Effects Of Id3-overexpressing On Biological Activities In Colorectal Cancer Cell SW480 By Down-regulation Of β-catenin

BackgroundColorectal cancer(CRC)is the third most common human malignancy and the second highest cause of cancer-related death worldwide.Although several kinds of treatment modalities have been developed recently for the patients with CRC,the clinical outcome of prognosis continues to be poor in patients with advanced CRC.Metastasis is responsible for the majority of cancer deaths.Despite the tremendous progress in treatment,the overall mortality of CRC is still approximately 40%.Surgery is the fundamental treatment,but 30%to 50%of patients with stage Ⅰ to Ⅲtumors relapse within 5 years following treatment.Additionally,oxaliplatin or 5-FU,the most widely used anticancer agent,has become ineffective against CRC due to the development of intrinsic or acquired drug resistance.The aberrant activation of Wnt/β-catenin signaling pathway is considered to be an essential issue in tumorigenesis and progression of CRC.The Wnt/β-catenin pathway has key functions in cell differentiation,proliferation,cell destination,organ formation,embryo development and tumor initiation.β-catenin is the core component of the canonical Wnt signal cascade.The hallmark of the Wnt/β-catenin pathway is the accumulation and nuclear localization of β-catenin.Cytoplasmic β-catenin is controlled by the degradation complex composed of adenomatosis polyposis coli(APC),Axin,protein phosphatase 2A(PP2A),glycogen synthase kinase 3 beta(GSK3P)and casein kinase 1α(CK1α).When Wnt signaling is activated aberrantly,β-catenin is discharged from the degradation of complex resulting in the translocation of β-catenin into nucleus,where it associates with the T-cell factor/lymphoid enhancer factor(TCF/LEF)family of transcription factors to activate specific Wnt target genes,such as c-myc、cyclinDl and COX.There exist the upregulation of β-catenin when Wnt/β-catenin signaling pathway is aberrantly activated.Therefore,so targeted gene treatment of β-catenin has become a research hotspot.Inhibitors of differentiation/DNA binding(Id)proteins,which belong to the group helix-loop-helix(HLH)transcription factor family members,play a negative gulatory role to basic helix-loop-helix(bHLH).Id proteins are members of basic helix-loop-helix(HLH)family of transcription factors but lack a DNA binding domain.The Ids are essential for embryogenesis,and they have been functionally implicated in proliferation,differentiation and cell survival.Id3 is one of four members of Id family(Id1,Id2,Id3 and Id4).In recent years,Id3 plays a important regulatory role in tumor cell growth,differentiation,apoptosis,invasion,metastasis and signal transduction.Our previous results showed that Id3 mRNA and pretein had low expression level in human lung adenocarcinoma cell line(A549)and resistant cell line A549/DDP.Additionally,Id3 can reverse the drug resistance to cisplatin of A549/DDP.Recent studies have shown that Id3 mediates apoptosis and influences cell proliferation of keratinocytes through the regulation of mRNA and protein level ofβ-catenin.However,the regulatory mechanism research of Id3 on P-catenin expression in colorectal cancer cell has not been reported.Part 1:The expression of Id3 and β-catenin different tumor cellsObjective:This study aimed to evaluate the expression of these two molecules in different tumor cells.Methods:Total RNA was extracted using Trizol Reagent according to the manufacturer’s instructions.The relative mRNA expression levels of Id3 and β-catenin in tumor cells were detected by quantitative real-timePCR(qRT-PCR)techniques.Results:qRT-PCR showed that Id3 had different levels of expression in different malignant cells and the lowest expression of Id3 was found in the colorectal cancer cell lines such as,SW-480 and HT-29.The expression level of P-catenin in SW-480 was significantly higher than other malignant tumor cells.Conclusion:The expression levels of Id3 and β-catenin were different in different tumor cell lines.The abnormal overexpression of β-catenin in the colorectal cancer cell is one of the most important elements causing.Part 2:The regulation effect of Id3 on β-catenin in different tumor cellsObjective:This study was to evaluate the effects of overexpression of Id3 on β-catenin level in three tumor cells.Methods:Recombinant eukaryotic expression vector Id3/pEGFP with human Id3 gene was transfected into A549,A549/DDP and SW-480 cells by the non liposome-mediated method,respectively.The fusion protein Id3-EGFP was successfully expressed in cell lines confirmed by fluorescence microscopy.RT-PCR and Western blot were performed to demonstrate the mRNA and protein level,respectively.Results:β-catenin expression was significantly down-regulated in SW-480 cells transfeted with Id3-overexpression compared with empty vector(p<0.01).While,there was no significant difference in the A549 and A549/DDP cells.Conclusion:Overexpression of Id3 markedly reduces the P-catenin mRNA and protein level in SW-480 cells,which is expected to provide new ideas of target gene therapy in colorectal cancer.Part 3:Effects of Id3-overexpressing in colorectal cancer cell SW480 on proliferation、apoptosis and migrationObjective:Id3 is a tumor suppressor gene.It plays a role of proliferation,migration and apoptosis in many type tumor cells.This study was to evaluate the effects of Id3-overexpressing of biological activities on SW-480 cells.Methods:Cell proliferation assay and flow cytometry were experimented to evaluate cell proliferation and apoptosis,respectively.Cell migration was detected by holographic imaging technology.Results:Id3 significantly inhibits the proliferation,induces the apoptosis and suppresses the migration of SW-480 cells in a time-dependent manner,confirming the implication of Id3 in the negative control of tumour growth.Conclusion:These results suggest that Id3 may plays a role as a tumor suppressor gene in the progression of CRC.