The Effects Of STAT3and Survivin Silencing On The Growth Of Human Bladder Carcinoma Cells

Background:Bladder cancer is the malignant tumor that occurring to the bladdermucosa. The incidence of bladder cancer gains the second placeafter prostate cancer in the west. In our country, bladder cancer is amongthe ten major carcinomas，and it is the malignant tumor of urinary systemwith the highest morbidity and mortality. Bladder cancer can occur at anyage, even in children. The incidence of bladder cancer was6.61/10million by the National Cancer Registry of China in2012, taking theninth place. The incidence of bladder cancer in males is10/10millionwhich is3-4times higher than in females, taking the sixth malignanttumor in2013.Bladder cancer is difficult to deal with because of some malignantbiological characteristics, such as multifocal, multiple genes, multi-stageheterogeneity, high incidence rate and high recurrence rate, et al.The current treatments for bladder cancer are operation, radiotherapyand intravesical administration of anticancer drugs. Patients’ quality oflife declines dramatically with the high injury methods. These meanscannot solve the high relapse rate (more than60%) and high resistanceproblem, either. Now, small interfering RNA gene target have gainedgreat attention of scientific researchers in therapy to bBladder cancer.RNA interference is post-transcriptional gene silencing. SiRNA canrecognize and cleave the homologous gene and block its expression. Thismethod can restrain gene expression of cancer cell, but it does notinterfere with the normal physiological activity of cells. SiRNA has thepotential for gene therapy of carcinoma and virus infection. It provides anew reliable method to investigate gene therapy for bladder cancer.The key of gene therapy is to find the target, so we selected STAT3 and Survivin as our experimental object.STAT3is the connection and regulation with multiple tumor signaltransduction pathways. It participates in tumor invasion, metastasis,angiogenesis, apoptosis resistance and immune escape process. It is oneof the most important known carcinoma genes.Survivin is a new member of the inhibitor of apoptosis (IAP) family.Survivin, like the others, was discovered by its structural homology toIAP family of proteins in human B-cell lymphoma. It is proteaseinhibitory effecter through multiple ways.Survivin is expressed in all malignant tumors while it isn’t expressedin normal cells. Survivin can be a suitable target gene for antitumorimmunotherapy.Objective:Despite more and more evidence suggesting an important roleof signal transducer and activator of transcription3(STAT3) andSurvivin in human bladder cancer, the effects of silencing these geneseach or both on the proliferation of T24bladder carcinoma cell remainsunknown. Here, we investigated the inhibitory effects of STAT3and (or)Survivin silencing on the in vitro and in vivo growth of human T24bladder carcinoma cells. Our work can be basis for target selectionof gene interference therapy for bladder cancer.Methods:1Design two groups of small interfering RNA online, then verifythe specificity and effectiveness of small interfering RNA onbioinformatics methods.2Use Invitrogen kit to prepare expression vector, then transfectT24cells with vector.3The gene expression level final and before of STAT3and Survivin genes were detected by real-time fluorescence quantitative PCR.The difference in Protein content were compared before and after withwestern blotting. Determination of cell proliferation and cell activity wasdetected by MTT.4Compound small interference RNA was designed. Experimentsabove were carried out to detect the inhibition.5The murine model of bladder cancer was established. Theinhibition effects in vivo were determined by detecting tumor volume andweight.Data were analyzed using SPSS v17.0software. Comparisonbetween two groups was carried out by using a student’s t-test. Statisticalsignificance among multiple groups was determined using one-wayanalysis of variance. Data are presented as means±standard deviation(SD). P <0.05was considered to be statistically significant.Results:1Four STAT3and four Survivin siRNAs were designed andsynthesized.2Among these three plasmids, three siRNAs for STAT3wereefficient at knocking down STAT3expression. The STAT3-3constructwas the most efficient reduction by mRNA and protein expression ofSTAT3in T24cells. All of the Survivin siRNA constructed efficiently.The Survivin-4construct was the most efficient reduction by mRNA andprotein expression of Survivin in T24cells. Moreover, the STAT3-3andSurvivin-4reduced STAT3and Survivin respectively. Their complexdecreased the expression of STAT3and Survivin critically. There was nosignificant difference between the three siRNAs in nucleic acids and celllevel.3Significant reduction in weight and size of mice bladder cancer tumor was found. The difference was statistically significant.Conclusion:We found that silencing STAT3or Survivin in vitro and in vivosignificantly suppressed the proliferation of the T24bladder cancer cellline. The series compound siRNA could inhibit both STAT3and Survivindramatically. Our work provide a new way for the gene drug discovery.At the same time, simultaneous inhibition of these two genes could notsignificantly increase the anticancer effect in T24cells. So STAT3andSurvivin may work in the same signal pathway in T24cells. These resultsprovide valuable materials for the study on occurrence and developmentof bladder cancer. It also indicates the target for clinical therapy ofbladder cancer.