The Correlation Between APC Gene Silencing And TGF-β's Effect On Human Colorectal Cancer Cell Adhesion

ObjectiveInsufficiency of cell adhesion is an important factor of the malignant tumor's metastasis, which cause the most death related to tumor. E-cadherin is an important regulator of cell cell adhesion within the epithelial cell. Transforming growth factor-β(TGF-P) signaling pathway has an impact on the tumorigenesis and progression of colorectal cancer, so it is important to investigate the effects of TGF-β. WNT signaling pathway may be the most intensive research spot in genetics, it has significant effects in both embryo and tumor, especially the factors like Wnts, APC, axin, TCFs are involved with tumorigenesis.MethodsConstruction of APC-shRNA plasmid:Design APC-shRNA template and ligate it with pSliencerTM 4.1-CMV neo plasmid; transfect the plasmid into E.coli competent cells DH5a for selection and amplification; identify APC-shRNA plasmid by PCR and sequencing. Establishment of HCT-116-APC-RNAi cell model:Purify APC-shRNA plasmid and transfect it into HCT-116 cells with LipofectamineTM2000; confirm APC gene silence at mRNA and protein level by RT-PCR and western blot. After APC gene silence, investigate the invasion of HCT-116 cell by Transwell assays; investigate the mRNA and protein expression of E-cadherin by RT-PCR and western blot.Results1. Successful Construction of APC-shRNA plasmid:PCR product of bacteria suspension with APC-shRNA plasmid is about 250bp; Sequencing results demonstrate the insert is correct.2. Successful establishment of HCT-116-APC-RNAi cell model:Compared to negative control, the cells transfected with APC-shRNA had a down regulation of APC in both mRNA and protein level. The mRNA expression of APC gene is reduced by 63.31±6.84%,77.03±5.61%(P<0.05) in the 48 and 72 hours, and the protein expression of APC gene is reduced by 73.03±9.33%,79.51±10.13%(P<0.05) accordingly. It confirmed the silence of APC gene.3. The different effects of TGF-β1 on HCT-116 cells after APC gene silence: Compared to negative control, APC gene silence could inhibit the E-cadherin of HCT-116 cells by 66.35±3.65%,71.32±4.57%(P<0.05)in mRNA level in the 48 and 72 hours, and in the protein level, the inhibition rate is 71.10±5.76%和77.54±9.16% (P<0.05) accordingly.4. Effect of APC gene silence on invasion of HCT-116 cells:Compared to negative control, After APC gene silence, the lOng/ml TGF-β1 effect switch from a 16.6% inhibiting to a 13.2%introducing of tumor cell invasion.Conclusion1. RNAi is competent to establish the APC silencing cell model in human colorectal cancer HCT-116.2. APC gene silence could inhibit the introduction of TGF-βon E-cadherin in HCT-116 cell.3. APC gene silence could switch TGF-βfrom inhibiting invasion to introducing invasion in HCT-116.4. There may be a cross talk between TGF-βand WNT signaling pathways.