Exosomal MiR-141-3p Facilitates Transformation Of Nomal Fibroblasts To Carcinoma-associated Fibroblasts In Oral Cancer And Its Mechanism

[Purpose] To study the facilitating effect of OSCC exosomes in the transformation of normal fibroblast(NF)to carcinoma-associated fibroblasts(CAF).Then perform high-throughput sequencing on OSCC exosomal micro RNA and analyse the results.To explore the mechanism of miR-141-3p induced transformation of NF to CAF.[Methods] 1.OSCC cell line: CAL-27?HSC-3?SCC-4;Human normal oral epithelium cell line: HGEC.Isolate exosomes from cell culture supernatant and identify by Transmission Electron Microscope(TEM)?particle size analysis and western blot.OSCC tissues and paired para-carcinoma normal tissues were collected to isolate carcinoma-associated fibroblasts(CAF)and normal fibroblast(NF),and identification performed by immunofluorescence staining?Real-time fluorescent quantitative PCR and western bolt.Taking CAF as control,apply tumor exosomes with gradient protein concentration of 0?5?10?20mg/ml to NF,and detect NF protein expression and cell morphology changes by real-time fluorescence quantitative PCR,immunofluorescence staining and western blot.2.Perform high-throughput miRNA sequencing of OSCC cell and HGEC exosome and analyse the results,discovering the miR-141-3p that may potentially facilitate the transformation of NF to CAF by target PTEN.3.Transfect miR-141-3p mimics?inhibitor and paired negative control into NF,and detect NF cell migration by wound-healing and transwell culture.NF protein expression and cell morphology changes are detected by real-time fluorescence quantitative PCR? immunofluorescence staining and western blot.[Results] 1.High-purity exosomes were isolated by ultracentrifugation.The morphology was approximately ovoid with obvious double membrane under TEM.The diameter of exosomes was about 100 nm by particle size analysis.Exosome marker proteins TSG101 and CD9 were strongly positive by western blot.2.The OSCC tumor tissues and adjacent normal tissues were collected for isolation of paired CAF and NF.Immunofluorescence staining was used to mark ?-SMA.The expression of ?-SMA in CAF was significantly stronger than that in NF.Real-time fluorescence quantitative PCR and western blot were used to detect the expression of CAF markers,such as ?-SMA and FAP-?,and they were both found to possese higher expressions in CAF than NF respectively in transcription and protein levels.3.Apply OSCC cell exosomes on NF respectively according to 0,5,10 and 20 mg/ml protein concentration gradient.Western blot was used to inspect the CAF marker protein expression,finding that the expression of FAP-? in NF increased significantly with the increase of concentration of the tumor exosomes.These results indicated that OSCC exosomes could promote the transformation of NF to CAF,and this effect was positively correlated with exosome concentration.4.In order to explore the underlying mechanism of exosomes in promoting NF to CAF,we performed high-throughput sequencing on exosomes from OSCC cell lines HSC-3,CAL-27,SCC-4 and human oral mucosa normal epithelial cell lines HGEC,and performed bioinformatics analysis on the results to screen out that mir-141-3p may be involved in promoting NF transformation to CAF.Combined with the miRNA database,the possible target gene was analyzed to be PTEN.5.We performed transient transfection of mimics,inhibitor,and the corresponding negative control(NC)of miR-141-3p into NF.After mimics transfection,expression of FAP-???-SMA?PDGFR-?both improved in NF by real-time fluorescent quantitative PCR and western blot,indicating phenotype change of NF.Wound-healing and Transwell experiment found that mimics transfection promoted cell mobility and migration.Immunofluorescence staining indicated that the NF morphology was more similar to that of CAF,and the cell body was larger with more protrusions.On the contrary,these indexes of NF did not change significantly after inhibitor transfection.6.WB indicates that the target gene is PTEN is suppressed after mimics transfection,thus facilitating transition of NF to CAF,showing a significant increase of PDGFR-?.[Conclusion] OSCC cell exosomes possess effect of inducing transformation of NF to CAF,and the inducing effect partly depends on exosomal miR-141-3p.miR-141-3p targets PTEN m RNA to suppress translation after entering the NF.By binding to PTEN m RNA,miR-141-3p finally increase the expression of PDGFR-?,promoting the formation of CAF.