ABSTRACT
Emerging evidence has indicated that circular RNAs (circRNAs) play pivotal roles in the regulation of cellular processes and are found to be aberrantly expressed in a variety of tumors.However,the clinical role of circRNAs in bladder cancer (BC) and the molecular mechanisms have yet to be fully understood.In this study,the clinical specimens were obtained and the expression level of a circRNA BCRC4 was detected by real-time PCR in both BC tissues and cell line.The circular RNA over-expression plasmid was constructed and transfected into BC cells and related cell line.The cell cycles and apoptosis were observed using inverted microscope and flow cytometry.Western blotting was used to compare the relative protein expression of groups with different treatments.It was found that circRNA BCRC4 expression was lower in BC tissues than in adjacent normal tissues.Furthermore,consequences of fomed-expression of BCRC4 promoted apoptosis and inhibited viability of T24T and UMUC3 cells,and up-regulated BCRC4-inereased miR-101 level,which suppressed EZH2 expression in both RNA and protein levels.In addition,gambogic acid (GA) is a promising natural anticancer compound for BC therapy,and GA treatment increased the BCRC4 expression in T24T and UMUC3 cells in a dose-dependent manner.Altogether,our findings suggest that BCRC4 functions as a tumor suppressor in BC,and mediates anticancer function,at least in part,by up-regulating the expression of miR-101.Targeting this newly identified circRNA may help us develop a novel strategy for treating human BC.
ABSTRACT
The effect of Smac gene on the TRAIL-induced apoptosis of the prostate cancer cell line PC-3 and the molecular mechanism were investigated.The Smac gene was transfected into PC-3 cells under the induction of liposome.The intrinsic Smac gene expression was detected by Western blotting.After treatment with TRAIL as an apoptosis inducer,in vitro cell growth activity was assayed by MTT colorimetry.The apoptosis rate of PC-3 cells was determined by annexin V -FITC and propidium iodide staining flow cytometry.The expression of cellular XIAP and caspase-3 genes was examined by Western blotting.Smac-transfected cells (PC-3/Smac group) had significantly increased Smac protein level as compared with PC-3 controls (P＜0.01).After induction with 100-200 ng/mL TRAIL for 12-36 h,cellular proliferation rate in PC-3/Smac group was significantly lower than in PC-3 controls (P＜0.05).After induction with 100 ng/mL TRAIL for 24 h,the apoptosis rate in PC-3/Smac group was significantly enhanced as compared with that of PC-3 controls (P＜0.05).Accordingly,the XIAP expression level was down-regulated significantly (P＜0.05) and caspase-3 subunit P20 was up-regulated significantly (P＜0.05).It is suggested that the over-expression of cellular Smac can inhibit inhibitor of apoptosis proteins (IAPs),enhance caspases activity and the apoptosis rate of PC-3 cells induced by TRAIL,which may provide a useful experimental basis for prostate cancer therapy.
ABSTRACT
The growth inhibition and pro-apoptosis effects of dracorhodin perchlorate on human prostate cancer PC-3 cell line were examined.After administration of 10-80 μmol/L dracorhodin perchlorate for 12-48 h,cell viability of PC-3 cells was measured by MTT colorimetry.Cell proliferation ability was detected by colony formation assay.Cellular apoptosis was inspected by acridine orange-ethidium bromide fluorescent staining,Hoechst 33258 fluorescent staining,and flow cytometry (FCM) with annexin V-FITC/propidium iodide dual staining.The results showed that dracorhodin perchlorate inhibited the growth of PC-3 in a dose- and time-dependent manner.IC50 of dracorhodin perchlorate on PC-3 cells at 24 h was 40.18 μmol/L.Cell clone formation rate was decreased by 86％ after treatment with 20 μmol/L of dracorhodin perchlorate.Some cells presented the characteristic apoptotic changes.The cellular apoptotic rates induced by 10-40 μmol/L dracorhodin perchlorate for 24 h were 8.43％ to 47.71％ respectively.It was concluded that dracorhodin perchlorate significantly inhibited the growth of PC-3 cells by suppressing proliferation and inducing apoptosis of the cells.
ABSTRACT
In our previous study,we identified a novel testis-specific expressed gene 2(TSEG-2)from mouse testis.To further investigate its functions,35 male Balb/c mice(8 weeks old)were divided into cryptorchidism group(n=20),sham group(n=10),and control group(n=5).In cryptorchidism group,the right testes were anchored to the inner lateral abdominal wall.In situ hybridization(ISH)was applied to measure the localization of TSEG-2 in mouse testis.Real-time quantitative PCR was performed to detect the expression of TSEG-2 gene.Meanwhile,under the mediation of polyethylenimine(PEI),the recombinant vector pEGFP-TSEG-2(n=5)or empty vector(mock,n=5)was transfected into the testis of male mice.The transfection efficiencies were measured under a fluorescence microscope.The apoptosis of spermatogenic cells was detected by terminal deoxynuleotidyl-mediated nick end labeling(TUNEL).The results showed that TSEG-2 was expressed in convoluted seminiferous tubules,more precisely,in spermatogonia and spermatocytes.As compared with sham and control groups,the TSEG-2 transcription was significantly enhanced(P<0.05)and was correlated with apoptosis of spermatogenic cells in cryptorchid testes(P<0.05).PEI was efficient in mediating transfection of TSEG-2 into seminiferous tubules of testis.One week post-transfection,intratesticular injection of TSEG-2 resulted in increased apoptosis of spermatogenic cells in vivo (P<0.05).These results indicate that TSEG-2 may participate in the apoptosis of spermatogenic cells and the pathogenesis of cryptorchidism.
ABSTRACT
In this study,RNA interference technique was employed to silence the expression of DNMT1 and/or DNMT3b in human bladder cancer T24 cells.The expression levels of their mRNA and protein were greatly decreased by up to 75% and 65% respectively after T24 cells were transfected with lipofectamine2000 for 72 h,indicating RNA interference is an effective tool in gene knockdown.Proliferation and apoptosis of T24 cells were detected by MTT,and annexin-V-FITC and propidium iodide staining flow cytometry,respectively.It was found that loss of the DNMT1 or DNMT3b expression could inhibit the cell growth and promote the cell apoptosis to some extent.However,combined treatment with shRNA targeting both DNMT1 and DNMT3b mRNA could ob-viously enhance the above effects.It was concluded that simultaneously silencing both genes could result in strong suppressing effect on tumor proliferation and promoting ceil apoptosis than separate use,suggesting combined use of DNMT1 and DNMT3b can achieve a synergistic effect in the CpG island methylation in human bladder tumorigenesis.
ABSTRACT
ysis, which had important reference values for further studying biological functions of UP Ⅱ gene and targeted therapeutic strategy for TCC of bladder.
ABSTRACT
The present study was aimed at finding an effective method to isolate and purify the subtype of type A spermatogonial stem cells (SSCs) in juvenile rats. Testes from 9-days-old rats were used to isolate germ cells by using two-step enzymatic digestion. The expression of c-kit in the testes of the rats was immunohistochemically detected. After isolation, cell suspension was enriched further by discontinuous density gradient centrifugation. Then type A1-A4 spermatogonia was isolated from the purified spermatogonia with c-kit as the marker by using fluorescence-activated cell sorting(FACS). Electron microscopy was used to observe their ultrastructure. Finally, highly purified and viable subtype of SSCs was obtained. Cells separation with discontinuous density gradient centrifugation significantly increased the concentration of c-kit positive cells [(18.65±1.69)% after the centrifugation versus (3.16±0.84)% before the centrifugation, P＜0.01]. Furthermore, the recovery and viability were also high [(65.9±1.24)% and (85.6±1.14)%]. It is concluded that FACS with c-kit as the marker in combination with discontinuous density gradient centrifugation can well enrich type A1-A4spermatogonia from the testes of 9-days-old rats.
ABSTRACT
It has been suggested that progression of bladder transitional cell cancer (BTCC) may be regulated at the molecular level by a typical pattern of expression of genes involved in apoptosis. Re- cently Livin, belonging to the inhibitors of apoptosis (IAP) family, has been found to be expressed in most solid tumors, where its expression is suggested to have clinical significance. In order to explore the significance of Livin expression in the development of BTCC, immunohistochemistry and RT-QPCR were used to detect the expression of Livin mRNA in tumor tissues and adjacent normal tissues of 30 cases of BTCC. The results showed that the positive rate of Livin expression in adjacent normal tissues and tumor tissues was 0 and 60% (18/30) respectively. The -△△CT value of Livin in BTCC tissues was 8.0454 (7.4264-8.6644) times of that in adjacent normal tissues. The expression of Livin mRNA had no correlation with tumor pathological grades and clinical stages. It was sug- gested that there was weak expression ? Livin mRNA in adjacent normal tissues, but strong in tumor tissues.
ABSTRACT
To investigate the effects of diethylstilbestrol (DES) in reestablishing spermatogenesis and the mechanism by which estrogen works on spermatogenesis, rats were exposed to 1% 2,5-HD for 5 week. Then 0.1 mL of DES was given (s.c.) at a rate of 0.3 μg/kg, 30 μg/kg, 3 ms/kg every other day for 2 weeks respectively (DES group) while the other rats received ethyldeate only. Plasma testosterone (T) and LH were measured on the 8th week after the treatment. The rats were killed at the 18th week. The left testis was histopathologieally examined. In all the rats in the DES groups, spermatogenesis was re-established and the rats in the 30 μg/kg group showed the best results. Serum T was suppressed markedly in rats of 30 μg/kg and 3 mg/kg groups while T was only mildly inhibited in 0.3 μg/kg group, without significant difference found in serum LH. It is concluded that the nearly complete testicular atrophy could be reversed by DES treatment in rats. Estrogen plays an important part in spermatogenesis, and the role of estrogen in spermatogenesis is more than suppressing the hypothalamo-pituitary-testis axis.
ABSTRACT
The effects of synthetic Smac peptide (SmacN7) on chemotherapeutic sensitivity of bladder cancer cells were investigated. SmacN7 penetratin peptide was synthesized and delivered into T24 cells. MTT assay was used to evaluate the viability of T24 cells induced by low-dosage of MMC. Flow cytometry was used to analyze the proportions of apoptosis. Western blot was used to detect the expression of XIAP and Caspase-3. The activity of Caspase-3 was measured and the effect of SmacN7 combined with MMC on T24 cell lines was also determined. The results showed that SmacN7 penetratin peptide could successfully interact with endogenous XIAP, increase the proportions of apoptosis of T24 cell lines induced by low-dosage of MMC in a dose-and time-dependent manner. An obvious down-regulation of XIAP expression and up-regulation of Caspase-3 was identified by Western blot. The activity of Caspase-3 in experimental group was significantly increased as compared with that in the control group. As compared with MMC group, the viability of T24 cells in SmacN7 penetratin peptide + MMC group was markedly decreased to 2.22 and 3.61 folds at 24h and 48h respectively. It was concluded that SmacN7 penetratin peptide could act as a cell-permeable IAP inhibitor, inhibit the proliferation, induce apoptosis and enhance the chemo-sensitivity of bladder cancer cells to MMC. These findings indicate that SmacN7 penetratin peptide may be a very promising ageut for bladder cancer treatment when used in combination with chemotherapy.