SCRN1在口腔鳞状细胞癌肿瘤相关成纤维细胞中的表达及对HSC3细胞株生物学特性的影响

Expression of SCRN1 in oral squamous cell cancer-associated fibroblasts and its effect on biological characterization of HSC3 cell line

目的:探究胞吐作用相关蛋白(SCRN1)在口腔鳞状细胞癌(OSCC)肿瘤相关成纤维细胞(CAFs)中的表达及对CAFs分泌MMP2功能的影响,进一步研究CAFs对HSC3细胞株迁移、侵袭能力的影响。方法:通过免疫组织化学染色(IHC)检测SCRN1在OSCC组织及癌旁组织中的表达。原代培养、分离、纯化、验证正常成纤维细胞(NFs)和CAFs,用免疫细胞化学染色法(ICC)、蛋白免疫印迹(WB)、qRT-PCR检测NFs、CAFs中SCRN1的表达情况。CCK-8、Transwell实验检测NFs、CAFs及CAFs-shSCRN1增殖、迁移及侵袭能力。收集NFs、CAFs上清并用ELISA和明胶酶谱法检测基质金属蛋白酶2(MMP2)水平及活性,然后分别与HSC3细胞株共培养,探究两种上清共培养对HSC3细胞株迁移、侵袭能力的影响。沉默CAFs的SCRN1基因后,收集CAFs、CAFs-shSCRN1和CAFs-Vector上清并检测MMP2水平及活性,然后分别与HSC3共培养,探究三种上清培养条件对HSC3细胞株迁移、侵袭能力的影响。结果:SCRN1在OSCC组织中阳性表达率及表达水平较高。成功分离、培养、鉴定NFs和CAFs, CAFs较NFs表达更高水平的SCRN1,且其增殖、迁移、侵袭能力均较NFs强。沉默SCRN1能够降低CAFs增殖、迁移及侵袭能力。CAFs上清分泌更高水平和活性的MMP2,与HSC3细胞株共培养可明显促进HSC3细胞株迁移、侵袭能力。沉默SCRN1后的CAFs上清中MMP2水平和活性降低,其促进HSC3细胞株迁移、侵袭能力也明显减弱。结论:SCRN1可调控CAFs分泌MMP2,在肿瘤微环境中,促进HSC3细胞株迁移和侵袭能力。

Objective:To investigate the expression of secernin 1(SCRN1)in cancer-associated fibroblasts(CAFs)of oral squamous cell cancer(OSCC)and its effect on the secretion of MMP2,and to further study the effect of co-culture with CAFs on migration and invasion ability of HSC3 cells.Methods:The expression of SCRN1 in OSCC and adjacent tissues was detected by immunohistochemistry(IHC).Through primary cultivated,isolated,purified and identified normal fibroblasts(NFs)and CAFs,the SCRN1 expression was detected by immunocytochemistry(ICC),Western Blot(WB)and qRT-PCR.The proliferation,migration,and invasion capabilities of NFs,CAFs and CAFs-shSCRN1 were detected by CCK-8 and Transwell assays.The supernatant of NFs and CAFs was collected and the matrix metalloproteinase 2(MMP2)levels and activity were detected by ELISA and gelatin zymography.Then they were co-cultured with HSC3 cells respectively to explore the effects of the above two supernatant culture conditions on the migration and invasion ability of HSC3 cell line.After silencing the SCRN1 gene of CAFs,the supernatant of CAFs,CAFs-shSCRN1 and CAFs-Vector was collected and MMP2 level and activity were detected,and then the supernatant was co-cultured with HSC3 cells respectively,to investigate the effects of the three supernatant culture conditions on the migratory and invasive abilities of HSC3 cell line.Results:SCRN1 was highly expressed in OSCC tissue.NFs and CAFs were successfully isolated,cultured and identified.CAFs expressed higher levels of SCRN1 than NFs,and their proliferation,migration,and invasion abilities were stronger than those of NFs.Silencing of SCRN1 reduced proliferation,migration and invasion ability of CAFs.Co-culture with CAFs supernatant significantly promoted the migration and invasion ability of HSC3 cells and secreted higher levels and activity of MMP2.Co-culture with CAFs-shSCRN1 resulted in a markedly diminished ability to promote migration and invasion of the HSC3 cells,as well as a reduction in MMP2 levels and activity.Conclusion:SCRN1 can modulate the secretion of MMP2 by CAFs,which,in the tumor microenvironment,promotes the migration and invasion ability of HSC3 cells.