The Effects Of △Np63 Specific ShRNA On Bladder Cancer

PART ONE EXPRESSION OF DELTA NP63 MRNA AND PROTEIN IN TRANSITIONAL CELL CARCINOMA OF THE BLADDERObjective:to explore the expression and clinical significance of delta N p63 mRNA and protein in transitional cell carcinoma of the bladder(TCCB).Methods:The reverse transcriptase-polymerase chain reaction(RT-PCR) assay was conducted to detect expression of delta Np63 mRNA in 24 cases of TCCB and 10 cases of control bladder epithelium. The immunohistochemical staining assay was conducted to detect expression of delta Np63 protein in 43 cases of TCCB and 11 cases of control bladder epithelium. The relationships between delta Np63 mRNA and protein expression, pathological types and clinical stages of TCCB were also analyzed.Resultes:The expression rate of delta N p63 mRNA and protein in TCCB were 75.0%(18/24)and 100%(43/43) respectively, The expression rate of delta Np63 mRNA and protein in control bladder epithelium were 18.2%(2/11)and 0.0%(0/10)respectively. The differences expression rate of delta Np63 mRNA and protein in different pathological types and clinical stages of TCCB were no significant .Conclusion:There were high expression of delta N p63 mRNA and protein in TCCB, that means delta Np63 maybe associated with tumorigenesis in TCCB, but may not be associated with pathological types and clinical stages of TCCB. Additional studies will be required to explore the function of delta N p63 in the progression of TCCB.PART TWO CONSTRUCTION OF DELTA N P63 SPECIFIC SMALL HAIRPIN RNA EXPRESSING PLASMIDObjective : To construct delta Np63 specific small hairpin RNA(shRNA) expression plasmid, and exam the inhibition effects of delta Np63-shRNA in 5637,a cell line of transitional cell carcinoma of the bladder(TCCB).Methods: Delta Np63-shRNA oligonucleotides were synthesized and ligated into genesil-1 plasmid.The recombinant Pgenesil-1 plasmid was transfected into 5637 conducted by transfection reagent.The delta Np63 mRNA levels were examed by semi-quantitative reverse transcriptase-polymerase chain reaction(RT- PCR).Resultes: The delta Np63-shRNA expression plasmid was confirmed by using PstI+SalI double digestion and by sequencing. The delta Np63 mRNA level in 5637 cells treated with delta Np63-shRNA was much lower than it in 5637 cells treated with control vector or PBS(p<0.05).Compared to the control vector and PBS group, the inhibition rate of delta Np63-shRNA onΔNp63 mRNA was 57.3% and 63.0%. Conclusion:Our research demonstrated that delta Np63-shRNA can effectively inhibit the expression of delta Np63 mRNA in 5637 cell line.PART THREE RESEARCH OF DELTA N P63 SPECIFIC SMALL HAIRPIN RNA EXPRESSING PLASMID IN TCCB CELL LINEObjective: Transfected deltaN p63 specific small hairpin RNA(shRNA) expression plasmid into 5637 cells,to exam its inhibit the expression of delta N p63 protein and mRNA in TCCB, its effect on TCCB cells cycle and proliferation,its affect on cyclin D1 and cdk4.Methods: The fluorescence was used to confirm the success of transfection in 5637 cells under the fluorescence microscope. The inhibitory effect of delta Np63-shRNA construct was examed with RT-PCR and the immunohistochemical staining assay. The cell cycle of TCCB cells was assayed by flow cytometry(FCM) .The cellular proliferation of 5637 cells was assayed by tetrazolium bromide (MTT) colorimetry,delta N p63 and ckd4 protein was examed with the immunohistochemical staining assay.Results: The delta Np63-shRNA expression plasmid was successfully constructed, and was successfully transfected into 5637 cells, it can effectively reduce the expression of delta N p63 protein and mRNA, the reduce rate of delta N p63 mRNA was 63.0%, the G₀/G₁ cycle was increased and S cycle was decreased in transfected TCCB cells, the cellular proliferation was also lower in transfected 5637 cells compared with no-transfected 5637 cells,delta N p63-shRNA directly down-regulated The expression of delta N p63 protein and mRNA , and the ckd4 protein was also reduced.Conclusion:The delta N p63-shRNA expression plasmid which was constructed by Pgenesil-1 plasmid can successfully transfected into 5637 cells and can effectively inhibit the expression of delta N p63 protein and mRNA, it also can control the cell cycle and inhibit the cellular proliferation of 5637 cells,it also can inhibit the cellular proliferation of TCCB through inhibit cyclinD1 and ckd4.PART FOUR INHIBITORY EFFECT OF THE TUMOR GROWTH BY SILENCING DELTA NP63 GENE WITH SHORT HAIRPIN RNA ON TCCB IN XENOGRAFT NUDE MICEObjective:To explore the inhibitory effect of tumor growth by silencing delta Np63 gene with short hairpin RNA on human TCCB in xenograft nude mice through RNAi technique.Methods:Human TCCB 5637 cells were transplanted into nude mice to establish xenograft tumors, delta Np63 specific shRNA expression plasmid was transfected into the tumors, the tumor volume was observed, tumor morphology was observed with HE staining, The inhibitory effect of delta Np63 gene was examed with semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR), delta N p63 protein was examed with the immunohistochemical staining assay,delta Np63,cyclin D1 and ckd4 protein was examed with the immunohistochemical staining assay.Results: After 8 weeks of delta Np63 specific shRNA expression plasmid transfected,the tumor volume was significantly smaller in delta N p63 - shRNA group than in control - shRNA and normal saline group(p<0.01), the inhibition rate was 74.44% in delta N p63-shRNA group, there was no significant difference in control-shRNA and normal saline group(p>0.05). In delta Np63-shRNA group the necrosis and decrease of karyokinesis were fond under light microscope inflammatory cell were also fond, there was no such change in control-shRNA and normal saline group. delta Np63-shRNA directly down-regulated The expression of delta Np63 protein and mRNA,delta Np63-shRNA directly down-regulated The expression of delta Np63 protein and mRNA , and the cyclinD1 and ckd4 protein was also reduced.Conclusion:The delta Np63-shRNA expression plasmid which was constructed by Pgenesil-1 plasmid can significantly inhibit the growth of TCCB in nude mice,it also can inhibit the cellular proliferation of TCCB through inhibit cyclinD1 and ckd4.