| BackgroundHypopharyngeal squamous cell carcinoma(HSCC)is an aggressive malignancy located in the lower pharynx,which occurs in the pyriform sinus,posterior wall of the hypopharynx and postcricoid region,accounting for 2%-6%of all head and neck carcinoma.For highly aggressive by invasion to adjacent structures and enlarged cervical lymph node,HSCC is one of the worst prognoses among head and neck carcinoma.Although the diagnosis and treatment of HSCC have made great progress,the prognosis and survival rate of patients with HSCC is still not very satisfactory.There is urgently need for a novel biomarker that could provide better prognosis and to be useful in guiding treatment.Ras-related C3 botulinum toxin substrate 1(RAC1)is a member of the Rho family small GTPases,the total length is 29kb,including 7 exons,located on the human chromosome 7p22,It cycles between the GTP-and GDP-bound forms.RAC1 is activated by guanine nucleotide exchange factors through promoting the exchange of GDP to GTP and inactivated by GTPase activating proteins by accelerating GTP hydrolysis and plays an important role in cytoskeleton reorganization,migration,invasion,proliferation,cell division,apoptosis,reactive oxygen species production and inflammatory responses.However,deregulation of RAC1 is also involved in a number of pathological conditions,including cancer,cardiovascular diseases,neurodegenerative disorders,pathological inflammatory responses,kidney disorders and infectious diseases.Currently,RAC1 has been widely explored in many cancers,and as a biomarker to predict prognosis,RAC1 plays a crucial role in mediating tumor tumorigenesis and metastatic via many signal pathways and was associated with chemo-radioresistance of the tumor.Therefore,RAC1 and molecules involved in its signaling pathway can provide multiple effective targets for tumor therapy.But the expression and mechanism of RAC 1 in HSCC is still little known.In this study,we first explore the expression of RAC1 in HSCC,investigate its associations with clinicopathological parameters and analyze its prognostic value.Further investigation the effects of RAC 1 downregulation on cell biological behavior was analyzed by RNA interfering technology.In addition,the model of HSCC xenograft nude mice was established to reveal the effects of RAC 1 downregulation on the growth of HSCC xenograft nude mice.Finally,the mechanism of RAC1 in HSCC was to explore.Part I Expression of RAC1 in HSCC tissues and its clinical significanceObjectiveTo investigate the expression of RAC1 in HSCC and paracancerous tissues,investigate its associations with clinicopathological parameters and analyze its prognostic value,preliminarily detect its potential role in HSCC.Methods1.Quantitative real-time PCR(qRT-PCR)and Western blot were performed to examine the expression of RAC1 mRNA and protein in HSCC and paracancerous tissues.Immunohistochemical(IHC)was employed to assess RAC1 expression in 93 cases paraffin-embedded archived HSCC samples.2.Analyze the association between RAC1 expression and clinicopathologic factors,the prognostic value of RAC 1 in HSCC patients was researched by Kaplan-Meier survival curve and prognostic factors were detected by Cox proportional hazards model.Results1.The results of qRT-PCR and Western blot showed that overexpression of RAC1 mRNA and protein in HSCC tissues(P<0.05).2.RAC1 expression was significantly associated with T classification,lymph node metastasis,clinical stage,and disease recurrent in HSCC patients(P<0.05).3.Kaplan-Meier curve revealed that high RAC1 expression was related to worse OS and DFS(P<0.05).4.The multivariate analysis identified RAC1 overexpression as an independent prognostic factor for OS(P<0.05),poor differentiation and RAC1 overexpression as an independent prognostic factor for DFS(P<0.05).Part II Effects of RAC1 on proliferation and invasion in HSCC cellsObjectiveTo investigate the effects of RAC1 on proliferation,cell cycle,apoptosis,migration and invasion in FaDu cells.Methods1.The potential biological role of(?)in HSCC was analyzed by GSEA enrichment.2.RAC1 shRNA and control shRNA were transfected into FaDu cells.3.Expression of RAC1 mRNA and protein were detected by qRT-PCR and Western blot before and after transfected.4.MTT and plate clone formation assay were used to assess the effect of RAC 1 downregulation on FaDu cell proliferation.5.The effects of RAC 1 downregulation on cell cycle of FaDu cells was investigated by flow cytometry,the expression of cell cycle-related protein was detected by Western blot.6.The effects of RAC 1 downregulation on apoptosis of FaDu cells were investigated by hoechst and flow cytometry,the expression of apoptosis-related protein was detected by Western blot.7.Transwell assay was used to assess the effect of RAC 1 downregulation on FaDu cell migration and invasion.8.The effects of RAC1 downregulation on EMT of FaDu cells was detected by Western blot.9.The effects of RAC1 downregulation on MMP-2,MMP-9 protein expression and activity were detected by Western blot and Gelatin Zymography.Results1.GSEA plot showing that RAC1 overexpression positively correlated with cell cycle and cell adhesion molecules cams(P<0.05).2.The expression of RAC 1 mRNA and protein were significantly decreased in shRAC1 group(P<0.01).3.The results of MTT assay showed that the OD number was significantly suppressed 24 hours later in the shRAC1 group(P<0.05).4.The colony formation number of the shRAC1 group was significantly reduced in the shRAC1 group(P<0.01).5.The flow cytometry result showed that G0/G1 phase cell number of the shRAC1 group was higher than Scramble group(P<0.01).6.The protein level of cyclin D1,cyclin B and cyclin E detected by Western blot in the shRAC1 group were lower than Scramble group(P<0.05).7.The hoechst assay and flow cytometry showed that apoptotic cells of the shRACl group were higher than Scramble group(P<0.01).8.The protein level of pro-caspase-3,pro-caspase-9,PARP and Bcl-2 detected by Western blot in the shRAC1 group were lower than Scramble group(P<0.01),the protein level of cleaved caspase-3,cleaved caspase-9,cleaved PARP,and Bax were higher than Scramble group(P<0.01).9.Transwell assay results showed that migration and invasion cells of the shRAC1 group were lower than Scramble group(P<0.01).10.The protein level of Vimentin detected by Western blot in shRAC1 group was lower than Scramble group(P<0.01),the protein level of N-cadherin and E-cadherin were higher than Scramble group(P<0.01).11.The protein level and activity of MMP-2,MMP-9 detected by Western blot and gelatin zymography in shRAC1 group were lower than Scramble group(P<0.05).Part III Effects of RAC1 on the xenograft of HSCC cell nude miceObjectiveTo observe the effects of RAC 1 on HSCC xenograft nude mice.Methods1.Animal groups:The 18 healthy nude mice were randomly divided into 3 groups.Blank,Scramble,and shRAC1 group cells were injected subcutaneously into axilla of mice.2.Mice and tumor bodies were observed.The longest diameters and shortest diameters of tumor bodies in each group were recorded every 3 days.3.Thirty days later,the mice were killed and the tumor bodies were obtained.4.HE staining was carried out to assess the histopathologic changes in each group.5.The expression of shRAC1 protein in xenograft tumor tissues was detected by Western blot.6.Apoptosis in xenograft tumor tissues was detected by TUNEL,IHC was employed to assess Ki67 and E-cadherin expression.Results1.Tumor nodules were observed at 9 days after injection,the average size of tumors in the shRAC1 group was significantly smaller than Scramble group after 12 days of injection(P<0.01).2.The weight of xenograft tumors from nude mice in the shRAC1 group was lower than Scramble group(P<0.01).3.HE staining showed more obvious necrotic cores and nucleolus shrunk in the shRAC1 group.4.The protein level of RAC 1 detected in xenograft tumor tissues by Western blot in shRACl group was lower than Scramble group(P<0.01).5.Downregulation of RAC 1 effect the Ki67,E-cadherin protein and apoptosis in xenograft tumor tissues.Part ? Mechanism of RAC1 effect proliferation and invasion in HSCCObjectiveTo investigate the mechanism of RAC 1 effect growth and invasion in HSCC.Methods1.Western blot was performed to examine the expression of p-p38,p38 protein in HSCC and paracancerous tissues.2.The effects of RAC 1 downregulation on p-p38 protein of FaDu cells was observed by immunofluorescence,p-p38 and p38 protein expression of FaDu cells were detected by Western blot.3.The effects of RAC1 downregulation on MKK3,p-MKK3 protein expression of FaDu cells were detected by Western blot.4.The effects of RAC1 downregulation on p-p38,p38,p-MKK3,MKK3 protein expression of xenograft tumor tissues were detected by Western blot.5.P38 activator was applied to shRACl cells,MTT and plate clone formation assay were used to assess the proliferation of FaDu cells and transwell assay was used to assess the migration and invasion of FaDu cells.Results1.The results of Western blot showed that overexpression of p-p38 protein in HSCC tissues(P<0.05).2.Immunofluorescence showed downregulation of RAC1 effect the expression of p-p38.The protein level of p-p38 and p-MKK3 of FaDu cells detected by Western blot in the shRAC1 group were lower than Scramble group(P<0.01).3.The protein level of p-p38 and p-MKK3 of xenograft tumor tissues detected by Western blot in the shRAC1 group were lower than Scramble group(P<0.01).4.The growth rate of shRAC1+activator group was higher than shRAC1 + control group(P<0.05).5.The colony number of shRACl+activator group was higher than shRAC1+control group(P<0.05).6.The migration and invasion cells detected by transwell assays of shRAC1+activator group were higher than shRACl+control group(P<0.05).Conclusions1.High RAC1 expression in HSCC tissues is tightly associated with tumor size,lymph node metastasis,clinical stage,disease recurrent and prognosis,and RAC 1 overexpression as an independent prognostic factor for OS and DFS.2.RAC1 downregulation induce cell cycle distribution and apoptosis to suppress cell proliferation,this is correlated with cell cycle-related protein and apoptosis-related protein.3.RAC 1 downregulation suppressed cell migration and invasion,this is closely correlated with EMT and MMP-2,MMP-9.4.RAC1 downregulation suppressed the growth of xenograft of FaDu nude mice.5.P38 was activated in HSCC tissues,the machanism of proliferation and invasion mediated by RAC 1 partly achieved via p38 MAPK signaling pathway. |
PDF Full Text Request