Overexpression of CircRNA BCRC4 regulates cell apoptosis and MicroRNA-101/EZH2 signaling in bladder cancer.

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 forced-expression of BCRC4 promoted apoptosis and inhibited viability of T24T and UMUC3 cells, and up-regulated BCRC4-increased 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.

Overexpression of CircRNA BCRC4 Regulates Cell Apoptosis and MicroRNA-101/EZH2 Signaling in Bladder Cancer / 华中科技大学学报（医学）（英德文版）

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.

Ultrasonography-guided percutaneous nephrolithotomy with Chinese one-shot tract dilation technique based on stimulated diuresis: A report of 67 cases.

The safety and effectiveness of a novel Chinese one-shot dilation technique based on stimulated diuresis for percutaneous nephrolithotomy (PCNL) were investigated. After the feasibility of the Chinese one-shot dilation based on stimulated diuresis was verified by an animal study, this technique was applied in the clinical practice. A total of 67 patients in our department underwent the modified PCNL from July 2014 to June 2015. After the renal infundibulum was distended by stimulated diuresis, the kidney was punctured under the ultrasonographic guidance via the fornix of the target calyx. The working channel was dilated using a special designed pencil-shaped fascial dilator. The successful access rate, nephrostomy tract creation time, pre- and postoperative hemoglobin values and serum creatinine concentrations, stone-free rate and complications were recorded and analyzed. The renal infundibulum was successfully distended in all of the patients by the diuresis treatment. Under the ultrasonographic guidance, the successful access rate was 100% and the mean tract creation time was 2.0 min (range: 1.5-5.0 min). The stone-free rate right after surgery was 91.0%. Although the postoperative hemoglobin was significantly reduced (P<0.01), transfusion was not clinically necessary. There was no significant difference in serum creatinine concentrations before and after operation (P>0.05). No severe complication occurred during or after the PCNL. It was suggested that this Chinese one-shot dilation technique based on stimulated diuresis is an efficient and safe innovation for PCNL, and is even helpful for those patients with non-dilated pelvicaliceal systems.

Long non-coding RNA MEG3 induces renal cell carcinoma cells apoptosis by activating the mitochondrial pathway.

This study aimed to examine the effect of long non-coding RNA (LncRNA) MEG3 on the biological behaviors of renal cell carcinoma (RCC) cells 786-0 and the possible mechanism. MEG3 expression levels were detected by RT-qPCR in tumor tissues and adjacent non-tumor tissues from 29 RCC patients and in RCC lines 786-0 and SN12 and human embryonic kidney cell line 293T. Plasmids GV144-MEG3 (MEG3 overexpression plasmid) and GV144 (control plasmid) were stably transfected into 786-0 cells by using lipofectamine 2000. Cell viabilities were determined by MTT, cell apoptosis rates by flow cytometry following PE Annexin V and 7AAD staining, apoptosis-related protein expressions by Western blotting, and Bcl-2 mRNA by RT-qPCR in the transfected cells. The results showed that MEG3 was evidently downregulated in RCC tissues (P<0.05) and RCC cell lines (P<0.05). The viabilities of 786-0 cells were decreased significantly after transfection with GV144-MEG3 for over 24 h (P<0.05). Consistently, the apoptosis rate was significantly increased in 786-0 cells transfected with GV144-MEG3 for 48 h (P<0.05). Furthermore, overexpression of MEG3 could reduce the expression of Bcl-2 and procaspase-9 proteins, enhance the expression of cleaved caspase-9 protein, and promote the release of cytochrome c protein to cytoplasm (P<0.05). Additionally, Bcl-2 mRNA level was declined by MEG3 overexpression (P<0.05). It was concluded that MEG3 induces the apoptosis of RCC cells possibly by activating the mitochondrial pathway.

The effects of intravesical therapy with hyaluronic acid for painful bladder syndrome: Preliminary Chinese experience and systematic review.

OBJECTIVE: To present the preliminary results of treating a series of Chinese patients with painful bladder syndrome/interstitial cystitis (PBS/IC) using intravesical hyaluronic acid (HA). MATERIALS AND METHODS: A series of 13 patients with PBS/IC received first-line therapy followed by HA once-a-week for 4 weeks and then once monthly for 4 months. Outcomes measured included O'Leary-Sant Interstitial Cystitis Symptom Index (ICSI) and Interstitial Cystitis Problem Index (ISPI) scores, voiding frequency, and bladder capacity. RESULTS: ISPI and ICSI scores were significantly (p < 0.001) decreased after treatment [median change (interquartile range): ISPI = 2 (2-3); ICSI = 3 (2-3)]. Voiding frequency and functional bladder capacity were significantly (p < 0.001) decreased [median change: 7 (6-8) times/d] and increased [median change: 190 (116-233) mL], respectively after treatment. CONCLUSION: Our case series supports the efficacy of intravesical HA in the treatment of PBS/IC.

Over-expression of testis-specific expressed gene 1 attenuates the proliferation and induces apoptosis of GC-1spg cells.

The effects of over-expression of testis-specific expressed gene 1 (TSEG-1) on the viability and apoptosis of cultured spermatogonial GC-1spg cells were investigated, and the immortal spermatogonial cell line GC-1spg (CRL-2053™) was obtained as the cell model in order to explore the function of TSEG-1. We transfected the eukaryotic vector of TSEG-1, named as pEGFP-TSEG-1 into cultured spermatogonial GC-1spg cells. Over-expression of TSEG-1 inhibited the proliferation of GC-1spg cells, and arrested cell cycle slightly at G0/G1 phase. Transfection of TSEG-1 attenuated the transcript levels of Ki-67, PCNA and cyclin D1. In addition, over-expression of TSEG-1 induced early and late apoptosis, and reduced the mitochondrial membrane potential of GC-1spg cells. Moreover, transfection of TSEG-1 significantly enhanced the ratio of Bax/Bcl-2 and transcript levels of caspase 9, and decreased the expression of Fas and caspase 8 in GC-1spg cells. These results indicated over-expression of TSEG-1 suppresses the proliferation and induces the apoptosis of GC-1spg cells, which establishes a basis for further study on the function of TSEG-1.

[MiR-124 suppresses the proliferation of human prostate cancer PC3 cells by targeting PKM2].

OBJECTIVE: To explore the mechanism of miR-124 inhibiting the proliferative activity of prostate cancer PC3 cells. METHODS: Luciferase reporter gene assay was used to examine the specific binding ability of miR-124 to PKM2 mRNA 3'-UTR. After miR-124 was transfected mimic to PC3 cells, the expression levels of PKM2 mRNA and protein were detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot, respectively. The effects of miR-124 mimic and PKM2 siRNA on the proliferative activity of the PC3 cells were determined by MTT assay. RESULTS: The expressions of PKM2 mRNA and protein were upregulated (5.12 +/- 0.35) times and (4.05 +/- 0.20) times respectively in the PC3 cells as compared with those in the RWPE-1 cells (P < 0.05). Luciferase reporter gene assay demonstrated that miR-124 targeted PKM2 3'-UTR. At 24 hours after transfection with miR-124 mimic, the PKM2 protein expression in the PC3 cells was downregulated (0.16 +/- 0.04) times (P < 0.05), while the PKM2 mRNA level was not changed significantly (P > 0.05), as compared with the control group. MTT assay showed that both miRNA-124 mimic and PKM2 siRNA could inhibit the proliferation of the PC3 cells, but the former exhibited a greater inhibitory effect than the latter. After transfection with miR-124 mimic and PKM2 siRNA, the cell growth rates were (66.20 +/- 5.10)% vs (82.10 +/- 6.35)% at 24 hours (P < 0.05) and (49.34 +/- 2.37)% vs (70.10 +/- 5.80)% at 48 hours (P < 0.05). CONCLUSION: miR-124 can suppress the proliferation of PC3 cells by regulating the PKM2 gene.

[Application of one-stop diagnosis and treatment plan in percutaneous nephrolithotomy for patients with complex renal calculi].

OBJECTIVE: To evaluate the safety and efficacy of preoperative computed tomography urography (CTU) three-dimensional reconstruction, intraoperative radiology and ultrasound guidance followed by percutaneous nephrolithotomy (PCNL) in the treatment of complex renal calculi. METHODS: We summarized the clinical data of 210 patients with complex renal calculi treated at our hospital from December 2008 to December 2011 in this retrospective study. In the one-stop diagnosis and treatment group (n = 119), the optimal puncture approach was designed according to CTU imaging and three-dimensional reconstruction. Percutaneous track was established by ultrasound and radiology guided puncture. PCNL was performed with EMS system. The control group (n = 91) underwent PCNL without radiological guidance. The success rate of puncture, mean accessing time, mean operative duration, intraoperative volume of blood loss and stone-free rate after one operative session were observed. Post-operative follow-ups were conducted until June 2012. RESULTS: Compared to the control group, the one-stop diagnosis and treatment group showed a higher success rate of puncture [98.3% (117/119) vs 92.3% (84/91), P = 0.037], a shorter operative duration [97.8 ± 13.20 vs 110.0 ± 14.73 min, P = 0.043] and a higher stone-free rate after one operative session [92.4% (110/119) vs 83.5% (76/91), P = 0.037]. No significant difference was detected in the mean accessing time[15.3 ± 3.7 vs 13.9 ± 3.9 min, P = 0.398] or intraoperative volume of blood loss [195.8 ± 84.15 vs 263.3 ± 82.06 ml, P = 0.059]. No severe complications occurred. No recurrence of calculi was noted during the follow-up period. CONCLUSION: One-stop diagnosis and treatment plan (CTU 3-D reconstruction plus radiology, ultrasound guidance followed by PCNL) may identify the puncture path, improve the successful rate of puncture and stone-free rates and reduce the complications of PCNL.

[Silencing pyruvate kinase M2 sensitizes human prostate cancer PC3 cells to gambogic acid-induced apoptosis].

OBJECTIVE: To study the effect of silencing pyruvate kinase M2 (PKM2) on gambogic acid (GA)-induced apoptosis of human prostate cancer PC3 cells. METHODS: Three specific PKM2 siRNAs and one negative control siRNA (si-NC) were transfected into PC3 cells. The silencing effect of PKM2 siRNAs was determined by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot, and the effects of PKM2 siRNA on the vitality and apoptosis of GA-stimulated PC3 cells detected by MTT and AO/EB double staining, respectively. The mRNA and protein levels of c-myc and cyclin D1 were analyzed by qRT-PCR and Western blot, respectively. RESULTS: All the 3 PKM2 siRNAs effectively reduced the mRNA and protein expressions of PKM2, and PKM2 siRNA-1 exhibited the strongest silencing effect. At 24 h after transfection, the expression levels of PKM2 mRNA and protein were reduced by 70% and 85%, respectively (P < 0.05). Twenty-four hours of treatment with GA (0.5 micromol/L) following transfection with PKM2 siRNA-1 inhibited the vitality of the PC3 cells by 68%, increased their apoptosis, and significantly down-regulated the mRNA and protein levels of c-myc (50% and 35%) and cyclin D1 (60% and 20%) (P < 0.05). CONCLUSION: Inhibition of PKM2 sensitized PC3 cells to GA-induced apoptosis, suggesting that PKM2 may be a potential therapeutic target for sensitizing human prostate cancer to GA.

Gambogic acid inhibits TNF-α-induced invasion of human prostate cancer PC3 cells in vitro through PI3K/Akt and NF-κB signaling pathways.

AIM: To investigate the mechanisms underlying the inhibitory effect of gambogic acid (GA) on TNF-α-induced metastasis of human prostate cancer PC3 cells in vitro. METHODS: TNF-α-mediated migration and invasion of PC3 cells was examined using migration and invasion assays, respectively. NF-κB transcriptional activity and nuclear translocation were analyzed with luciferase reporter gene assays, immunofluorescence assays and Western blots. The ability of p65 to bind the promoter of Snail, an important mesenchymal molecular marker, was detected using a chromatin immunoprecipitation (ChIP) assay. After treatment with Snail-specific siRNA, the expression of invasiveness-associated genes was measured using quantitative real-time PCR and Western blot. RESULTS: GA significantly inhibited the viability of PC3 cells at 1-5 µmol/L, but did not produce cytotoxic effect at the concentrations below 0.5 µmol/L. GA (0.125-0.5 µmol/L) dose-dependently inhibited the migration and invasion of PC3 cells induced by TNF-α (10 ng/mL). Moreover, the TNF-α-mediated activation of phosphatidylinositol-3-OH kinase/protein kinase B (PI3K/Akt) and NF-κB pathways was suppressed by GA (0.5 µmol/L). Furthermore, this anti-invasion effect of GA was associated with regulation of Snail. Snail expression was significantly down-regulated by treatment with GA (0.5 µmol/L) in the TNF-α-stimulated PC3 cells. CONCLUSION: GA inhibits TNF-α-induced invasion of PC3 cells via inactivation of the PI3K/Akt and NF-κB signaling pathways, which may offer a novel approach for the treatment of human prostate cancer.

Urodynamic investigation of cyclophosphamide-induced overactive bladder in conscious rats.

BACKGROUND: Overactive bladder (OAB) can be caused by many factors such as inflammation, bladder outlet obstruction, neurogenic factors. We performed an intraperitoneal (ip) injection of cyclophosphamide to induce cystitis in rats, which causes their detrusors to overact, to provide a valuable disease model for discussing OAB pathogenesis and to study effective curing methods. METHODS: Female Sprague-Dawley rats were induced to form cystitis by cyclophosphamide (200 mg/kg, ip). The day after the injection, two catheters were inserted into each rat's bladder to study its urodynamics. The BL-410 model bio-function experimental system was used to monitor bladder pressure while the rats were conscious. Unstable detrusor contractions appear in the urine storage period as a standard to determine OAB, and the positive rate was calculated. Urodynamic parameters such as bladder basal pressure (BP), maximum voiding pressure (MVP), intercontraction interval (ICI), spontaneous activity (SA), maximum cystometric capacity (MCC), and bladder compliance (BC) were recorded in each group, and a light microscope was used to observe the pathological changes in the rat bladder tissue. RESULTS: The detrusor instability rate of the model group was 83.33%. The MVP, MCC and BC of rats in the model group were lower than the control group (P < 0.01), and the BP, ICI and SA of the model group rats were higher than the control group (P < 0.01). The difference between the control group and the model group is statistically significant. The model group rats' bladder walls swelled and bled, the submucosa thickened and leukocyte infiltration became serious. CONCLUSIONS: Acute cystitis and OAB symptoms can be induced by ip injections of cyclophosphamide in rats. This can provide a valuable animal model to study OAB in human beings.

Evaluation of thymosin ß4 in the regulation of epithelial-mesenchymal transformation in urothelial carcinoma.

OBJECTIVES: To study the underlying alteration in the expression of epithelial markers involved in epithelial-mesenchymal transition (EMT), and elucidate the potential mechanism(s) for Tß4-induced EMT-like phenotypic changes in bladder cancer cells. MATERIALS AND METHODS: All tissue samples in this study were obtained from clinical patients of the Union Hospital of Tongji Medical College, and were confirmed by surgery and pathology. Of these, normal bladder tissues (control), primary urothelial carcinoma of different grades (Stage pTa, Stage pT3), bladder paracancerous tissues, accompanied with 2 bladder cancer cell lines (BIU-87 and T24), were divided into 6 groups. Quantitative RT-PCR, Western blotting, and immunohistochemical study of adhesion molecules Tß4, ILK, E-cadherin, and ß-catenin involved in EMT were carried out. A lentiviral gene transferring vector containing the RNA polymerase III-dependent U6 promoter to express short hairpin RNA (shRNA) directed against Tß4 was also applied. In the present study, all agents were evaluated using commercial kits. RESULTS: A strong correlation between the expression levels of Tß4, ILK, E-cadherin, and ß-catenin was found in the bladder transitional cell carcinoma (TCC) patients. In the BIU-87 and T24 bladder cancer cells overexpressing Tß4, which were accompanied by a loss of E-cadherin as well as a cytosolic accumulation of ß-catenin, up-regulation of ILK was also revealed. The inhibition of the Tß4 expression with lentiviral shRNA vector could raise EMT-like phenotypic changes, significantly depressed motility, and subsequent invasiveness of bladder cancer cells. CONCLUSIONS: Our results imply that the Tß4 is likely to play a crucial role in EMT progression, and that inhibition of the Tß4 expression or interactions with other genes should be novel therapeutic targets for bladder cancers with high invasive and metastatic potential.

Predominant enhancement of apoptosis induced by methyl jasmonate in bladder cancer cells: therapeutic effect of the Antp-conjugated Smac peptide.

Methyl jasmonate (MJ) has recently attracted attention as a promising antitumoral compound because of its highly specific proapoptotic properties in a wide range of malignancies. However, the high doses required to achieve a therapeutic benefit have limited its clinical development. Here, we hypothesize that the family of inhibitor of apoptosis proteins (IAPs) may inhibit MJ-mediated apoptosis in cancer cells. We combined MJ with the IAPs inhibitor, the second mitochondria-derived activator of caspases (Smac) peptide to treat bladder cancer cells. The results showed that the combination of MJ and Smac peptide enhanced the apoptosis-inducing effect in a synergistic manner by releasing and activating IAPs-bounding caspase-3. These findings suggest that the inhibition of IAPs could overcome the resistance of cancer cells to MJ.

Silencing USP22 by asymmetric structure of interfering RNA inhibits proliferation and induces cell cycle arrest in bladder cancer cells.

The ubiquitin specific peptidase 22 (USP22) is a positive regulator of the growth of tumors. However, little is known about the impact of USP22 knockdown on the growth of human bladder cells. In the present study, we designed a series of asymmetric interfering RNAs (aiRNAs) and compared the efficacy of aiRNA and conventional symmetric interfering RNA (siRNA) in the silencing of USP22 expression and the growth of human bladder EJ cells in vitro and in vivo. In comparison with transfection with the USP22-specific siRNA, transfection with 15/21 aiRNA was more potent in down-regulating the USP22 expression and inhibiting EJ cell proliferation in vitro. Furthermore, transfection with 15/21 aiRNA induced higher frequency of EJ cells arrested at the G0/G1 phases, but did not trigger EJ cell apoptosis. Moreover, transfection with either the siRNA or 15/21 aiRNA up-regulated the expression of p53 and p21, but down-regulated the expression of cyclin E and Mdm2 in EJ cells. The up-regulated p53 expression induced by the specific siRNA or aiRNA was abrogated by induction of Mdm2 over-expression. In addition, treatment with the specific siRNA or aiRNA inhibited the growth of implanted human bladder tumors in mice and the aiRNA had more potent anti-tumor activity in vivo. Therefore, our data suggest that knockdown of USP22 expression by the aiRNA may down-regulate the expression of Mdm2 and cyclin E, resulting in the up-regulated expression of p53 and p21 and leading to cell cycling arrest and inhibition of human bladder EJ cell proliferation. Our findings indicate that the USP22-specific aiRNA may be a novel approach for the intervention of human bladder tumors.

[Endourological treatment of aged high-risk patients with benign prostate hyperplasia: a report of 283 cases].

OBJECTIVE: To evaluate the safety and effectiveness of endourological techniques in the treatment of benign prostate hyperplasia (BPH) in aged high-risk patients. METHODS: We used endourological techniques in the treatment of 283 BPH patients aged over 70 years and complicated with hydronephrosis, renal failure, heart failure, cerebral infarction, respiratory dysfunction, anemia, diabetes, bladder tumor, or prostate weight over 80 g, TURP (transurethral resection of the prostate) for 112 cases and PKRP (transurethral plasmakinetic resection of the prostate) for the other 171. All the patients were followed up for 1-30 months. RESULTS: In the TURP group, the scores on IPSS and QOL were decreased from 27.5 +/- 2.8, 5.5 +/- 1.0 to 5.8 +/- 1.2, 1.0 +/- 0.5, and the residual urine volume (RUV) from (75.0 +/- 20.0) ml to (8.0 +/- 3.0) ml, but the maximal flow rate (Qmax) increased from (6.5 +/- 2.0) ml/s to (18.5 +/- 1.5) ml/s (P < 0.05), while in the PKRP group, the scores on IPSS and QOL were decreased from 28.2 +/- 2.2, 5.5 +/- 1.0 to 5.4 +/- 1.6, 1.0 +/- 0.5, and RUV from (80.0 +/- 20.0) ml to (7.0 +/- 3.0) ml, and Qmax increased from (6.8 +/- 2.1) ml/s to (20.0 +/- 1.5) ml/s (P < 0.05). There were no statistically significant differences in IPSS, QOL, Qmax and RUV after treatment between the two groups (P > 0.05), but significantly less complications were found in the PKRP than in the TURP group (P < 0.05). CONCLUSION: Endourological treatment, especially PKRP, with comprehensive perioperative preparations, unerring operative skills, well-controlled operation time, and intensive postoperative monitoring and nursing, has the advantages of high safety, less bleeding, fewer complications and definite effectiveness for aged high-risk BPH patients.

[Cloning and expression of a novel mouse testis gene TSEG-2].

OBJECTIVE: To clone the mouse testis specific gene TSEG-2 via a bioinformatic approach. METHODS: The expressed sequence tags (EST) in the normal mouse testis were obtained from the online EST database ZooDDD. Their highly homologous EST sequences were retrieved through the dbEST database to construct contigs and spliced with the biomedical software Biolign. The corresponding exons and introns within the genome sequences were predicted with the software GeneScan. Primers were designed according to the open reading frame. RT-PCR was applied in cloning the cDNA of the novel gene from the mouse testis tissue and analyzing its expression patterns in the undescended testis and various organ tissues as well as in different developmental stages of the mouse testis. The sequencing results of TSEG-2 underwent bioinformatic analyses. RESULTS: The novel mouse testis gene TSEG-2 was successfully cloned, with full-length sequence of 451 bp. The open reading frame was 267 bp, coding a protein of 88 amino acid residues, and demonstrated to be correct by RT-PCR. The expression of TSEG-2 was high in the mouse testis, regular in the testis cDNA samples of different postnatal days, and down-regulated in the cryptorchidism model. No obvious homology with other mouse cDNA was found for TSEG-2. The GenBank accession number EU079025 was achieved. Function prediction showed that mouse TSEG-2 was probably a soluble non-secretary protein located at chromosome 15qE3, or a nucleoprotein with 2 phosphorylation sites of protein kinase C (PKC) and 1 of casein kinase II (CK2). CONCLUSION: A novel mouse testis specific gene TSEG-2 was successfully cloned, which could be down-regulated by cryptorchidism-inducible 17-beta estradiol. This has prepared the ground for further researches on the biological function and expression regulation of TSEG-2.

[Methyl jasmonate induces apoptosis of human neuroblastoma cell line BE(2) -C and its mechanism].

This study is to explore the inhibitory effect of methyl jasmonate on cell proliferation and expression of XIAP and survivin of human neuroblastoma cell line BE(2)-C. After cultivation of 1 - 2 mmol x L(-1) jasmonates with BE (2) -C cells for 6 - 24 h, the growth inhibiting rates of BE (2) -C cells were studied by MTT colorimetry. Cell proliferation was detected by colony formation assay. Cell cycle phases were assayed by propidium iodide staining flow cytometery. Cell apoptosis was inspected by acridine orange-ethidium bromide fluorescent staining, Hoechst 33258 fluorescent staining, and Annexin V-FITC and propidium iodide staining flow cytometry. Expressions of cyclin D1, XIAP and survivin were determined by RT-PCR and real-time RT-PCR. Methyl jasmonate inhibited the growth of BE(2)-C cells in a dose- and time-dependent manner. After addition of 1, 1.5 and 2 mmol x L(-1) of methyl jasmonate for 24 h, the inhibiting rates of cell growth reached 20.6% - 85.5% (P < 0.01), and the IC50 was 1.35 mmol x L(-1). The cell cycles were arrested at S phase. A part of cells presented the characteristic morphological changes of apoptosis. The early apoptotic rates were 13.51%, 17.32%, 24.59% (P < 0.01) and the cell death rates were 29.36% , 54.73% , 75.52% (P < 0.01), respectively. The expression of XIAP and survivin mRNA were downregulated by 18.5% - 68.9% , 22.4% - 48.7% (P < 0.05), respectively, without change in that of cyclin D1. The results indicated that methyl jasmonate could significantly inhibit the growth of BE(2) -C cells through inducing cell cycle arrest and apoptosis, downregulating the expression of XIAP and survivin might be one of its molecular mechanisms of action.

Natural jasmonates of different structures suppress the growth of human neuroblastoma cell line SH-SY5Y and its mechanisms.

AIM: Recent evidence has indicated that members of natural jasmonates, a family of plant stress hormones, exhibit anticancer activity. The current study was undertaken to investigate the effects of jasmonates on the in vitro growth of human neuroblastomas, one of the most common solid tumors in children. METHODS: Cellular proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide colorimetry and colony formation assay. Apoptosis was detected by Hoechst 33258 staining and flow cytometry. Western blotting was applied to assay gene expression. RESULTS: The administration of natural jasmonates, methyl jasmonate, cis-jasmone, and jasmonic acid to cultured neuroblastoma cell line SH-SY5Y, resulted in a decrease of cell proliferation in a doseand time-dependent manner. However, the in vitro growth of cultured human embryonic kidney (HEK) cell line HEK 293 was not affected by jasmonates. The cell cycles of jasmonate-treated SH-SY5Y cells were arrested at the G2/M phase. The incubation of SH-SY5Y cells with jasmonates resulted in characteristic changes of apoptosis. The anticancer activities of natural jasmonates on SH-SY5Y cells are as follows: methyl jasmonate>cis-jasmone>jasmonic acid. In addition, the expressions of proliferating cell nuclear antigen and N-myc were downregulated by methyl jasmonate. Moreover, methyl jasmonate decreased the expression of the Xlinked inhibitor of apoptosis protein and survivin, critical members of inhibitors of the apoptosis protein family, in SH-SY5Y cells. CONCLUSION: Jasmonates suppress the growth of human neuroblastoma cell line SH-SY5Y via inhibiting cell proliferation and inducing apoptosis, which lays the groundwork for further investigation into the anticancer activities and its mechanisms of natural jasmonates on human neuroblastomas.

Methyl jasmonate downregulates expression of proliferating cell nuclear antigen and induces apoptosis in human neuroblastoma cell lines.

Recent evidence indicates that methyl jasmonate, a plant stress hormone, exhibits anticancer activity on human cancer cells. Whether methyl jasmonate could inhibit the growth of human neuroblastoma cells still, however, remains largely unknown. In this study, administration of methyl jasmonate to cultured neuroblastoma cell lines, SK-N-SH and BE(2)-C, resulted in a decrease of cell viability in a dose-dependent and time-dependent manner as demonstrated by MTT colorimetry and colony formation assay. The results from RT-PCR indicated that the expression of proliferating cell nuclear antigen, but not of cyclin D1, was downregulated by methyl jasmonate. Accordingly, the cell cycle of methyl jasmonate-treated neuroblastoma cells was arrested at the G0/G1 phase. Moreover, incubation of SK-N-SH and BE(2)-C cells with methyl jasmonate resulted in characteristic changes of apoptosis, as demonstrated by acridine orange-ethidium bromide (AO/EB) staining, Hoechst 33258 staining and flow cytometry. Moreover, methyl jasmonate decreased the expression of the X-linked inhibitor of apoptosis protein and survivin, critical members of the inhibitors of apoptosis protein family, in neuroblastoma cells. These findings indicate that methyl jasmonate suppresses the growth of cultured human neuroblastoma cells associated with downregulation of proliferating cell nuclear antigen, and induces apoptosis accompanied by downregulation of the X-linked inhibitor of apoptosis protein and survivin, which lays the groundwork for further investigation into the mechanisms of methyl jasmonate-mediated anticancer activities.

Establishment of CXCR4-small interfering RNA retrovirus vector driven by human prostate-specific antigen promoter and its biological effects on prostate cancer in vitro and in vivo.

PURPOSE: CXC chemokine receptor-4 (CXCR4) is closely involved in bone metastasis of prostate cancer, and CXCR4 levels are frequently increased in prostate cancer cells and tissues. In the present study, its biological effects on prostate cancer in vitro and in vivo and feasibility to be a therapy target were investigated using a RNA interfering retrovirus vector targeting CXCR4 gene driven by human prostate-specific antigen promoter (pPSA). METHODS: We established a pPSA-siCXCR4 retrovirus vector and transfected prostate cancer cell PC-3m, LNCaP and breast cancer cell MCF-7, respectively. The expression of CXCR4 mRNA and protein was detected by RT-PCR and western blot, and the ability of adhesion, migration, invasion of prostate cancer cells was assessed using Transwell chamber. A metastasizing model using BALB/cA mice with human bone tissue implantation was established too, and transfected prostate cancer cells were via caudal vein. Survival time of mice suffering bone metastatic tumor as well as the weight and volume of these tumors were recorded and analyzed. RESULTS: The expression of CXCR4 mRNA and protein in androgen-responsive LNCaP cells was blocked by the pPSA-siCXCR4 vector, but it could not work in non androgen-responsive PC-3m cell and breast cancer cell MCF-7. The results of experiments in vitro also showed that the adhesion, transendothelial migration and invasive ability of transfected LNCaP cells were impaired, while there was no change in PC-3m and MCF-7 cells after transfection. pPSA-siCXCR4 represented a similar inhibitory effect in fluorescent bone metastasis model of LNCaP cells compared with PC-3m cells. CONCLUSION: These results suggest that the downstream siRNA controlled by PSA promoter in retrovirus system can express selectively in androgen-responsive prostate cancer in vitro and in vivo, and CXCR4 plays an important role in prostate cancer metastasis. We believe that the pPSA-siCXCR4 retrovirus vector is a potential choice in gene therapy for androgen-responsive prostate cancer.