Identification of novel protein biomarkers from the blood and urine for the early diagnosis of bladder cancer via proximity extension analysis.

BACKGROUND: Bladder cancer (BC) is a very common urinary tract malignancy that has a high incidence and lethality. In this study, we identified BC biomarkers and described a new noninvasive detection method using serum and urine samples for the early detection of BC. METHODS: Serum and urine samples were retrospectively collected from patients with BC (n = 99) and healthy controls (HC) (n = 50), and the expression levels of 92 inflammation-related proteins were examined via the proximity extension analysis (PEA) technique. Differential protein expression was then evaluated by univariate analysis (p < 0.05). The expression of the selected potential marker was further verified in BC and adjacent tissues by immunohistochemistry (IHC) and single-cell sequencing. A model was constructed to differentiate BC from HC by LASSO regression and compared to the detection capability of FISH. RESULTS: The univariate analysis revealed significant differences in the expression levels of 40 proteins in the serum (p < 0.05) and 17 proteins in the urine (p < 0.05) between BC patients and HC. Six proteins (AREG, RET, WFDC2, FGFBP1, ESM-1, and PVRL4) were selected as potential BC biomarkers, and their expression was evaluated at the protein and transcriptome levels by IHC and single-cell sequencing, respectively. A diagnostic model (a signature) consisting of 14 protein markers (11 in serum and three in urine) was also established using LASSO regression to distinguish between BC patients and HC (area under the curve = 0.91, PPV = 0.91, sensitivity = 0.87, and specificity = 0.82). Our model showed better diagnostic efficacy than FISH, especially for early-stage, small, and low-grade BC. CONCLUSION: Using the PEA method, we identified a panel of potential protein markers in the serum and urine of BC patients. These proteins are associated with the development of BC. A total of 14 of these proteins can be used to detect early-stage, small, low-grade BC. Thus, these markers are promising for clinical translation to improve the prognosis of BC patients.

RTN2, a new member of circadian clock genes identified by database mining and bioinformatics prediction, is highly expressed in ovarian cancer.

Increasing evidence suggests that core circadian genes have major roles in the carcinogenic mechanisms of multiple human malignancies. Among these genes, the role of reticulon 2 (RTN2) in ovarian cancer (OV) has so far remained elusive. In the present study, circadian clock gene (CCG) aberrations were systematically assessed across malignancies by using Gene Expression Omnibus and The Cancer Genome Atlas data. The results indicated that various core clock genes (ULK1, ATF3, CRY2, CSF3R, DAAM2, GAS7, NPTXR, PPPIR15A and RTN2) had elevated levels in tumors in comparison with normal tissues and their low expression levels were associated with a better prognosis in OV, indicating that they may be potential candidates for novel investigational approaches. The mRNA and protein expression levels of RTN2 in OV were then further analyzed by reverse transcription­quantitative PCR and immunohistochemistry, respectively. The results indicated that RTN2 mRNA and protein levels were increased in OV specimens in comparison with control samples. Differentially expressed CCGs, such as RTN2, were suggested as indicators of asynchronous circadian rhythms in cancer, which may provide a theoretical basis for chrono­therapy.

The Circadian Gene NPAS2 Act as a Putative Tumor Stimulative Factor for Uterine Corpus Endometrial Carcinoma.

BACKGROUND: Mounting evidence indicates altered circadian rhythm represents a critical factor affecting carcinogenesis and tumor progression. The circadian gene neuronal PAS domain protein 2 (NPAS2) constitutes a newly discovered prognostic biomarker. NPAS2 dysregulation is found in multiple malignancies, although its functions in uterine corpus endometrial carcinoma (UCEC) remain largely unknown. OBJECTIVE: To evaluate NPAS2's roles in UCEC and to explore the underlying mechanisms. METHODS: NPAS2 transcription levels, patient prognosis, different clinical stages and target microRNAs in UCEC cases were comparatively assessed based on public databases, including UALCAN, GEPIA, TIMER, Kaplan-Meier plotter, starBase database, LinkedOmics and String. Then, qRT-PCR and immunohistochemical analysis were applied to analyze the expression of NPAS2 in UCEC tissue samples. CCK-8, clonogenic assay and flow cytometry were carried out for detecting cell viability, colony formation ability and cell apoptosis, respectively. RESULTS: NPAS2 was upregulated in tissue samples from UCEC cases compared with the corresponding control specimens. NPAS2 overexpression was associated with decreased overall (OS), disease free (DFS) and relapse free (RFS) survival in UCEC. In addition, NPAS2 overexpression was associated with clinical stage, tumor grade, estrogen receptor status, myometrial invasion in UCEC. Furthermore, NPAS2 knockdown or overexpression altered cell proliferation and apoptosis in UCEC. Moreover, NPAS2 showed significant negative correlations with miR-17-5p and miR-93-5p, and positive correlations with miR-106a-5p and miR-381-3p in UCEC. CONCLUSION: NPAS2 overexpression is associated with poor prognosis and clinicopathological characteristics in UCEC and promotes proliferation and colony formation while inhibiting apoptosis. Finally, NPAS2 is associated with several miRNAs in UCEC.

Long non-coding RNA MIR4435-1HG promotes cancer growth in clear cell renal cell carcinoma.

BACKGROUND: Long non-coding RNAs (lncRNAs) play important roles in cancer development, yet their roles in renal carcinoma remain unclear. OBJECTIVE: We performed this study in order to investigate the expression and roles of lncRNAs in renal cell carcinoma. METHODS: In this study, we investigated the expression of lncRNAs in renal cell carcinoma through microarray analysis. Quantitative real-time PCR was performed to measure the expression of lncRNAs. Gain- or loss-of-function experiments were performed to investigate the roles of lncRNAs in cell proliferation and apoptosis. RNA pull-down and western blotting were performed to explore the underlying mechanism. RESULTS: The microarray analysis identified an upregulated lncRNA MIR4435-1HG in renal carcinoma. The expression level of MIR4435-1HG was correlated with TNM stage, tumor size, and Fuhrman grade. High expression of MIR4435-1HG indicated poor prognosis. MIR4435-1HG knockdown inhibited cell proliferation, and suppressed the migrating and invasive capacity of renal carcinoma cells. RNA pull-down followed by mass spectrometry revealed an interaction between MIR4435-1HG and pyruvate carboxylase, which was later corroborated by western blotting. CONCLUSIONS: MIR4435-1HG plays a critical role in the oncogenesis of renal cell carcinoma and may serve as a potential biomarker for renal cell carcinoma.

Correction to: Anti-tumor effects of Atractylenolide I on bladder cancer cells.

In the publication of this article [1], there is an error in Fig. 4b. The Cyto c western blot image in Fig. 4b is misrepresented. This has now been included in this correction. The authors declare that the correction does not change the results or conclusions of this paper.

RON is overexpressed in bladder cancer and contributes to tumorigenic phenotypes in 5637 cells.

Tyrosine kinase receptor macrophage stimulating 1 receptor (MST1R, also known as RON) contributes to the transformation and malignant progression observed in epithelial cells. The purpose of the present study is to assess the value of RON as a potential target in bladder cancer (BC) therapeutics. The expression profile of RON in BC tissues and adjacent noncancerous tissues was detected via immunohistochemistry. The rate of positive RON expression differed significantly between bladder urothelial cancer tissues (54.7%) and paraneoplastic tissues (29.4%) (P<0.05). RON expression was positively associated with the number of tumors per patient, histological grading, pathological stage and distant metastasis (all P<0.05). Downregulation of RON expression using small interfering RNAs inhibited cell growth, cell migration and promoted cell apoptosis in the 5637 cell line. RON inhibition induced cell cycle arrest at the G1/S boundary following an increase of cyclin-dependent kinase inhibitor 1B and cyclin-dependent kinase inhibitor 1A, and a decrease of cyclin D1, cyclin D3 and cyclin-dependent kinase 4 expression. Furthermore, knockdown of RON significantly blocked signal transduction, including downstream protein kinase B and mitogen-activated protein kinase pathways. These results indicated that RON serves a notable role in BC and is a potential target of therapeutic intervention.

Knockdown of DUXAP10 inhibits proliferation and promotes apoptosis in bladder cancer cells via PI3K/Akt/mTOR signaling pathway.

DUXAP10 is a member of long non-coding RNAs (lncRNAs) and has been reported to be upregulated in bladder cancer (BC) tissues. However, the biological functions of DUXAP10 in BC are largely unknown. The present study detected the expression of DUXAP10 in human normal bladder cell SV­HUC­1 and BC cell lines. Subsequently, cell proliferation, cell cycle, and apoptosis were analyzed by knockdown of the DUXAP10 expression. Results suggested that the expression level of DUXAP10 was significantly enhanced in cancer cells. After knockdown of DUXAP10, cell proliferation was inhibited, cell cycle was arrested at G0/G1 phase, and apoptosis was increased in T24 and 5637 cells. Western blot analysis detected that knockdown of DUXAP10 decreased the expression of Bcl-xL, cyclin D and CDK4. This increased the expression of Bad, cleaved caspase­3, cleaved caspase-9, and p27. Further studies indicated that knockdown of DUXAP10 inhibited PI3K/Akt/mTOR signaling pathway. Combining these results, our study suggests that DUXAP10 plays an important role in BC and DUXAP10 inhibition is a potential therapeutic target for BC.

Monoclonal antibody Zt/g4 targeting RON receptor tyrosine kinase enhances chemosensitivity of bladder cancer cells to Epirubicin by promoting G1/S arrest and apoptosis.

Epirubicin (EPI) is one of the most used intravesical chemotherapy agents after transurethral resection to non-muscle invasive bladder tumors (NMIBC) to prevent cancer recurrence and progression. However, even after resection of bladder tumors and intravesical chemotherapy, half of them will recur and progress. RON is a membrane tyrosine kinase receptor usually overexpressed in bladder cancer cells and associated with poor pathological features. This study aims to investigate the effects of anti-RON monoclonal antibody Zt/g4 on the chemosensitivity of bladder cells to EPI. After Zt/g4 treatment, cell cytotoxicity was significantly increased and cell invasion was markedly suppressed in EPI-treated bladder cancer cells. Further investigation indicated that combing Zt/g4 with EPI promoted cell G1/S-phase arrest and apoptosis, which are the potential mechanisms that RON signaling inhibition enhances chemosensitivity of EPI. Thus, combing antibody-based RON targeted therapy enhances the therapeutic effects of intravesical chemotherapy, which provides new strategy for further improvement of NMIBC patient outcomes.

Anti-tumor effects of Atractylenolide I on bladder cancer cells.

BACKGROUND: Atractylenolide I (ATR-1), an active component of Rhizoma Atractylodis Macrocephalae, possesses cytotoxicity against various carcinomas. However, little is known about the effects of ATR-1on bladder cancer. In the present study, the anti-tumor activity of ATR-1 was examined on bladder cancer cells both in vivo and in vitro. METHODS: MTT assay was used to assess the cytotoxic effect of ATR-1. Cell cycle distribution and apoptosis levels were evaluated using flow cytometry. Western blotting assay was applied to measure the levels of proteins associated with the apoptotic pathway, cell cycle progression and PI3K/Akt/mTOR signaling pathway. Tumor models in nude mice were induced by injection of T-24 and 253J human bladder cancer cells. RESULTS: ATR-1 inhibited bladder cancer cell proliferation, arrested cell cycle in G2/M phase through up-regulation of p21 and down-regulation of cyclin B1, CDK1 and Cdc25c. Meanwhile, ATR-1 also triggered cellular apoptosis depending on the activation of mitochondrial apoptotic pathway. Mechanism investigation indicated that ATR-1 exerts its anti-tumor effect also relies on the inhibition of PI3K/Akt/mTOR signaling pathway. Finally, mice studies showed that ATR-1 blocked the T-24 or 253J-induced xenograft tumor growth without noticeable toxicity. CONCLUSIONS: ATR-1 may be served as a potential therapeutic agent for the treatment of bladder cancer.

Attitudes and knowledge about naloxone and overdose prevention among detained drug users in Ningbo, China.

BACKGROUND: To date there has been limited research on both the prevalence of overdose and drug user knowledge about overdose prevention and response methods in China. In addition, there has been no effort to integrate naloxone information and distribution into pre-release services for drug users detained in isolated compulsory detoxification facilities in China. METHODS: The authors conducted a survey of 279 heroin users in isolated compulsory detoxification centers in Ningbo, China in an attempt to evaluate the possibility of conducting prelease peer naloxone programs in Ningbo isolated compulsory detoxification centers. Respondents' demographic background, history of heroin overdoses, and attitudes/knowledge about overdose prevention and response were collected. RESULTS: While drug users in Ningbo's compulsory detoxification centers have limited understandings of how to effectively respond to overdoses, they expressed concern about the possibility of overdose, interest in participating in overdose prevention and response programs, and a willingness to help their peers. In general, there was no significant difference in history and attitudes/knowledge of overdose between male and female participants. CONCLUSION: Based on the findings of this research, our survey provides preliminary evidence that detained drug users have considerable interest in overdose prevention and response information and willingness to help peers. However, drug users in Ningbo isolated compulsory detoxification centers currently have limited understandings of effective ways of helping to prevent overdose deaths.