The Role And Mechanism Of CLIC1 In The Progression Of Oral Squamous Cell Carcinoma

Background and ObjectivesOral cancer is one of the most common malignant tumors in the head and neck.Including the cancer of tongue,cheek,gingiva,palate and jaws,etc.There is more than 90% of oral cancer is squamous cell carcinoma.At present,the incidence of oral cancer in China accounts for 1.5% ? 5.6% of all malignant tumors,while the five-year survival rate is only about 50%.Postoperative recurrence and cervical lymph node metastasis are the important factors affecting the prognosis of oral cancer.CLIC1,a member of the chloride channel protein family,is located at the human chromosome 6p21.3 locus in the form of soluble monomer proteins.Soluble dimer protein oligosaccharide membrane binding protein and membrane protein,CLIC1 plays an ion channel role in lipid bilayer.On the other hand,CLIC1,as a receptor and effector of oxidative stress,can be involved in the regulation of intracellular oxidation,which is related to neurodegenerative diseases(such as Alzheimer's disease).In addition,CLIC1 is related to the proliferation,metastasis and drug resistance of a variety of malignant tumors,and can be involved in tumor cell cycle regulation,apoptosis,cell invasion and other processes,and the role of CLIC1 in the process of different tumor diseases is not the same.In the previous study,we found that CLIC1 was highly expressed in pathological specimens and plasma of patients with OSCC,and the overexpression of CLIC1 was significantly correlated with histopathological grade,TNM stage,tumor size and survival rate.In order to further analyze the relationship between CLIC1 and the development of oral squamous cell carcinoma(OSCC),this study focused on the effect of CLIC1 on the biological behavior of OSCC cells in vitro,and discussed its potential molecular mechanism.Methods1.In this study,oral squamous cell carcinoma cell line(SCC-15)was transfected with lentivirus targeting CLIC1,and CLIC1 knockdown,overexpression and no-load SCC-15 cell lines were observed by antibiotic screening.Western blot and RT-PCR experiments were used to verify the transfection effect.CCK-8 assay(cell proliferation test/ cisplatin cytotoxicity test),colony formation test,flow cytometry(Alexa Fluor 647/PI double staining method),wound healing assay and Transwell invasion and migration assay and tube formation assay were performed to analyzed the effects of CLIC1 on the proliferation,survival,cisplatin sensitivity,apoptosis,migration and invasion of human oral squamous cell carcinoma cells and the ability to induce the tube formation of vascular endothelial cells.2.The subcutaneous transplanted tumor model of nude mice was established by subcutaneous injection of SCC-15 cells with different CLIC1 expression levels into the back of nude mice.The growth and size of transplanted tumor were observed and compared in order to analyze the effect of CLIC1 on the growth of oral squamous cell carcinoma(OSCC)in vivo.HE staining was used to determine the type of tumor and the lesion of liver and kidney.The expression levels of CLIC1,phosphorylated ERK1/2 and phosphorylated p38 protein in transplanted tumor tissues with different CLIC1 expression levels were detected by western blot.The expressions of CLIC1,phosphorylated ERK1/2,phosphorylated p38,MMP2,MMP9 and MMP13 in tumor tissues with different CLIC1 expression levels were detected by immunohistochemistry.3.The possible relationship between integrin protein Family(ITG?v,ITG?1),CLIC1 and MAPK1(ERK and p38)was analyzed by GeneMANIA.Western blot and RT-PCR experiments were performed to analyze the expression of ITG?v,ITG?1,caspase3,caspase9,MMP2,MMP9,E-cadherin,vitmentin and MAPK signaling pathway proteins ERK1/2,p38 and phosphorylated ERK1/2,p38 after the regulation of CLIC1 in the OSCC cells.Associated with the experimental results of cell apoptosis and migration,we could preliminary couclude the possible mechanism of Integrins/MAPK signaling pathway and CLIC1 in the regulation of apoptosis and migration of OSCC cells.Results1.Through cell immunofluorescence,Western blot and RT-PCR experiments,we found that the expression of CLIC1 in SCC-15 transfected cell lines was signifcant different.The subcellular localization of CLIC1 mainly includes nucleus,nuclear membrane,cytoplasm and cell membrane.Further experiments showed that after CLIC1 silencing,the abilities of proliferation,invasion,migration and angiogenesis of SCC-15 cells were significantly inhabited,and the level of apoptosis and drug sensitivity to cisplatin were promoted.On the contrary,the up-regulation of CLIC1 promoted the cell proliferation,invasion and migration and angiogenesis.2.The transfected SCC-15 cells with different CLIC1 expression were injected into the back of nude mice to construct the transplanted tumor models?The tumor volume increased over time.One month later,the tumor was dissected and identified as squamous cell carcinoma by HE staining,which means that the transplanted tumor animal model was successfully established.By comparing the growth rate,volume and weight of xenografts in nude mice,we found that CLIC1 overexpression promoted the growth of OSCC in vivo,but silenced CLIC1 inhibited its growth.The expression of CLIC1,p-ERK,p-p38,MMP2,MMP9 and MMP13 in tumor tissues was analyzed by immunohistochemistry and Western blot.We found that the expression of phosphorylated ERK was significantly decreased in CLIC1 knockdown groups,while the expression of p-p38 protein increased significantly,and the protein staining of phosphorylated ERK,MMP2,MMP9 and MMP13 was also lighter by immunohistochemical staining assay.However,the expression of p-ERK was significantly increased,the expression of p-p38 was decreased in CLIC1 overexpression group,and the staining of MMP2,MMP9 and MMP13 were significantly deepened in immunohistochemical staining.These results suggested that CLIC1 affect the growth of oral squamous cell carcinoma in vivo,and the expression of CLIC1 will affect the expression of phosphorylated ERK,phosphorylated p38,MMP2,MMP9 and MMP13.CLIC1 may be involved in the growth of oral squamous cell carcinoma in vivo by working on MAPK signaling pathway.3.GeneMANIA prediction showed that there was a gene correlation between integrin proteins ITG ? v,ITG ? 1,CLIC1 and MAPK.The results of Western blot and RT-PCR showed that after down-regulating the expression of CLIC1 in SCC-15 cells,the expression of ITG ? v and ITG ? 1 decreased,the expression of phosphorylated ERK also decreased,and the expression of EMT related protein MMP2,MMP9,vimentin decreased.The expression of E-cadherin,phosphorylated p38 and apoptosis-related protein caspase3,caspase9 increased.In contrast,the expression of ITG ? v and ITG ? 1 increased,the expression of phosphorylated ERK also increased,while the expression of downstream proteins MMP2,MMP9,vimentin increased and E-cadherin decreased in CLIC1 overexpression SCC-15 cells.The results of RT-PCR analysis of CLIC1,MMP2,MMP9,caspase3 and caspase9 showed that the similar trends of WB experiment.Associated with the phenomenon of invasion and migration assay and apoptosis experiment,we suggested that CLIC1 may be involved in the regulation of migration and apoptosis of OSCC through ITGs/ERK and ITGs/p38 signal pathways.Conclusions1.In vitro,CLIC1 can significantly promote the proliferation,invasion and migration of human OSCC cells and induce the ability of tube formation of vascular endothelial cells.Silening CLIC1 could promote the apoptosis and improve the sensitivity of OSCC cells to cisplatin.2.Animal experiments have confirmed that CLIC1 could promote the OSCC tumorigenesis.At the same time,the regulation of CLIC1 would affect the expreesion of phosphorylated EKR and p38 proteins and MMPs.CLIC1 may be involved in the growth of OSCC by interaction with MAPK signaling pathway.3.The upregualtion of CLIC1 promoted the expression of ITG?v and ITG?1,which triggered the activation of MAPK/ERK signaling pathway and promoted cell invasion and migration by regulating the expression of EMT related proteins(MMP2,MMP9,vimentin,E-cadherin).Similarly,the down-regulation of CLIC1 significantly decreased the expression of ITG?v and ITG?1,then activating the p38 MAPK signaling pathway and promoting the apoptosis of OSCC cells by increasing the expression of caspase3 and caspase9.