The Biological Effect Of Exosomes Derived From Cal27 On Normal Oral Mucosa Fibroblasts

Background: Oral cancer is a general term for malignant tumors occurring in the oral mucosa,characterized by rapid progress,extensive invasion and poor prognosis,and the 5-year survival rate is only 50%~55%.The occurrence and development of tumors are not determined unilaterally by the epithelium or the stroma,but by the balance state of the tumor-host interface microenvironment.Cancer-associated fibroblasts(CAFs)are one of the main host cells in the tumor microecology,which promote the invasion and metastasis of oral cancer.But the origins of CAFs remain controversial.Studies have shown that tumor cell regulation causes fibroblasts to be activated into CAFs,but the mechanism is unclear.Recent studies have shown that tumor cells secrete exosomes which contain a large amount of protein RNA and lipids that promote tumor growth,invasion and metastasis.By releasing exosomes,cells affect the state and function of recipient cells.Studies on pancreatic cancer have shown that pancreatic cancer cells can secrete exosomes which deliver mir-155 to fibroblasts,inducing their conversion to CAFs.However,the role of exosomes derived from oral cancer cell in fibroblast activation has not been reported.Objectives: This study was to isolate the exosomes derived from Cal27(Cal27-exo)and co-cultured Cal27-exo with normal oral mucosal fibroblasts(NOMFs)to detect the uptake of Cal27-exo by NOMFs,and to study the proliferation capacity,migration capacity and the expression of related proteins of NOMFs co-cultured with Cal27-exo(NOMFs+Cal27-exo).The further aims are to investigate whether Cal27-exo can induce NOMFs activation and its effect on the biological behavior of NOMFs;and to investigate the role of NOMFs+Cal27-exo in the invasion and metastasis of oral cancer.Methods: 1.The primary culture method of NOMFs was explanting enzymaticdigestion,and then the transmission electron microscopy(TEM)and immunofluorescence staining(IF)were used to identify cell origin;2.Cal27-exo was extracted by ultracentrifugation,and it was identified by Western blotting,transmission electron microscopy and particle size detection;3.Cal27-exo was labeled with PKH67 and co-cultured with NOMFs to identify it's uptake of Cal27-exo;4.The proliferation activity of NOMFs+Cal27-exo was detected using CCK8;5.The migration ability of NOMFs+Cal27-exo was evaluated using a wound healing test;6.Quantitative Real-Time PCR was used to evaluate the expression of NOMFs+Cal27-exo related proteins.Results: 1.NOMFs was successfully cultured by explanting enzymatic digestion,and light microscope showed the cells were long flat spindle shaped,with single oval nucleus which located in the center of the cytoplasm.The cells are arranged in a spiral pattern,and it has contact inhibition.Transmission electron microscopy(TEM)showed the NOMFs has large oval nucleus with no obvious sag,the intracytoplasmic organelles were relatively developed,which riched in rough endoplasmic reticulum,ribosome and Golgi bodies.Immunofluorescence staining showed anti-vimentin staining was positive and anti-keratin staining was negative,that proved the cells originated from mesenchymal.2.Cal27-exo could be extracted by ultracentrifugation.Western blotting showed that the expression of Alix,TSG101 and CD63 were positive in both cells and exosomes.Transmission electron microscopy showed the exosome pellets presented a circular double layer membrane vesicle and the size diameter was between 70 and 120 nm.And nano particle size analysis demonstated that the particle size of Cal27-exo was between 30 and 150 nm.3.Cal27-exo labeled with PKH67 entered NOMFs successfully after co-culture.4.CCK8 results showed that compared with NOMFs,the proliferation activity of NOMFs+Cal27-exo was significantly enhanced after co-culture for 24 h,48h,72 h and 96h(P<0.05),with time-dependent.5.The results of wound healing test showed that compared with NOMFs,the migration ability of NOMFs+Cal27-exo was not significantly changed at 12 h after scratch(P>0.05),but it was enhanced at 18 h,24h,36 h and 48h(P<0.05),until the scratch was healed.6.Quantitative Real-Time PCR results showed that compared with NOMFs,mRNA expression of PDGFR-? ? VEGF and Tn-C were increased in NOMFs+Cal27-exo(P<0.05),and ?-SMA mRNA decreased(P<0.05),TGF-?mRNA increased(P>0.05),and FAP mRNA decreased(P>0.05),that indicated NOMFs was activated after co-culture with Cal27-exo.Conclusion: Normal oral mucosa fibroblasts were successfully cultured by explanting enzymatic digestion method;Cal27 can secrete abundant exosomes which can be separated by ultracentrifugation effectively;After co-culture,the proliferation and migration of NOMFs+Cal27-exo were significantly enhanced,and mRNA increased,all the results indicated that Cal27-exo could activate NOMFs.This suggests potential significance for tumor invasion and metastasis.