| Background:Esophageal cancer (EC),is highly aggressive, ran king as the sixth most commoncancer among males and the ninth most common cancer among females globally. ECpresents as two main types, each with distinct etiological and pathological characteristics.Esophageal squamous cell carcinoma (ESCC) is the predominant type of EC worldwide,accounting for more than90%of all cases. The5-year overall survival rate (12%) of ECremains poor[1].Due to early metas tasis and the invasion of surrounding tissues of thisdisease, Surgery remains to be the standard primary treatment and the best palliation forpatients with EC; however, the survival rate is still poor and lots of patients developmetastatic disease or locoregional recurrence soon after surgery. As an alternative toresection for the locoregional treatment of EC, there is evidence supporting the use ofcombined chemoradiotherapy over radiotherapy alone. Squamouscell carcinoma andadenocarcinoma of the esophagus are both responsive to chemotherapeutics, which makingchemotherapy feasible for treatment of EC.As one of the most widely used anticancer chemotherapy drugs for EC among thosecontaining platinum drugs, cisplatin (CDDP) is believed to induce tumor cell death resultingfrom the formation of CDDP-DN A adducts, which in hibit DN A replication andtranscription. However, the intrinsic or acquired resistance to CDDP in EC cells remains amajor obstacle to successful chemotherapy. Therefore,clarifing the precise mechanisms ofCDDP resistance—and reversing this resistance—would offer new strategies for cancertherapy. It is definedthat the mechanism of resistance to CDDP is multifactorial; manygenes or gene products have been affirmed to be responsible for CDDP resistance. Inaddition, it is well known that a number of cellular adaptations could cause the drug-resistance, including reduced uptake of the drug, inactivation by glutathione and otheranti-oxidants,and increased levels of DNA repair or DNA tolerance, all of these may lead tothe poor survival of EC.In1993, Wang found that the wild type p53expressed in the temperature sensitivestructures (temperature sensitive p53gene),which can trigger cell apoptosis of p53negativeT cell lymphoma cell line J3D induced by v-myc retrovirus[2].1995, Wang found a newp53induced gene--WIG-1[3]. Whole body gamma irradiation induced formation of bothwig-1transcripts in mouse brain, testis, kidney, spleen and lung. A basal wig-1expressionwas detectable in brain, testis and kidney. The WIG-1protein bears three zinc finger motifsand a putative nuclear localization signal[4]. Human WIG-1could bind with smallinterfering RNA and microRNA, as well as different types of double-stranded RNA,andplay a critical role in cell growth inhibition[5].Another studies showed that WIG-1couldinhibit significantly the proliferation of esophageal squamous cell carcinoma cell line[6].And some researchers found WIG-1might have relationship with cell growth and survivalregulation by microRNA, and could start cell growth arrest or apoptosis induced by p53[7].Because the WIG-1gene could effect on cellgrowth cycle and apoptosis, the researchersbelieved WIG-1may also play a role in the regulatory of drug resistance in tumor cells.Objective:The aim of this study is to construct the C DDP-resistant subline EC109/CDDP andexplore the effect of overexpr ession/knockd own WIG-1on cis platin resistance ofEsophageal squamous cell carcinoma and its possible mechanisms.Methods:1. Exposing the parental EC109cells to CDDP intermittently to develop theCDDP-resistant subline EC109/CDDP.2. Constructing Lentiviral Vectors for the Expression of Small Interfering HairpinRNAs (shRNAs) and the Human WIG-1Gene.3. By lentiviral vectors WIG-1/shWIG-1were expressed in EC109/CDDP cells. UsingCCK assay to evaluate sensitivity of WIG-1/shWIG-1EC109/CDDP cells to cisplatin..4. Detect expressions of WIG-1, Bcl-2, Bax, GST-, ERCC1and some cell signaltransduction gene by Western blot analysis.Apoptosis rate and cell cycle were examinedby FACS. 5. Establish the xenografts model of EC109/CDDP cell on the nude mice. Evaluate thesensitivity of WIG-1/shWIG-1EC109/CDDP cells to cisplatin ed in the model.Results:1. EC109/CDDP line was obtained.2. Lentiviral Vectors for the Expression of shRNAs and the Human WIG-1Gene weresuccessfully constructed. The WIG-1gene was stablely down or upregulated after theWIG-1/shWIG-1-containing lentiviral vectors was transferred into cell strain EC109/CDDP.3. Up-regulation of WIG-1significantly decreased the tolerance of EC109/CDDP cellsto cisp latin and down-regulation of WIG-1sign ificantly increased the tole rance ofEC109/CDDP cells to cisplatin.4. Over-expression of WIG-1in EC109/CDDP cells resulted in more apoptotic cells.Hypo-expression of WIG-1in EC109/CDDP cells led to less cells in apoptotic status.5. The xenografts model of EC109/CDDP cell were established successfully on thenude mice.6. In the xenografts model we also found that up-regulation of WIG-1significantlydecreased the tolerance of EC109/CDDP cells to cisplatin and down-regulation of WIG-1significantly increased the tolerance of EC109/CDDP cells to cisplatin.7. Over-expression of WIG-1in EC109/CDDP cells significantly decreased ERCC1and increased Bax protein levels. Less-expression of WIG-1in EC109/CDDP cellssignificantly increased ERCC1and decreased Bax protein levels.Conclusion:1. Lentiviral Vectors for the Expression of shRNAs and the Human WIG-1Gene weresuccessfully constructed. The sub cell lines EC109/CDDP/WIG-1and EC109/CDDP/shWIG-1were established after the lentiviral vectors was transferred into cell EC109/CDDP.2. Up-regulation of WIG-1significantly decreased the tolerance of EC109/CDDP cellsto cisplatin, possibly due to1) the inhibition of ERCC1expression;2) the up-regulation ofBax expression.And vice versa.3. Up-regulation of WIG-1significantly decreased the tolerance of EC109/CDDP cellsto cisplatin in the xenografts model and vice versa. It uncovered that WIG-1gene could reverse drug resistance of tumor cells, and it is a novel gene regulating the tumor cell drugresistance.4. GFP-based fluorescent optical tumor imaging system can also be utilized to evaluatethe sensitivity of tumors to chemotherapeutics in vivo.which is more accurate, intuitive andreliable。... |
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