Gene Expression Assay In Colorectal Cancer And Empirical Study On Therapeutics By SiRNA-c-myc And SiRNA-VEGF

Colorectal cancer is one of the most frequently incident malignant tumor at present. In the past 20 years, the incidence of colonrectal cancer has increased markedly over the most of countries and areas in the world. The mortality rate of colorectal cancer was the second place of malignant tumor in the western countries. With the aging of population and the change of life style, the incidence of colorectal cancer increased in the developed regions such as Shandong, Shanghai, Guangdong, Zhejiang and Hubei etc. And now, it is the fourth malignant tumor in city and the fifth in countryside.The main therapy of colorectal cancer was surgical treatment. The 5-year disease-free survival (DFS) rate with surgery alone for stageⅠpatients is 90%, and it is 70%–75% for stageⅡpatients, 35-50% for stageⅢpatients and less 5% for stageⅣpatients. Approximately 50% of the patients after surgery treatment would recurred, and most of them would lost the chance of retreatment. Appearance of new dosage modality, new chemotherapeutics and new adjuvant therapy elevated the therapeutic efficacy of colorectal cancer.Molecular targeted therapy was the new pathway to cure tumor. It target the marker molecular witch over express in the tumor cells, select the corresponding blocking agent to interfere in the regulation of marker molecular effectually and ultimately suppress the tumor growth, progression and metastasis. RNA interference (RNAi) is an initial defend mechanism that cells counteracted the expression of exogenous genes. It is an RNA dependent postranscription gene silencing phenomenon with sequence specific. With the well stability, better specificity and low cytotoxicity, It has become the powerful tool for gene function studding and oncotherapy.The c-Myc oncogene is the most frequently studied in colorectal cancer research. It was a transcriptional factor with double function to induce cell proliferation or to induce apoptosis. The expression of c-Myc will increase in most human tumor tissues. It was verified that the expression of c-Myc gene increased in the initial stage of colorectal cancer and had more important function in that period.The vascular endothelial growth factor (VEGF) level in blood plasma of colorectal cancer patients is higher than health human and significant correlated with the Dukes staging. It has been reported that the relapse rate of colorectal cancer with VEGF positive expression was over 50%, but less 11.7% recurred in VEGF negative patients. It is thus clear that the VEGF level in blood serum is more important to predict tumor's growth, invasion and metastasis. Objective:To screen the abnormal expression genes fit with the early diagnosis in colorectal cancer. Study the therapeutic effect of siRNA-c-Myc and siRNA-VEGF to colorectal cancer cell.Methods:(1) Clinical pathological assay and genes expression analysis. Analyze the pathological typing and Dudes's staging by HE dyeing and pathological section. Study the expression of some of the oncogenes or anti-oncogenes, apoptosis related genes, metastasis related genes and part of the transcription factors by using RT-PCR. Verify the protein expression of c-Myc and VEGF by western blot assay.(2) Constructed and identified the psilencerTM U6-siRNA-c-Myc and psilencerTM U6 -siRNA-VEGF expression plasma.Using human c-Myc and VEGF gene sequences from GenBank, selected suitable target site, synthesized oligonucleotides as DNA template encoding siRNA-c-Myc and siRNA-VEGF, annealed and ligated into psilencerTM U6 siRNA expression vector to construct the plasmid psilencerTM U6 -siRNA-c-Myc and psilencerTM U6 -siRNA-VEGF respectively. Identified the recombinant plasmids by endonuclease digested process and DNA auto sepuencing.(3) Transfected psilencerTM U6 -siRNA-c-Myc and psilencerTM U6 -siRNA-VEGF plasmids to the colorectal cancer cell line VoLo and observe the silence effect respectivesy.To transfect the psilencerTM U6 -siRNA-c-Myc and psilencerTM U6 -siRNA-VEGF plasmids to VoLo cell lines respectively by eukaryocyte transfection technique. Detect the c-Myc or VEGF gene's and protein's expression by RT-PCR and Western blot respectively.(4) The effects to the colorectal cancer cell line VoLo by gene silence of the c-Myc and VEGF respectively.Select the VoLo cell lines with c-Myc or/and VEGF gene silence, analyze the ability of cell proliferation and apoptosis character change by MTT, FCAS and TUNEL assay; study the migration ability of cells by Boyden chamber migration assay.Result:(1) Collect the 48 experiment samples of colorectal cancer, 64.6% in which is men and 35.4% is women. The mean age of these patients are 59.6±5.1. The number of Colon cancer is 30, rectal cancer is 25 and colorectal cancer is 7. With the clinical pathohistological assay that there are 4 papillary adenocarcinomas, 28 tubular adenocarcinomas, 12 mucus adenocarcinomas, 4 signet-ring cell carcinomas and 25 lymphaden metastasis. Eight patients in Dukes'A, 15 in Dukes'B, 18 in Dukes'C and 7 in Dukes'D.(2) The results from semi-quantitative RT-PCR analysis suggested that 16 genes appeared abnormal expression, and the expression of c-Myc, AKT and VEGF significant increased in the early stage of cancer. Western blot assay showed that the protein expressions of c-Myc and VEGF were increased in colorectal cancer tissues and the increasing expressions were positive correlated with the Dukes'staging.(3) The results of endonuclease digested and DNA auto sepuencing suggested that the construction of .psilencerTM U6 -siRNA-c-Myc and psilencerTM U6 -siRNA-VEGF expression plasma was successfully. RT-PCR and Western blot assay showed that the c-Myc or VEGF gene's and protein's expression were silenced by the siRNA respectively in the transfected VoLo cell lines.(4) The MTT assay showed that the proliferation ability of cells with c-Myc or VEGF genes silenced decreased significant and appeared time dependent relation. FCAS and TUNEL assay suggested that the cells with c-Myc or VEGF genes silenced appeared apoptosis. Boyden chamber migration assay showed that the migration ability of the cells with c-Myc or VEGF genes silenced decreased.Conclusion:In these studies, we screen the c-Myc, AKT and VEGF genes as the target molecules marker for the early diagnosis by the semi-quantitative RT-PCR analysis. Constructed the psilencerTM U6 -siRNA-c-Myc and psilencerTM U6 -siRNA-VEGF expression plasma successfully. And we show for the first time that a combined gene therapy with co-expression of siRNA-c-Myc and siRNA-VEGF made a synergistic effect to inhibit cancer cell growth and induce the cell apoptosis and suppress the metastasis of the colorectal cancer cell lines.