ABSTRACT
Objective To investigate the gene expression of PIWIL2 in the bladder urothelial carcinoma (BTCC) and siRNA interact on PIWIL2 gene expression in human bladder cancer cell line BIU-87.Methods Semi-quantitative reverse transcription polymerase chain reaction (qRT-PCR) was applied to detect the PIWIL2 expressions in tissues of BTCC (46 cases),cystitis glandularis(21 cases),adjacent non-cancerous tissues (17 cases) and normal bladder tissues (7 cases). 3 specific siRNA targeted PIWIL2 gene were synthesized after designed and transferred. After siRNA was transferred into BIU-87 cells, MTI and TUNEL methods were applied to detect the proliferation inhibitory rate (IR) and apoptosis index (AI) in BIU-87 cells,qRT-PCR and Western blot were used to examine effects of siRNA on the expressions of the PIWIL2 gene and protein,respectively.Results The expression rate of PIWIL2 mRNA in BTCC tissues was 76.08 ％(35/46) and significantly higher than those in the cystitis glandularis tissues (42.86 ％,9/21),adjacent non-cancerous tissues (41.17 ％,7/17) and normal tissues (7.14 ％,1/14) (P =0.008,P =0.010,P =0.000).The IR [(37.52±8.84) ％,(64.36±9.64)％] and (62.94±8.43) ％] and AI [(26.18±5.42) ％,(38.75±6.19) ％ and (40.02±5.64) ％] of BIU-87 cells in the siRNA 1～3 groups were respectively significantly higher than those [(1.97±0.02) ％ and (3.35±0.47) ％] in the control group(P=0.000),and expressions of PIWIL2 mRNA and protein in the siRNA groups were both lower than those in the control group. Moreover, the effects of siRNA 2 group and siRNA 3 group on inhibiting PIWIL2 expression, IR and AI of BIU-87 cells were stronger than siRNA 1 group. Conclusion The over-expression of PIWIL2 suggested that it played an important role in the mechanism of development and malignant progression of BTCC. The siRNA of transcription can significantly inhibit its expression, induce cell apoptosis and inhibit the growth of BIU-87 cells which might provide the experimental evidence for the gene targeting therapy of bladder tumor.
ABSTRACT
Objective To observe the effects of ( - )-epigallocatechin-3-gallate (EGCG) on human prostate cancer xenografted tumor growth and connexin43 expression in nude mice,and explore the mechanism of the EGCG on prevention for prostate cancer.Methods The methyl thiazolyl tetrazolium and annexin-V/PI double-labeled flow cytometry methods were used to observe the growth inhibiting rate (IR)and apoptosis rate (AR) of human prostate cancer cell line PC-3 which was treated by EGCG at different concentration (10,20 and 40 mg/L,respectively).The scrape-loading fluorescence dye transfer method was applied to assess the gap junction intercellular communication (GJIC) through fluorescence microscope.PC-3 cells were subcutaneously transplanted to establish tumor-bearing nude mice model.A total of 32 mice were randomly divided into four groups,both control group and three treatment groups were treated with different doses of EGCG ( 10,20 and 40 mg/kg,respectively).After two weeks,the mice prostate tumor tissues were taken out.The tumor wet weight was measured and tumor growth inhibiting rate was calculated.The tumor microvascular density (MVD) and apoptosis index (AI) were detected by the immunohistochemical techniques and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling techniques,respectively.Semi-quantitative reverse transcription polymerase chain reaction was used to examine the expression level of the Cx43 mRNA.Results EGCG at concentration ( 10 and 20 mg/L) could significantly inhibit the proliferation[(22.33 ±4.62)％,(38.67 ±5.67)％ vs (3.47 ±0.31 )％,P ＜0.01],induce the apoptosis [(7.84 ± 1.37 ) ％,( 24.53 ± 2.28 ) ％ vs ( 2.17 ± 0.70 ) ％,P ＜ 0.01] and enhance the GJIC of PC-3 cells.EGCG of different doses could inhibit prostate cancer xenografted tumor growth,induce tumor cells apoptosis and inhibit angiogenesis.EGCG ( 20 and 40 mg/kg) could effectively up-regulate Cx43 mRNA expression in xenografted tumor (0.58 ± 0.08,0.80 ± 0.07 vs 0.42 ± 0.04,P ＜ 0.0 ).The effects had significant correlation with the dose-dependent of EGCG ( P ＜ 0.05 ).Conclusions EGCG could up-regulate the Cx43 expression and enhance the gap junction intercellular communication mediated by Cx43 in the prostate tumor,which provide the experimental evidence for the mechanism of its effectively inhibiting the prostate cancer growth.