| Gastrointestinal cancer is one of the most common malignant tumor and the mortality rate is rank at the top of cancer. Colon cancer is the most common gastrointestinal cancer, with a high degree of malignancy, recurrence rate and poor prognosis. Most current clinical cases are the advanced cases of colon cancer, and surgical resection rate and 5-year survival rate is low, the overall curative effect is poor.The occurrence and development of Colon cancer is a multi-factor and multi-step process.With the development of theory and technology of molecular biology, the molecular mechanisms and gene therapy of colon cancer has became a hotspot.RNA interference is a mechanism for eukaryotic gene regulation in vivo. RNA interference means the degradation of homologous gene sequences by endogenous or exogenous double-stranded RNA.The gene silencing effect caused by RNAi technology can inhibit the expression of oncogene, tumor suppression gene and gene mutations. The short hairpin RNA(shRNA),which can be steadily expressed by vector transfected into target cells,can inhibit gene expression permanently.In recent years, a small protein of about 12kD called SUMO1 has been found, and causes widespread concern. SUMO1 and SUMO 1-mediated ubiquitination has been a hotspot of signal transduction research areas. The role of the SUMO1 gene in occurrence and development process of colon cancer is still unknown.The function of SUMO1 and the correlation with the prognosis of colon cancer need further validation, which will undoubtedly contribute to the clinic application of SUMO1 and the treatment of colon cancer. This study is to investigate the expression of SUMO1 in human colon cancer cell lines and detect the inhibition effect of SUMO1 on the cell proliferation and mobility in vitro and the tumor-forming ability in nude mice. SUMO1 is expected to become the new molecular targeted to colon cancer in gene therapy. However, the function and application of SUMO 1 gene in human tumor therapy need further investigation. Together with our results, it provides new ideas and references for the functional mechanism of SUMO1 and its therapeutic applications for colon cancer.Objective:1 To investigate the expression of SUMO1 gene in colon cancer cell lines;2 To elucidate the effect of SUM01 gene on the cell proliferation and mobility, RNAi technology was applied to silence SUMO1 gene in colon cancer cell lines Hct116. Moreover, to further confirm the effect of decreased SUMO1 expression on tumor formation in nude mice.Methods:1 The expression of SUMO 1 mRNA and protein in the colon cancer cell lines SW480, SW620, Hct8, Hct116 were detected by semi-quantitative RT-PCR and Western blot.2 In accordance with the principle of siRNA design, we designed three shRNA sequences and cloned into the pSilencer TM 3.1-H1 neo vector. By liposome-mediated transfection, the recombinant plasmid was transfected into the colon cancer cells Hct116.And we detected endogenous SUMO1 protein expression using Western blot and immunofluorescence staining.3 Using liposome-mediated transfection,the MTT assay and colony formation assay was performed to evaluate the cell proliferation.Scratch healing experiment was performed to measure the cell mobility.The expression of P53 protein was detected by Western blot, in order to confirm the biological function of SUMO1 in Hctl 16 cell and to analyze its possible mechanism. 4 The Hct116 cells transfected pshRNA-SUMO1 were inoculated subcutaneously in nude mice to establish tumor models. After tumors grown the weight and size of tumors were measured.The inhibition rate was used as an index to evaluate the effect of SUMO1 gene silence on the colon cancer tumorigeneisis capacity in vivo. The expression of SUMO1 of the tumors grown in nude mice was detected by immunohistochemistry.Results:1 It is proved that colon cancer cell lines SW480, SW620, Hct8, Hct116 highly expressed SUMO1 by RT-PCR, Western blot.2 The recombinant expression vector of shRNA targeted SUMO1 was successfully constructed and transfected into Hct116 cells.Western blot and immunofluorescence showed that the three recombinant plasmids have endogenous SUMO1 gene silencing effect (p<0.05) and the pshRNA-SUMO1-1 is the most significant.3 The cell growth, which transfected by pshRNA-SUMO1-1, slowed significantly (p<0.05). MTT assay and colony formation assay showed significantly decreased cell proliferation (p<0.05); scratch healing experiments showed significantly decreased cell mobility (p<0.05). And pshRNA-SUMO1-1 significantly inhibited the expression level of P53 in Hct116 cell,4 In the xenografts assays in nude mice,after tumor cell inoculation tumor formation was 100% and the tumor size and weight were significantly inhibited following the gene silencing of SUMO1 gene,the tumor inhibition rate was 56.8%(p<0.05). Immunohistochemistry showed that expression of SUMO1 of implanted tumor was significantly weaker than the control group (p<0.05).Conclusions: 1 It is the first confirmed that colon cancer cell line SW480, SW620, Hct8, Hct116 can highly expresse the SUMO1 gene;2. The expression of SUMO1 gene in colon cancer cells can be effectively down-regulated by liposome-mediated RNA interference. Inhibition of the SUMO1 expression can inhibit the cell proliferation and mobility in vitro and the tumor-forming ability in nude mice.3. This study demonstrate that SUMO1 is an important molecular in the development of colon cancer and suggest that SUMOl may serve as a potential target for cancer gene therapy. |
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