Effects Of Recombinant PEGFP-N3-APC Vectors Carrying Various APC Functional Domains On The Expression Of β-catenin In Human Colorectal Cancer Cells

The remarkable thing about Colorectal cancer(CRC)-the most common cause of non-smoking-related cancer deaths in the world-is that the molecular mechanisms underlying virtually all of these cases are uniform.Recent studies on molecular carcinogenesis have highlighted that greater than 90%of all CRCs will have an activating mutation of the canonical Wnt/β-catenin signaling pathway,ultimately leading to the stabilization and accumulation ofβ-catenin in the hallmark of an active canonical Wnt pathway;the presence of nuclearβ-catenin is evident in even the smallest detectable lesions resulting from Wnt mutations.The consistency of mutations at some level of the Wnt signaling pathway in CRC makes this cancer an attractive model for molecular intervention.In CRC cells,Wnt/β-catenin signaling is activated mainly by mutations in the tumor suppressor gene APC.＞70%of sporadic CRCs contain APC gene mutations,which result in inefficientβ-catenin degradation and up-regulation ofβ-catenin-mediated transcription.The majority of somatic mutations in the APC gene occur within a segment called the mutation cluster region(codons 1,250-1,500) and almost all of the mutations result in truncated APC protein.APC mutations occurring in the mutation cluster region produce abnormal APC proteins that retain only the three 15-amino-acid repeats and the first one or two 20-amino-acid repeats while losing the other domains and failing to effectively down-regulateβ-catenin.Recent research have highlighted that wild type APC could degradeβ-catenin level in colorectal cells,while using wild type APC for gene therapy is hard to obtain because the length of gene is longer than 10 kb, which couldn't be expressed stably in vivo and unwell to be used for gene therapy.ObjectivesThe present study aimed to construct several recombinant eukaryotic expression vectors,containing various functional domains of APC protein, and investigated their functions and impact onβ-catenin level in human colorectal cancer cells. Methods1.APC gene fragments were amplified by PCR with pBluescript (involving full-length APC cDNA) as template and primers designed according to APC cDNA sequence and mutation cluster domain, containing BamH I and Xho I restriction site for directed cloning into pEGFP-N3 vector.2.The recombinant plasmids were identified by sequencing and analyzed by ORF finder and protein BLAST.3.The recombinant plasmids were transfected into colorectal cancer cells HCT 116 and HT-29 mediated by lipofectamine^TM 2000.Green fluorescence was observed via fluorescence microscope to investigate the expression of recombinant plasmids in cells.4.RT-PCR was employed to validate the expression of recombinant vectors in HT-29 cells.5.Western blot technique was applied to detect the influence onβ-catenin expression level of recombinant plasmids,gray scales of electrophoresis strips were analyzed by SPSS 13.0 via one-way ANOVA.Results1.Five recombinant expression vectors were constructed,named pEGFP-N3-APC1(including APC peptide from aa 6 to aa 767), pEGFP-N3-APC2(aa 1020-1169),pEGFP-N3-APC3(aa 1262-2033), pEGFP-N3-APC4(aa 1020-2033),pEGFP-N3-APC5(aa 1020-1698).2.Bioinformatics analysis showed that the recombinant plasmid could express a fusion protein involving APC protein domains and GFP.3.The stably transfected colorectal cancer cells were selected by neomycin resistance using 300μg/ml G418.Optimizing transfection parameters were determined by experiments.Results illustrated the five recombinant vectors could all be expressed in HCT 116 cells,green fluorescence detection showed transfection efficiency of pEGFP-N3-APC 1 was 50%,pEGFP-N3-APC2's was 50%, pEGFP-N3-APC3's was 30%,pEGFP-N3-APC4's was 30%, pEGFP-N3-APC5's was 50%,pEGFP-N3's was 80%,respectively, which certificated successful construction of recombinant vectors. Nevertheless,fluorescence intensity of GFP might be influenced by multiple factors such as property of cell itself,green fluorescence was hard to be observed in HT-29 cells. 4.Results of RT-PCR made clear that vectors constructed could be expressed in HT-29 cells,transfection efficiency in HT-29 was no less than that in HCT-116.5.Results of Western blot showed,in HCT 116 cells,discrepancy of gray scales of electrophoresis strips wasn't significant(p＞0.05),that was to say,β-catenin level wasn't effected a change when recombinant plasmids were transfected;in HT-29 cells,the situation wasn't so, discrepancy of gray scales of electrophoresis strips wasn't significant between the anterior four ones,which were normal cells,cells transfected with pEGFP-N3-APC1,cells transfected with pEGFP-N3-APC1,cells transfected with pEGFP-N3-APC2 and cells transfected with pEGFP-N3-APC3,respectively,discrepancy of gray scales of electrophoresis strips was significant between the posterior two ones(cells transfected with pEGFP-N3-APC4 and cells transfected with pEGFP-N3-APC5) and the anterior four ones,that was to say, recombinant plasmid pEGFP-N3-APC4 and pEGFP-N3-APC5 could degradeβ-catenin level obviously.As for pEGFP-N3-APC4 and pEGFP-N3-APC5,the length of pEGFP-N3-APC5 was shorter than that of pEGFP-N3-APC4.Conclusions1.Five recombinant pEGFP-N3-APC vectors carrying various APC functional domains were succefully constructed2.The recombinant plasmids were identified by sequencing and analyzed by ORF finder and protein BLAST,transfected into colorectal cancer cells HCT 116 and HT-29 mediated by lipofectamine^TM 2000.Detection of green fluorescence and RT-PCR illustrated the successful expression of recombinant plasmid in human colorectal cancer cells.3.Recombinant plasmid pEGFP-N3-APC4 and pEGFP-N3-APC5 could degradeβ-catenin level obviously in HT-29 cell lines.APC fragment No.5 was just the target gene we need,which could degradeβ-catenin level and possess short length relatively.