Effect Of Down-regulation Of Sp1 By RNA Inference On The Proliferation And Migration Of Colorectal Cancer SW620 Cell

Backgroud:Colorectal cancer (CRC) is one of the most common gastrointestinal malignancies that seriously threaten the human's health. In western countries, the morbidity of colorectal cancer ranks the second among the cancer-related death, which is next to lung cancer. In China, it ranks from the 4th to 6th among all the cancer-related death. Because of the 5 years survival rates of CRC was lower, it is important to improve CRC patient survival through early detection and early therapy. It is bad in tumor specificity and sensitivity with traditional ways, and it has serious side-effect and bad effect. In recent years, a breakthrough has been made on the molecular mechanisms of colorectal cancer with the development of molecular biology technology. Therefore, the gene therapy for colorectal cancer has been a research hotspot.Transcription factor special protein1 (Sp1) is a nuclear transcription factor. Its abnormal expression and activation in the tumor tissues and cells. Sp1 can improve the mechanisms of tumor-associated growth factor gene expression and angiogenesis which can create the suitable micro-environment for the tumor growth and regulate the proliferation, metastasis potential and angiogenesis of tumor. The current study showed that transcription factor Sp1 was abnormal expression in liver cancer, breast cancer, pancreatic cancer, gastric cancer and other tumor. It is closely related to their occurrence, development, and prognosis of tumor. Howerve, in our country, there is no report on Sp1 expression and its function in CRC. Thus, the research of Sp1 gene expression and biological function in CRC could contribute to clarify the role of Sp1 in colorectal carcinogenesis,which maybe beneficial to the diagnosis and treatment of CRC. It has great application prospects in the Cancer gene therapy.Objective:1. To construct RNA inference eukaryotic expression vectors targeting to transcription factor special protein 1(Sp1) gene and investigate its inhibitory effects on Sp1 expression and its activity in SW620 cell line.2. To study the effects of down-regulation of Sp1 on biological behaviors of colorectal cancer SW620 cells and detect the states of the related proliferation genes after transfected recombinant plasmid.Methods:1. Sense and antisense oligonucleotides targeting to transcription factor special protein 1(Sp1) gene were designed and synthesized. After annealing, it was inserted into pGenesil-1 vector, the recombined plasmid was identified by enzyme digestion and DNA sequence analysis. The mRNA and protein levels of Sp1 was detected by real time PCR and Western blot respectively after recombinants transfected into SW620 cell.The transcriptional activity of Sp1 was analyzed by EMSA after 48h transfection.2. The effects of down-regulation Sp1 on cell proliferation, cell cycle and apoptosis, migration were assayed by the cell growth curve, MTT, flow cytometry (FCM) and scratch method respectively.3. The recombinant plasmid of Sp1 RNAi was transfected into SW620 cells by Lipofectamin, The mRNA and protein expression levels of VEGF were measured by real time PCR,Western blotting and Immunofluoresence staining respectively. Nuclear factors binding to the VEGF promoter were analyzed by electrophoretic mobility shift assay (EMSA).Results:1. The expression plasmid against Sp1 was successfully constructed. Recombinant vectors could reduce the expressions of Sp1 mRNA and protein in SW620 cell, the relative expression of Sp1 mRNA were 0.549±0.034,0.311±0.075,0.406±0.041 in 24h, 48h, and 72h. the ratio of inhibition were 42.44%,68.47%,57.70% respectively, the ratio of inhibition against the expression of Sp1 protein was 56.82%in 48th hour. EMSA in our study showed that the transcriptional activity of Sp1 was decreased after 48h transfection. Compared with control group, the difference was significant (P<0.05). RNAi expression vectors can effectively inhibit the expression and activity of Sp1 in SW620 cell line.2. The proliferation and migration ability of transfected cells was inhibited which was detected by the cell growth curve, MTT analysis, flow cytometry (FCM) and scratch method respectively. Cell proportion of G0/G1 phase were (67.47±2.51)%,(42.19±2.13)% and (48.52±1.86)% in experimental group, negative controlled group and blank cell group, The apoptosis rates of SW620 cells was 22.54% and 4.31% in experimental group and controlled group respectively, the difference was significant (P<0.05).3. The expression of VEGF in the transfected cells was significantly down-regulated at both mRNA and protein levels(P<0.05). The capacity of nuclear factors Sp1 binding to the VEGF promoter was decreased.Conclusion:1. RNA interference expression vector of targeting to Sp1is successfully constructed and it can inhibite the expression and activity of Sp1, it prepared for exploring the function of Sp1 gene in CRC with RNA interference(RNAi) technique. 2. Silencing the Sp1 expression by RNAi could inhibit the proliferation and promote apoptosis of SW620 cells, which was probably related with Down-regulating the expression of VEGF due to the decrease of its transcriptional activity. It may provide a novel therapeutic approach for color cancer therapy.