Molecular Mechanisms Study On Recombinant Plasmid-mediated RNA Interference CEMIP Gene Expression Effects On Human Colorectal Tumorigenesis Potentials

Background Colorectal cancer has been one of the most common malignancies and ranking third in China currently,seriously threatening human health [1].Although CRC has been considered to be related to the lifestyle of Western countries for a long time [2],the incidence of colorectal cancer in Asia has risen significantly in the past 10 years with the improvement of living standards and dietary patterns,a variety of complex molecular mechanisms leading to tumor metastasis and epithelial-mesenchymal transition(EMT)caused poor prognosis and death of patients[3];therefore it was necessary to find new biomarker and explore its molecular mechanism to guide colorectal cancer Individualized precise treatment?prognosis and the development of effective new targeted drugs.Cell migration-induced hyaluronic acid-binding protein(CEMIP)mediates the depolymerization of hyaluronic acid(HA)and was named CCSP1(colon cancer secretory protein 1)and KIAA1199 [4].It has been confirmed the abnormally expression in many malignant tumor cells such as gastric cancer,breast cancer and colon cancer.Endogenous decreased expression of CEMIP can induce cell cycle arrest or cell apoptosis in pancreatic ductal carcinoma cells and breast cancer by the interaction with glycogen phosphorylase kinase beta to regulate apoptosis in cancer cells [5-7].Studies showed that high CEMIP expression is positively correlated with poor clinical prognosis in colorectal cancer patients[8];E-cadherin bindintracellular conservative domainsof?-catenin protein [9].The E-cadherin/?-catenin complex is an essential complex for maintaining epithelial integrity [10].?-catenin is the main sensor in Wnt signaling,when Wnt signaling pathway is not activated,?-catenin was synthesized kinase-3 and casein phosphorylate kinase I by glycogen,which is ubiquitinated/degraded afterrecognition of ?-Tr CP(E3 ubiquitin A portion of the enzyme complex)[11].Stable ?-catenin enters the nucleus and forms the complex with T-cell factor(TCF)to drive transcription of the EMT initiation gene [12-14],therefore,CEMIP-induced tumor cell metastasis may be caused by EMT impaired recombinant epidermal growth factor;The Wnt/?-catenin signaling pathway also suggests its role on maintaining cell-cell junctions,its aberrant activation are necessary steps leading to EMT-mediated metastasis in colorectal cancer [15-16].The interaction of ER chaperonin GRP78 with CEMIP has been confirmed [17-18].GRP78-mediated unfolded protein response(UPR)may be involved in CEMIP-associated carcinogenesis,but the mechanism has not yet been elucidated.The mechanism of CEMIP gene action has not yet been fully revealedin colon cancer.How the genemediated the proliferation and metastasis of colon cancer cells by GRP78 and UPR,and whether it is regulated by Wnt/?-catenin/Snail signaling pathways are rarely reported in relevant studies.In this study,we designed and synthesized CEMIP-sh RNA plasmids,constructed the stable cell line with CEMIP down-regulation,and plan to systematically study the function and regulation of CEMIP in vitro and in vivo to reveal the role down-regulated CEMIP gene onbiological behavior and molecular mechanisms of colorectal cancer cells.1.Silencing of CEMIP suppresses Wnt/?-catenin/Snail signaling transduction and inhibits EMT program of colorectal cancer cellsPurpose: In the first section,our study is to explore the effection of silencing of CEMIP on colorectal cancer cells proliferation and metastasis,and elucidate its the underlying molecular mechanism in EMT process.Methods 1.CEMIP-sh RNA plasmid was designed and synthesized according to human homo-CEMIP sequence,SW480 cells and HCT116 cells were transfected with CEMIP sh RNA.2.RT-PCR and Western blotting were used to detect the CEMIP m RNA levels and protein levels after stably tansfection with CEMIP sh RNA 3.SW480 cells and HCT116 cells proliferation ability was detected by MTT assay after Silencing of CEMIP 4.The migration ability of SW480 cells and HCT116 cells were subjected to wound healing analysis by knockdown of CEMIP.5.Transwell assay were used to detect the invasion ability of SW480 cells and HCT116 cells by knockdown of CEMIP.6.The cellular localization of EMT biomarker(E-cadherin and Vimentin)and ?-catenin were detected by immunofluorescence staining after knockdown of CEMIP.7.The expression of EMT biomarker(E-cadherin and Vimentin)and ?-catenin were detected by western blot technology after knockdown of CEMIP.Results 1.Both the m RNA and protein expression levels of CEMIP were downregulated in HCT116 and SW480 cells transfected with CEMIP sh RNA-1 or sh RNA-2 plasmid;2.MTT assay showed a reduction in the proliferation of CEMIP-silenced CRC cells,compared with the non-transfected cells and NC-sh RNA;3.The HCT116 and SW480 cells migratory ability was suppressed when the endogenous CEMIP was knocked down;4.Transwell invasion assay was used to simulate the cells movement in vivo,the results showed that CEMIP sh RNA attenuated the invasion ability of HCT116 and SW480 cells;5.E-cadherin was mainly located in cell junctions and cell membranes in HCT116 and SW480 cells,while Vimentin was mainly expressed in cell cytoplasm.The fluorescence of E-cadherin in SW480 and HCT116 cells was enhanced on the cell membrane,but the fluorescence intensity of Vimentin in cytoplasm significantly decreased after knockdown CEMIP.6.The expression of E-cadherin was upregulated,whereas that of Vimentin was downregulated compared with the control group when CEMIP was inhibited;7.?-catenin protein localized in both cytoplasm and nucleus.The fluorescence intensity of ?-catenin in SW480 and HCT116 cells were decreased after silencing CEMIP,and western blot also showed that knockdown of CEMIP were able to induce a decrease in ?-catenin significantly compared with untreatment and negative control groups.Conclusion 1.sh RNA-mediated inhibition of CEMIP suppressed CRC cell proliferation;2.Knockdown of CEMIP inhibits CRC cell migration and invasion ability;3.CEMIP silencing suppresses the EMT process of CRC cells;4.Wnt/?-catenin/Snail pathway is suppressed by downregulated CEMIP in CRC cells.2.Knockdown of CEMIP suppresses proliferation and induces apoptosis in colorectal cancer cells: downregulation of GRP78 and attenuation of unfolded protein responsePurpose This study aimed at investigating the effecting of CEMIP on apoptosis and cell cycle in CRC cells and whether CEMIP affected the endoplasmic reticulum unfolded protein response(ER UPR)of CRC cells.Method 1.Cell proliferation ability was edetected by MTT assay and colony formation assay in CEMIP-silenced SW480 cells and Colo205 cell.2.SW480 cell and Colo205 cell cycle were analyzed by Flow cytometry when decreased CEMIP expression.3.Western blot analyzed Cyclin D1,Cyclin E1 and p-Rb protein levels in CEMIP-silenced SW480 cells and Colo205 cell;4.Calculating the mice tumor volume after construct xenograft tumor mouse model for 8 weeks and determine the tumor cells by HE staining;TUNEL assay is used to evaluate the level of apoptosis;IHC assay is used to detect the endogenous CEMIP expression in mouse tumor cells.5.The levels of CEMIP protein and GRP78 protein was detected in mouse tumor tissue by Western blot.6.Flow cytometry was used to analyze the effect of silencing CEMIP on apoptosis of colorectal cancer cell lines SW480 and Colo205,and the expression of Bcl-2,Bax,cleaved caspase 3 and GRP78 PERK,IRE1 and ATF6 protein was detected by western blot.7.The effect of CEMIP sh RNA on apoptosis of SW480 and Colo205 cells with or without thapsigargin was analyzed by flow cytometry and the expression of cleaved caspase 3 and GRP78 protein were analyzed by western blot;8.The localization and expression of GRP78 in SW480 cells and Colo205 cells were detected with or without treatment of 1 ?M TG for 24 h by Immunofluorescence assay;Results 1.Knockdown of CEMIP inhibited the proliferation of SW480 cells,The clone formation capability of CEMIP-silenced SW480 cells was impaired as compared cells,and SW480 cells were arrested at G1 phase;The protein expression levels of Cyclin D1 and Cyclin E1 were downregulated and the phosphorylation of retinoblastoma(Rb)was inhibited in SW480 cells.2.knockdown of CEMIP did not affect the tumor-forming rate of SW480 cells,it reduced the tumor size;HE staining showed a poor growth of CEMIP-silenced cells,TUNEL-positive cells presented in tumors formed by CEMIP-silenced cells,The expression levels of GRP78 were downregulated by CEMIP was inhibited;3.Silence CEMIP up-regulated anti-apoptotic protein Bcl-2 and down-regulatied pro-apoptotic protein Bax and cleaved caspase-3 in SW480 cells.4.CEMIP sh RNA inhibited the expression of UPR-related proteins GRP78,PERK,IRE1 and ATF6 compared with the control group;The knockdown of CEMIP can increase TG-induced apoptosis.The protein expression of cleaved caspase-3(apoptosis marker)with TG treatment was up-regulated.Furthermore,cleaved caspase-3 showed higher expression by the knockdown of CEMIP.5.The results of immunofluorescence showed that GRP78 protein mainly located in both cell membrane and cytoplasm.GRP78 protein increased in the cytoplasm with TG(1 ?M),but GRP78 protein Fluorescence was weakened in the cell membrane and cytoplasm after CEMIP was silenced.Conclusion Knockdown of CEMIP suppresses proliferation and induces apoptosis in colorectal cancer cells: downregulation of GRP78 and attenuation of unfolded protein response;The proliferation rates of SW480 cells at the indicated time points were determined with MTT assay...