by adding exosomes release inhibitors.MiR-382-5p inhibitors were transfected into CAL-27 cells to detect PTEN expression.QRT-PCR and Western blot experiments indicate that miR-382-5p inhibitors can significantly up-regulate PTEN mRNA and protein expression.Knockdown of PTEN can significantly reverse the decrease of cell migration and invasion induced by miR-382-5p inhibitors.PTEN 3’-UTR activity reporter was constructed and the putative binding site of miR-382-5p on the reporter was mutated.The activity of PTEN 3’-UTR reporter can be significantly down-regulated by miR-382-5p,while this down-regulation of the activity of PTEN 3’-UTR reporter by miR-382-5p can be blocked by mutation of the binding site of miR-382-5p on the reporter.ConclusionIn this study,we found that the CAFs exosomes of oral cancer can transfer miR-382-5p into oral cancer cells.MiR-382-5p inhibits the expression of PTEN in oral cancer cells,thus affecting the migration and invasion of OSCC cells.This study reveals a new mechanism of CAFs regulating the migration and invasion of OSCC cells in tumor microenvironment,and provides a theoretical basis for targeting CAFs to develop anti-oral cancer drugs.