Expression Of SCRN1 In Oral Squamous Cell Carcinoma Associated Fibroblasts And Its Effect On HSC3

ObjectiveTo investigate the expression of secerninl(SCRN1)in oral squamous cell carcinoma(OSCC)associated fibroblasts(CAFs)and its effect on the biological characteristics of HSC3 cell line.Methods1.Primary fibroblasts(NFs)and oral squamous cell carcinoma associated fibroblasts(CAFs)were obtained by tissue culture,the cell morphology was observed,and the cells were identified by ICC method.CCK8 method and Transwell cell migration and invasion experiment were used to detect the proliferation,migration and invasion of NFs and CAFs cells.2.The expression differences of SCRN1 mRNA and protein in NFS and CAFs were detected by ICC,RT-qPCR and WB.The SCRN1 gene was silenced by lentivirus,and the successful transfection was verified by fluorescence microscope and WB;Then CCK8 method and Transwell cell migration and invasion experiment were used to verify the effect of silencing scrn1 on the growth,proliferation,migration and invasion of CAFs.3.Preparation of the conditioned medium of NFs、CAFs co-cultured with HSC3 cell line based on tranwell system to study the effects of NFs and CAFs on the migration and invasion of HSC3 cell line.At the same time,the concentration and enzyme activity of matrix metalloproteinase-2(MMP2)in the supernatant medium of NFs and CAFs were measured by ELISA kit and gelatin zymography.4.The supernatant of CAFs medium silencing scn1 was collected to detect whether the concentration and activity of MMP2 changed.At the same time,the medium was co-cultured with HSC3 cell line to explore whether the silencing of SCRN1 gene in CAFs would affect its secretory function,and then affect the migration and invasion ability of HSC3 cell line.Results1.NFs and CAFs were successfully isolated,purified and identified.Vimentin was strongly positive,cytokeratin(CK)was negative,and it was found in NFs cells a-Smooth muscle actin(a-SMA)weak positive expression;CAFs cell α-SMA was strongly positive.2.The results of CCK8 and Transwell migration and invasion experiments showed that the proliferation,migration and invasion of CAFs were stronger than those of NFs(P<0.05).3.The results of ICC,RT-qPCR and WB showed that the expression of protein and mRNA of SCRN1 in CAFs was higher than NFs(P<0.05).4.CAFs transfected with lentivirus silenced the expression of SCRN1,and its growth,proliferation,migration and invasion ability were decreased significantly compared with CAFs in empty group and non-transfected group,and the difference was statistically significant(P<0.05).5.Transwell co cultured NFs and CAFs with HSC3 cell line respectively.The results showed that CAFs could promote the migration and invasion of HSC3 cell line;When CAFs silenced the expression of SCRN1 gene,its promoting effect was significantly weakened;At the same time,the concentration and enzyme activity of MMP2 in the supernatant of co culture medium were detected.CAFs group was higher than NFs group,and CAFs-shSCRN1 group was significantly lower than CAFs-Vector group(P<0.05).Conclusions1.There are significant differences in the biological behaviors of growth,proliferation,migration and invasion between NFs and CAFs.The biological characteristics of oral squamous cell carcinoma related fibroblasts are significantly different from normal fibroblasts,which makes them have a stronger cancer promoting effect.2.The expression of SCRN1 in CAFs was higher than that in NFs,and the growth,proliferation,migration and invasion of CAFs decreased significantly after silencing the expression of SCRN1 gene.3.Silencing the expression of SCRN1 gene in CAFs will reduce its ability to paracrine MMP2,and then inhibit the migration and invasion of HSC3 cell line.