ARID1A Inactivation Increases Expression of circ0008399 and Promotes Cisplatin Resistance in Bladder Cancer.

OBJECTIVE: Cisplatin (CDDP)-based chemotherapy is a first-line, drug regimen for muscle-invasive bladder cancer (BC) and metastatic bladder cancer. Clinically, resistance to CDDP restricts the clinical benefit of some bladder cancer patients. AT-rich interaction domain 1A (ARID1A) gene mutation occurs frequently in bladder cancer; however, the role of CDDP sensitivity in BC has not been studied. METHODS: We established ARID1A knockout BC cell lines using CRISPR/Cas9 technology. IC50 determination, flow cytometry analysis of apoptosis, and tumor xenograft assays were performed to verify changes in the CDDP sensitivity of BC cells losing ARID1A. qRT-PCR, Western blotting, RNA interference, bioinformatic analysis, and ChIP-qPCR analysis were performed to further explore the potential mechanism of ARID1A inactivation in CDDP sensitivity in BC. RESULTS: It was found that ARID1A inactivation was associated with CDDP resistance in BC cells. Mechanically, loss of ARID1A promoted the expression of eukaryotic translation initiation factor 4A3 (EIF4A3) through epigenetic regulation. Increased expression of EIF4A3 promoted the expression of hsa_circ_0008399 (circ0008399), a novel circular RNA (circRNA) identified in our previous study, which, to some extent, showed that ARID1A deletion caused CDDP resistance through the inhibitory effect of circ0008399 on the apoptosis of BC cells. Importantly, EIF4A3-IN-2 specifically inhibited the activity of EIF4A3 to reduce circ0008399 production and restored the sensitivity of ARID1A inactivated BC cells to CDDP. CONCLUSION: Our research deepens the understanding of the mechanisms of CDDP resistance in BC and elucidates a potential strategy to improve the efficacy of CDDP in BC patients with ARID1A deletion through combination therapy targeting EIF4A3.

Identification of Kidney Transplant Recipients with Coronavirus Disease 2019.

Coronavirus disease 2019 (COVID-19) is a novel and lethal infectious disease, posing a threat to global health security. The number of cases has increased rapidly, but no data concerning kidney transplant (KTx) recipients infected with COVID-19 are available. To present the epidemiological, clinical, and therapeutic characteristics of KTx recipients infected with COVID-19, we report on a case series of five patients who were confirmed as having COVID-19 through nucleic acid testing (NAT) from January 1, 2020 to February 28, 2020. The most common symptoms on admission to hospital were fever (five patients, 100%), cough (five patients, 100%), myalgia or fatigue (three patients, 60%), and sputum production (three patients, 60%); serum creatinine or urea nitrogen levels were slightly higher than those before symptom onset. Four patients received a reduced dose of maintenance immunosuppressive therapy during hospitalization. As of March 4, 2020 NAT was negative for COVID-19 in three patients twice in succession, and their computed tomography scans showed improved images. Although greater patient numbers and long-term follow-up data are needed, our series demonstrates that mild COVID-19 infection in KTx recipients can be managed using symptomatic support therapy combined with adjusted maintenance immunosuppressive therapy.

Efficacy and Safety of COX-2 Inhibitor Parecoxib for Rigid Cystoscopy-related Pain Management in Male Patients: A Prospective, Randomized and Controlled Study.

Using anesthetic gel may not sufficiently exclude pain perception during and after cystoscopy in male patients. To evaluate the analgesic efficacy and safety of intramuscular parecoxib (40 mg) for outpatient-based rigid cystoscopy, we performed a prospective, randomized and controlled study. Consecutive male patients requiring diagnostic cystoscopy in our hospital were divided into group A (1% tetracaine gel, n=50) and group B (parecoxib, n=51) at random. Patients received intramuscular injections of either 2 mL sterile saline in group A or 40 mg parecoxib in group B 30 min before the procedure. Tetracaine gel was injected into the urethra 3 min before the procedure in group A, with patients receiving plain lubricant gel in group B at the same time. Cystoscopy-associated pain levels were evaluated using the Visual Analog Score (VAS) during the procedure. Post-procedure urethral pain and complications were recorded and analyzed. The results showed that male patients experienced significantly less pain in group B than in group A (2.70±1.36 vs. 3.56±1.74, P=0.008). The percentage of patients with dysuria pain was not significantly different between the two groups. In addition, 24 h after cystoscopy, the patients with no previous experience of cystoscopy were more likely to declare urethral pain (59.2% vs. 33.3%, P=0.012, relative risk=1.78). No difference was observed in analgesic-related complications between the two groups. We conclude that intramuscular injection of 40 mg parecoxib may improve comfort for male patients undergoing rigid cystoscopy.

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.

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.

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.

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.