Research On The IGFBP4 Regulating Action With Wnt/?-catenin Signaling Pathways In Colorectal Cancer Cells

Colorectal cancer(CRC)was one of the most common forms of cancer,the morbidity and mortality accounted for second place and shown an increasing trend year by year in the world.In recently years,the relationship between abnormal intracellular signaling pathways and tumor development had become a hot topic in medical research.Insulin-like growth factor binding protein-4(IGFBP4)was a secreted protein,which was expressed in many normal tissues,but showed low expression in most of tumors,such as renal carcinoma,colorectal cancer,breast cancer and so on.IGFBP4 could regulate gene activity which played an important role in the development of a series of tumors,to inhibit tumor development.The mechanism may be independent of its IGF binding activity and also related to different intracellular signaling pathways,but it was rarely reported.Studies have found that IGFBP4 could inhibit the Wnt binding to its receptor through competitively binding to the Wnt receptor Frizzled8(Frz8),so that it could inactivate the classical Wnt/?-catenin signaling pathway;IGFBP4 could also play a role in kidney cancer by regulating the classical Wnt/?-catenin signaling pathway,but it has not been reported in colorectal cancer.Currently,studies had shown that Wnt/?-catenin signaling pathway was an important mechanism for the development of colorectal cancer,while abnormal activation of this pathway may mediate the carcinogenesis of colorectal adenomas.The preliminary histopathology findings had found that IGFBP4 descended orderly in normal colon mucosa,dysplastic tubular adenoma and adenocarcinoma.This phenomenon showed that IGFBP4 played a role in the carcinogenesis of sporadic colorectal tubular adenoma and had a negatively correlation with related factors on the Wnt/?-catenin signaling pathway.This experiment was aimed to further explore regulation and mechanism between IGFBP4 and Wnt/?-catenin signaling pathway.Firstly,we selected the colorectal cancer cell lines,then introduced in vitro cell culture method,Plasmid transfection technology,Western blot and Real time PCR method in the experiment,investigating how IGFBP4 plays an important role in regulating Wnt/?-catenin signaling pathway in human colorectal cancer cells,which provided new ideas for the development of mechanisms of colorectal cancer at the cellular level.Objective: At the cellular level,to explore how IGFBP4 plays a role in regulating the classical Wnt/?-catenin signaling pathway.Methods:1 Cell culture and treatmentChoose two human colon cancer cell lines(HT-29 and HCT116)in vitro.HT-29 and HCT116,respectively,were grown in McCoy?s 5A and RPMI-1640 medium supplemented with 10% new-born calf serum(NBCS),streptomycin(100?g/ml)and penicillin(100 U/ml).Cells were growing attached.2 Screening target cells for plasmid transfection experimentThe cells in logarithmic growth phase were digested with 0.25% trypsin +0.02% EDTA,then collected and detected related indicators.By Using Western blotting technology,IGFBP4 expression was detected in two colorectal cancer cells,the cells low expressed IGFBP4 was screened for plasmid transfection experiment.3 Plasmid transfectionBuilt for human IGFBP4 gene vector plasmid M90,transiently transfected into human colon cancer cell line HCT116,at the same time transfected empty vector as negative control.Detecting the effect of Wnt2,Frizzled8,?-catenin,TCF-4,CDK4 and CyclinB1 respectively after IGFBP4 gene was upregulated in cell.4 Western blotAfter transfection of HCT116 cells treated 48 h,extracted the total cellular protein,detected IGFBP4,Wnt2,?-catenin,TCF-4,CDK2 and CyclinB1 protein expression in colon cancer cell lines of HCT116 with Western blot method.Then,chromogenic assaywas visualized by Odyssey and quantitative analysis was subjected to Bio-LD software.5 Real time-PCRAfter transfection of HCT116 cells treated 48 h,detected IGFBP4 mRNA expression in colon cancer cell lines of HCT116 with Real time-PCR.Using Comparative Ct(??Ct)method,supplied by Applied Biosystems company,to calculate method to calculate the relative expression of the target gene.6 Statistical analysisSPSS17.0 software was applicated.Measurement data were represented as((?)±s).P<0.05,difference was statistically significant.Results: 1 Screening low expression IGFBP4 of colon cancer cell linesIGFBP4 protein expression in the two colorectal cancer cells was detected.Results showed that IGFBP4 protein was expressed and HCT116 had the lower level of IGFBP4 in both cells.Therefore,selected HCT116 cell as subjects of the experiment.2 The efficiency of plasmid transfection interferenceThe cells were collected after 48 hours transfection and their transfection efficiency were detected by green fluorescent protein(GFP).3 The expression of IGFBP4 at mRNA and protein level after transfection with IGFBP4 gene plasmidThe expressions of IGFBP4 at mRNA and protein level were detected 48 h after transfected with IGFBP4 gene plasmid or empty vector in HCT116 cells by Real-time PCR and Western Blot method,respectively.In comparison with control group,IGFBP4 mRNA expression was significantly increased in HCT116 cells.Consistant with Real-time PCR result,IGFBP4 protein was strengthened obviously by IGFBP4 gene M90 plasmid compared to the control group in the cell lines,suggesting that the IGFBP4 gene sequences could increase IGFBP4 expression effectively.4 Effects of Wnt/?-catenin signaling pathways on IGFBP4 plasmid transfection in HCT116 cells 4.1 Effects of Wnt2 and Frizzled8 protein on IGFBP4 plasmid transfection in HCT116 cellsDetecting the effects of Wnt2 rotein on IGFBP4 plasmid transfected within 48 h.Western blot results showed that in comparison with control group,Wnt2 and Frizzled8 protein expression had no significant change in IGFBP4 plasmid transfection group(P>0.05);indicating IGFBP4 didn't participate in regulating the expression of Wnt2.4.2 Effects of ?-catenin,TCF-4,CDK2 and CyclinB1 protein on IGFBP4 plasmid transfection in HCT116 cellsDetecting the effects of ?-catenin,TCF-4,CDK2 and CyclinB1 protein on IGFBP4 plasimd transfected within 48 h.Western blot results showed that in comparison with control group,?-catenin,TCF-4,CDK2 and CyclinB1 protein expression were significantly decreased in IGFBP4 plasmid transfection group(P>0.05);indicating IGFBP4 could be participate in regulating the expression of ?-catenin,TCF-4,CDK2 and CyclinB1.Conclusion:IGFBP4 could down-regulate markedly ?-catenin and ?-catenin/TCF downstream target genes expression at protein levels,suggesting IGFBP4 could negatively regulate of Wnt /?-catenin signaling pathway.