The Study Of Mechanism And Effects Of DPC4 Gene On Invasion And Metastasis Of Colorectal Carcinoma Cells

Preface The colorectal carcinoma is a frequent malignant tumor ofdigestive tract, and its incidence is going up year by year. TGF-β(Transforming growth factor-β) plays an important role in modulatingimmune response, regulating cell differentiation and growth, stimulatingextracellular matrix formation, and so on. However, epithelial derivedtumor cells has often lost their sensitivity to the growth inhibition ofTGF-β, and lost their growth inhibition. DPC4 was thought as animportant tumor suppressor gene.Despite the extensive research of DPC4,we little know of whether the loss of DPC4 function contributes to theinvasion and metastasis of colorectal carcinoma cells.Objective Our investigation attempts to study the effect oftransfected DPC4 gene on biological behavior of human colorectalcarcinoma cells and the role of DPC4 gene in colorectal carcinogenesis.Methods Expression of DPC4 was detected in 70 samplesincluding nomal mucosa membrane, colorectal adenoma and colorectalcarcinoma tissue by immunohistochemistry S-P method; PcDNA3.1-DPC4 plasmid was re-constructed by gene-recombination technology;The expression of DPC4 was detected by RT-PCR and Western blotanalysis in five colon carcinoma cell lines, HCT116, HT29, LS174T,SW480 and SW620. Then HCT116 cells which were absent of DPC4protein expression were transfected with PcDNA3.1-DPC4 plasmid bylipofectamine transfecting technique; Growth inhibition property andadherent ability of transfected cells were detected by MTT method; Theapoptosis rate and cell cycle percentage of transfected cells weredetermined by flow cytometry; The ability of migration and invasion oftransfected cells was examined by Millicell method; Nude micemetastasis models were established by abdominal cavity transfer method.The activity of MMP-9 and MMP-2 was detected by gelatin zymography.Results DPC4 positive signal was localized in cytoplasm andnucleus. The immunostaining of DPC4 was strong positive in normalcolon mucosa and adenoma. The positive cells distributed in the wholecolon mucosa The expression of DPC4 was positive in well- differentiated adenocarcinoma, and weakly positive or negative in poorlydifferentiated adenocarcinoma and metastatic focus. The expression ofDPC4 was associated with tumor stage, tumor differentiation degree andmetastasis; The recombinant plasmid was PcDNA3.1-DPC4; Theexpression of DPC4 wasn't detected in HCT116 cell line; The cellmodels(DPC4~+-HCT116) steadily expressing DPC4 were obtained;Compared with HCT116 and PcDNA3.1-HCT116 cells, the cellpopulation doubling time of DPC4~+-HCT116 cells was lengthenedobviously(P＜0.01). Moreover, the apoptosis rate of DPC4~+-HCT116cells was significantly increased(P＜0.01), the cloning efficiency, celladherence, migration and invasion ability of DPC4~+-HCT116 cells wasdropped obviously(P＜0.01); There were much more cancer nodules inabdominal cavity and liver of the nude mice inoculated with HCT116 andPcDNA3.1-HCT116 cells, compared with those inoculated withDPC4~+-HCT116 cells; In Comparison with HCT116 and PcDNA3.1-HCT116 cells, the levels of active of MMP-9 decreased in DPC4~+-HCT116 cells.Conclusion 1. Down-regulation of DPC4 expression may beassociated with the carcinogenesis of colorectal carcinoma.2. PcDNA3.1-DPC4 plasmid was successfully re-constructed andtransfected into SW620 cells, and cell model steadily expressing DPC4was obtained.3. DPC4 may inhibit invasion and metastasis of colorectalcarcinoma.4. DPC4 may inhibit the proliferation of colon cancer cell byrestraining growth and inducing apoptosis.5. MMP-9 may be one of the downstream target genes regulated byDPC4.