Silencing Of E2F3 Expression Using Hairpin SiRNA Expressing Vector Suppresses Growth Of Human Bladder Cancer

Bladder cancer is one of the most common urological malignant diseases in theworld. 70% of patients with bladder cancer have only noninvasive disease or tumorswith invasion no deeper than the lamina propria on initial presentation, and theremainders have muscle-infiltrating or deeper cancers. The most important features ofurothelial cancers of the bladder are metachronous or synchronous multifocaloccurrence with high frequency. Operation especially transurethral resection ofbladder tumor has been the main measure in treating bladder cancer, but afteroperation bladder cancer easily recurs and the recurrence cancer often increases itsprogression. For this reason the use of intravesical drugs such as chemical drug andimmune revulsive have been the most important part of treating. However thesetherapeutic methods we use can't control the progression and recurrence of bladdercancer and at the same time the side effects of drugs and drug fast in patients remainthe tough problem. Therefore human being is still exploring new effective treatmentsof bladder cancer. For more than twenty years research gene therapy has graduallybecome mature and is accepting as extremely promising approach to treating cancerafter regular treatment such as operation, chemotherapy and radiotherapy. Inhibitingthe expression of activated oncogene in cancer cells has gradually been one trend ofgene therapy. The discovery that RNAi works in mammalian cells has sparkedintense investigation into its role in normal mammalian cell function, its use as a toolto understand or screen for genes functioning in cellular pathways in healthy and diseased cells and animals, and its potential for therapeutic gene silencing.RNA interference (RNAi) is an evolutionarily conserved mechanism forsilencing gene expression. In primitive organisms, RNAi protects the genome fromviruses and other insertable genetic elements and regulates gene expression duringdevelopment. The antisense (guide) strand of short double-stranded RNAs isincorporated into an RNA-induced silencing complex that can either suppress proteinexpression or direct degradation of messenger RNAs that contain homologoussequence(s). Though what we know now about the molecular basis of RNAi and howit regulates gene expression is clearly just the tip of the iceberg, RNAi may providean important new therapeutic modality for treating illnesses especially for cancer.The tumorigenesis and development of tumor has intimate relationship with lossof control in cell cycle regulation. The E2F transcription factors control expression ofgenes that are essential for cell proliferation, including key components of both theDNA-replication and cell cycle control machinery. Retinoblastoma (Rb) isfunctionally inactivated in most, if not all, human tumors. The growth-suppressiveproperties of pRB are thought to be largely dependent upon its ability to regulate theE2F transcription factors. E2F3 have high transcriptional activity and is sufficient toinduce quiescent cells to enter S phase. Many E2F-responsive genes, including c-myc,cdc2, cyclin E, cyclin A, and E2f-1, are regulated primarily by E2F3. In this articlewe want to know if E2F3 play the key role in cell cycle regulation, if E2F3 can be anew target in bladder cancer gene therapy and if knocking-down E2F3 expression byusing RNAi can inhibit the proliferation of cancer cells in vitro and in vivo. Toanswer these questions we did the following jobs: we have developed potent RNAiplasmids corresponding to E2f3 gene, transfected the plasmid into human bladdercancer cancer cell lines 5637 and T24, assessed the specificity of shRNA byexamining the E2F3 protein levels and mRNA levels in 5637 and T24 cells. Our datashow that recombinant plasmids can specifically and efficiently inhibit E2f3 gene expression. So in the second and third phases of our research, we employed it to studythe response of cancer cells in vitro and mouse in vivo. Our results strongly suggestthat recombinant plasmid can effectively inhibit the proliferation and induce theapoptosis in 5637 and T24 cells. Thus, pRNA-siE2F3 targeting E2f3 gene mayprovide an innovative approach to cancer therapy.PartⅠStudy on the Expression of E2F3, C-myc, Bcl-2 in Tissue of Bladder TransitionalCell Carcinoma and Their Clinical SignificancesObjective: In this part we want to acquire the expression date of E2F3, C-myc andBcl-2 protein in bladder transitional cell carcinoma (BTCC) tissue and normalbladder epithelial tissue. At the same time we want to know whether there have somecorrelation among the expression of E2F3, C-myc, Bcl-2 protein and whether thesethree factors' expression have relationship with the biological behaviors of BTCC. Wecan understand the molecular mechanism of BTCC all the better from these dates andat the same time determine whether E2F3 can be used as a target gene in bladdercancer gene therapy.Methods: Immunohistochemistry was used to detect the expression of E2F3 andrelative factors (C-myc and Bcl-2) in BTCC (n=64) and normal bladder mucosa(n=10); Immunohistochemistry result were analysis by Image-pro Plus software andthe expression result was indicated by integrated optical density (IOD). E2F3 mRNAwas investigated using RT-PCR analysis in fresh bladder tumor tissues and normalbladder mucosa.Results: Immunohistochemistry indicated that the expression rate of E2F3 in BTCC(32.8%) was higher than normal bladder mucosa (0)(P＜0.01). The expression rate ofE2F3 was strongly correlatated with pathological grade. The C-myc expression levelbetween BTCC and normal bladder mucosa was 60.9% and 0, respectively(P＜0.01).The Bcl-2 expression rate between BTCC and normal bladder mucosa was 79.6% and 10% respectively(P＜0.01). Immunohistochemistry indicated that the IOD of E2F3 inBTCC was significantly higher than normal bladder mucosa (P＜0.01). The expressionlevel of E2F3 was strongly correlative with pathological grade and clinical stage(P＜0.01). The IOD of C-myc expression between BTCC and normal bladder mucosawas 62855±19007 and 0 respectively (P＜0.01). The expression of C-myc wasstrongly correlated with pathological grade and clinical stage (P＜0.01). The IOD ofBcl-2 in BTCC was higher than normal bladder mucosa, and at the same time itsexpression was strongly correlated with pathological grade (P＜0.01), but have norelationship with tumor clinical stage. RT-PCR indicated that the expression level ofE2F3 in BTCC (n=64) was 100% and in normal bladder mucosa (n=10) was 0%.Conclusion: Result indicated that E2F3 was the diagnostic and prognostic index ofBTCC, and it provided theory basis about the gene target therapy in BTCC. BecauseE2F3 can regulate the expression of C-myc and Bcl-2 we found that there is a closerelationship between E2F3 and C-myc or Bcl-2 expression in BTCC tissue. So thecooperation of the three factors may participate in the carcinogenesis and progress ofBTCC.PartⅡThe Silencing Effect of E2F3 Gene Using RNAi on the Proliferation andApoptosis of Bladder Cancer Cell Lines in VitroObjective: We want to construct three plasmids containing different short hairpinRNA (shRNA) of E2F3 and observe if they can suppress the expression of gene E2F3in human bladder cancer cell lines 5637 and T24. The silence effect of recombinatedplasmid on cell cycle, apoptosis and proliferation then is determined.Methods: siRNA oligo of E2F3 were screened from online siRNA design software,and then the secondary structure of the target mRNA were analyzed by the softwareof RNA-structure 3.2 edition. At last three siRNA oligos of E2F3 have beendetermined. The expression frame of shRNA was digested with BamHI and HindⅢat its two ends respectively and was synthesized by company. It was inserted into linedpRNAT-U6.1/Neo to construct pRNAT-U6.1-E2F3/Neo plasmid. Enzyme digestion,sequencing analysis and plasmids PCR has been used to determine the correction ofconstruction. Recombinant plasmids pRNAT-U6.1-E2F3/Neo were transfected withLipofectamine 2000 into human bladder cancer cell lines 5637 and T24 and thetransfection efficiency can be acquired by observing green fluorescence of cells.RT-PCT was used to determine if recombinant plasmids can downregulate theexpression of E2F3 mRNA and which shRNA has better result. The silencing effectcan also be detected by Western blot in protein levels. The change of cell morphologyand E2F3 protein expression level after transfection can also be detected byimmunocytochemistry and light microscope. The cell survival and proliferationcondition was determined by MTT study and cell clone. Cell cycle and apoptosis ofAGS cells after transfection were detected with Flowcytometry (FCM).Results: We can determine the successful construction of three recombinantpRNAT-siE2F3-1/-2/-3 by single enzyme digestion of KpnI, sequencing analysis andplasmids PCR. The recombinant pRNAT-siE2F3-1/-2/-3 without endotoxin wasextracted successfully and then was transfected into 5637 and T24 cells respectively.Green fluorescence can be seen after 12 hours in two cell lines and reach to maximumat 24 hours. Relative to control, three pRNAT-U6.1-E2F3/Neo induced (72.91±9.67)%, (69.75±13.61)%, (75.24±11.33)% inhibition of E2f3 mRNA at 48htreatment in 5637 cells (P＜0.01) and(65.4±7.76)%, (59±5.73)%, (33.6±1.72)%inhibition in T24 cells (P＜0.01). The first sequence was chosed to apply in laterexperiment because of the good silencing effect and secondary structure of targetmRNA. Comparing with control, E2F3 protein levels of 5637 and T24 cell.significantly reduced and inhibition rate is (85.07±2.05)% and (96.21±2.24)%respectively at 48h after transfecting pRNAT-siE2F3-1. MTT results showed that thecell survival rates of 5637 and T24 cells 96h after transfecting with pRNAT-siE2F3-1 were (46.15±5.06)% and (43.12±5.94)% respectively when comparing with blank.Cell cycle analysis showed that pRNAT-siE2F3-1 induced accumulation of cells inG1 phase by (26.53±4.55)% and (29.57±2.35)% with a significant decrease in thepercentage of cells in S-phase by (27.56±3.78)% and (26.99±2.26)% in 5637 andT24 cells relative to control. A significant apoptosis were also presented in 5637 andT24 cells transfected with pRNAT-siE2F3-1.Conclusion: The Lipofectamine 2000 was a better transfection reagent for RNAi byplasmid in 5637 and T24 cells. These sequence specific siRNAs showed a blockeffect in downregulation of E2f3 gene expression, inhibition of the cellularproliferation, arrestment of the cell cycle. Silencing E2F3 may be developed into apotential tool for bladder gene therapy.PartⅢThe Silencing Effect of E2F3 expression Using Plasmid RNAi Vector OnBladder Cancer Cells in VivoObjective: In this part we want to utilize bladder cancer cell lines 5637 to constructthe bladder cancer nude mice model and to investigate the effect induced by therecombinant pRNA-siE2F3-1 on the expression of E2F3 and the growth inhibitationof tumor in nude mice model. We can acquire more information about the therapyeffect of pRNA-siE2F3-1 in vivo and found the base for it using in patients in future.Methods: Balb-c nude mice, aged 4 weeks, were bred in the specific pathogen free(SPF) condition, and 2.0×10~6 5637 cells were inoculated intradermally on theventro-anterior limb surface of nude mice to establish xenograft tumor. After thediameter of xenograft tumor reach 8mm, 15 nude mice were divided into 3 groupsand injected with 200μl of 1×PBS, pRNAT-U6.1/Neo and lipofectamine 2000mixture, pRNAT-siE2F3-1 and lipofectamine 2000 mixture respectively in tumor.The xenograft tumors' diameter of three groups was measured every two days afteriniection, and so we can acquire inhibition ratio and growth curve of tumor. All the nude mice were sacrificed 7 days after treatment and every tumor was weighed andmeasured. RT-PCT and Western blot were used to determine if recombinant plasmidscan downregulate the expression of E2F3 mRNA and protein in xenograft tumor.Immunohistochemistry was used to detect the expression of E2F3 and Bcl-2 in threegroups.Results: In this part the nude mouse model of bladder cancer xenograft tumor wasconstructed successfully and 3 weeks later, the xenograft formation rate is 87.5%. Thetumor volume of recombinant pRNA-siE2F3-1 group decreased significantly whencomparing with PBS group and empty plasmid group 7 days after treatment. Theweight and volume of tumor body reduced after being transfected with therecombinant plasmid pRNA-siE2F3-1. From the tumor volume, the tumor growthinhibition rates in PBS group, empty vector group, and pRNAT-siE2F3-1 group were19.72%, 13.59% and 75.03% respectively. At the same time the weight of tumor inthe groups were 1.96±0.25g, 1.99±0.14g, 0.24±0.04g. All these dates can provedsRNA-EGFR can significantly inhibit the tumor growth in vivo both in size and inweight. Immunohistochemistry indicated that the IOD level of E2F3 and Bcl-2 inPBS group (210790±14405 and 98633±12248) was higher than pRNA-siE2F3-1group (38955±7439 and 30094±9886)(P＜0.01). We also displayed sequencespecific silencing of the E2F3 with (87.88±2.35)% of down-regulation of E2F3protein production, and (87.09±1.31)% of decrease of E2F3 mRNA level inpRNA-siE2F3-1 group when comparing with PBS control group.Conclusion: This study demonstrates pRNA-siE2F3-1 has anti-tumor function andcould inhibit the growth of tumor in nude mice. Injection of pRNA-siE2F3-1 can alsodecrease the expression of E2F3 and Bcl-2, all this perhaps be the reason of goodtherapeutic effect of the recombinant plasmid.