| Background:carcinoma-associated fibroblasts(CAFs) share a phenotype of fibroblasts whose origin is still unknown.CAFs are located in tumor stroma with vivacious biological behavior. It has always been a hotspot in research field to understand the connection between CAFs and tumorigenesis. Currently, CAFs targeted drugs have been used in clinical testing stage.Hyaluronan synthase2(HAS-2) exsists in vertebrate extensively and is one of three HAS which can catalyze the synthesis of hyaluronicacid (HA).HAS-2is closely related to embryonic development and diseases such as tumor and arthritis. Our experiment explored the role whichHAS-2played in OSCC on the perspective of CAFs with the hope of providing new clues to targeted drug therapy in OSCC.Part One:The analysis of HAS-2expression correlated with clinicopathological parameters in OSCC patientsObjective:To investigate HAS-2expression in paraffin embedding tissue of OSCC patients, and explore the relationship between HAS-2expression and clinicopathological parameters anda-SMAMethods:OSCC patients did not receive Chemotherapy or radiotherapy before surgery.48tumor tissue and16pair-matched normal mucosa from negative incisal margin were collected.Then paraffin embedding tissues were conventionally cut into5μm thick slices following by immunohistochemical staining.HAS-2and a-SMA expression in tumor tissue or normal tissue were scored according to distribution density. Finally, dates were analysed through statistical method. Results:The staining positive rate of HAS-2in tumor tissue was different from normal counterpart. Its positive rate in tumor cells was93.8%(45/48),52.8%(25/48) in tumor stromal tissue. While93.8%(15/16)in epithelial cells from oral mucosa,12.5%(2/16) in connective tissue.HAS-2expression in tumor stroml was significantly correlated with clinical stages (p=0.02),lymph node state (p=0.02) and a-SMA positive CAFs (p<0.01)Conclusion:HAS-2plays a pivotal role in tumor progression,metastasis and tumor-stromal activation. Part Two:The effect of HAS-2level in CAFs on in vitro biololgical characterisitics of Ca127Objective:To examine the role of CAFs in promoting the biological behavior of cell line Ca127from tongue squamous cell carcinoma,and to determine the impact of HAS2on the invasion of Ca127.Methods:CAFs and NFs were received from carcinoma and para-carcinoma tissue of the same tongue cancer patient respectively. The cell phenotype was verified by immunocytochemistry and Western blot.MTT assay,indirect coculture model and Transwell assays were used to compare the role of CAFs with NFs in promoting tumor cells proliferation,invasion and EMT. The expression of HAS in fibroblasts was measured by Western blot and real-time RT-PCR. The effect of drug4-MU on CAFs and tumor cells was observed. HAS2was knocked down in CAFs by siRNA and the invasiveness of Ca127in HAS2-interferenced group and control group was surveyed by transwell assays.Results:α-SMA was expressed in CAFs but not NFs.CAFs enhanced the proliferation,EMT and invasion of ca127as compared to NFs. The level of HAS2expression in CAFs was6times higher than that in NFs,which determined by real-time RT-PCR (P<0.01).At the concentration of0.4mM,4-MU reduced HAS2and HAS3expression in CAFS without changing cell proliferation. The promoting role on tumor cells of CAFS was largely inhibited by4-MU. Tumor cells cocultured indirectly with NFs experienced EMT while increasing HAS2expression was detected in NFs.The invasiveness of Ca127was largely decreased by down-regulation of HAS2in CAFs in contrast to control group (P<0.01)Conclusion:CAFs promoted the proliferation and invasion of oral tumor cells and induced EMTin vitro,mostly through expressing higher level of HAS2. inhibition of HAS2in CAFs may be a new therapy for anti-tumor invasion and metastasis. |
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