Effect Of Down-regulation Of Sp1 By RNA Inference On The Teloerase Activation Of Colonic Cancer SW480 Cells

Background:Colorectal cancer is one of the leading gastrointestinal malignancies in Western countries.The morbidity and mortality of colorectal cancer rising trend has attracted wide attention inChina in recent years. The molecular biological study of colorectal cancer has not onlytheoretical significance to understand the essence of cancer but also practical value to guide theprevention and treatment of colorectal cancer. Histological features of colorectal cancer andtumor biological characteristics decide that colorectal cancer is unique in that from normalmucosa to hyperplasia, carcinoma from benign to malignant transformation, carcinoma from situto invasion and metastasis and these stages all have clear distinctions. In the same individual, wecan obtain tissue samples from normal mucosa to metastatic carcinoma at different stages ofdevelopment for the Comparative Study. At the same time, the colorectal cancer has a cleardistinction between genotype of Susceptible to a genetic predisposition (Xiao cancerous polypsin familial and Familial colon cancer) and sporadic of Inclined not to Genetic susceptibility. Thisoffers the best basis for the study of colorectal cancer Cytogenetic and Molecular Genetics. Fromthe perspective of molecular biology, the study of colorectal cancer is the most extensive andpenetrating. The nature of the tumor is also the clearest. So this kind of tumor can be used as themodel of the process which tumor gene express and structural change.Tumor genesis and tumor development is the research focus and Telomerase activationhighly correlated with cancer which hints that the interaction among telomerase, telomere andtelomere's maintenance plays an important role in the process of cancer's transmutation. Studieshave indicated that telomerase activity has no expression or low expression in normal humancells and it exists in the reproductive cells, tumor cells, immortalized cell lines and tissue ofregeneration(for example, blood, skin and intestinal epithelium). Human telomerase complexconsists of 3 major subunits: telomerase RNA components (hTR), telomerase associated protein(TP1/TEP1) and telomerase catalytic subunit (hTERT). Sp1 is the key element binding tohTERT core promoter which rich with GC sites and activating hTERT transcription. We regardcolorectal cancer as the study object and apply the techniques of RNA interference to inhibit theexpression of Sp1 gene. It can provide a theoretical basis for a new method of gene therapy ofthe colorectal cancer through observing its influences on telomerase activation and inhibitions ontumor cells. ObjectiObjective:1. To construct eukaryotic expression vector of RNA interference to Sp1 gene(pGenesil-1-Sp1)and investigate its inhibitory effects on SP1 expression and its activity in SW480 cells line.2. To observe the inhibitions of pGenesil-1-Sp1 on Sp1 gene expression in SW480 cells anddetect the states of the related migration and invasive genes after transfected recombinantplasmid.3. To detect the inhibition of Sp1 silencing to hTERT and telomerase activation in SW480 cells.Methods:1. The eukaryotic expression vector of RNA interfering for Sp1 gene (pGenesil-1-SP1) as ashort hairpin structure was synthesized in our previous experiments, which was confirmed bysequencing.2. After transfected with eukaryotic expression vector p-shRNA and p-CON byLipofectamineTM 2000 Reagent, detecting the protein expression levels of Sp1 in SW480 cells.3. The variation of hTERT gene expression in transfected colorectal cancer SW480 cells wasexamined by RT-PCR.4. The variation of telomerase activation in transfected colorectal cancer cells SW480 wasevaluated by TRAP-silver staining.5. The MTT and trypan-blue staining was used to check out the viability of transfectedcolorectal cancer SW480 cells.6. Flow cytometry was used to analyses the rates of SW480 colon cancer cell apoptosis.Results:1. Enzyme identification and DNA sequencing proved that the eukaryotic expression vector ofRNA interference for the Sp1 gene was successfully constructed.2. Inhibition of Sp1 expression reduces the proliferation of SW80 cells may through inhibitingexpression of hTERT. The result of MTT showed that A490 value of the experimental group issignificantly lower than control group at same time and normal cells after transfecting plasmid inSW480 cells and the inhibition rates of 24h, 48h, 72h, 96h were 29.03%, 41.51%, 42.25%,45.16%.3. Sp1 gene silence may promote apoptosis of colorectal cancer by inhibiting the expression ofhTERT. Detected by flow cytometry, it was found that after silencing the Sp1 72 h SW480 cellsoccurred obvious apoptosis and its apoptosis rate is 15.2%±1.99% while the apoptosis rate is2.7%±1.07%.4. Recombinant vectors could reduce the expressions of SP1 protein. In SW480 the relative protein expression of Sp1 were 0.55±0.11 for SW480/p-shRNA, and 0.84±0.10 for SW480/p-CON, and 0.87±0.09 for SW 480 cells.5. Cells transfected with pGenesil-1-Sp1 (+) plasmid occurred the interference effect forhTERT mRNA. When compared with the control group and blank group, it was found that afterSp1 silencing the expression level of hTERT mRNA in colorectal cancer SW480 cells which isdetected by RT-PCR is significantly reduced.6. Transfected pGenesil-1-Sp1 (+) plasmid can reduce telomerase activity by inhibiting theexpression of Sp1 protein. Telomerase activity was detected by TRAP-PCR silver staining andthe result showed that TRAP analysis of characteristic bands in interference group SW480 cellsis not obvious when compared with the control group and blank group.ConclusiConclusion:1. RNA interference expression vector of targeting to Sp1 is successfully constructed and it caninhibit the expression and activity of Sp1, it prepared for exploring the function of Sp1 gene inCRC with RNA interference technique.2. Silencing the Sp1 expression by RNA interference could inhibit the migration and cellinvasion ability of SW480 cells, which was probably related with down-regulating theexpression of hTERT due to the decrease telomerase activation. It may provide a noveltherapeutic approach for color cancer therapy.