LncRNA SNHG4 promotes prostate cancer cell survival and resistance to enzalutamide through a let-7a/RREB1 positive feedback loop and a ceRNA network.

BACKGROUND: Prostate cancer threatens the health of men over sixty years old, and its incidence ranks first among all urinary tumors among men. Enzalutamide remains the first-line drug for castration-resistant prostate cancer, however, tumors inevitably become resistant to enzalutamide. Hence, it is of great importance to investigate the mechanisms that induce enzalutamide resistance in prostate cancer cells. METHODS: Bioinformatic analyzing approaches were used to identified the over-expressed genes in prostate cancer tumor tissues from three GEO datasets. qRT-PCR, western blotting and immunochemistry/In situ hybridization staining assays were performed to assess the expression of SNHG4, RRM2, TK1, AURKA, EZH2 and RREB1. Cell cycle was measured by flow cytometry. CCK-8, plate colony formation and EdU assays were performed to assess the cell proliferation. Senescence-associated ß-Gal assay was used to detect the cell senescence level. γ-H2AX staining assay was performed to assess the DNA damages of PCa cells. Luciferase reporter assay and RNA immunoprecipitation assay were performed to verify the RNA-RNA interactions. Chromatin immunoprecipitation assay was performed to assess the bindings between protein and genomic DNA. RESULTS: We found that RRM2 and NUSAP1 are highly expressed in PCa tumors and significantly correlated with poor clinical outcomes in PCa patients. Bioinformatic analysis as well as experimental validation suggested that SNHG4 regulates RRM2 expression via a let-7 miRNA-mediated ceRNA network. In addition, SNHG4 or RRM2 knockdown significantly induced cell cycle arrest and cell senescence, and inhibited DNA damage repair and cell proliferation, and the effects can be partially reversed by let-7a knockdown or RRM2 reoverexpression. In vitro and in vivo experiments showed that SNHG4 overexpression markedly enhanced cell resistance to enzalutamide. RREB1 was demonstrated to transcriptionally regulate SNHG4, and RREB1 was also validated to be a target of let-7a and thereby regulated by the SNHG4/let-7a feedback loop. CONCLUSION: Our study uncovered a novel molecular mechanism of lncRNA SNHG4 in driving prostate cancer progression and enzalutamide resistance, revealing the critical roles and therapeutic potential of RREB1, SNHG4, RRM2 and let-7 miRNAs in anticancer therapy.

ncRNA-mediated overexpression of ubiquitin-specific proteinase 13 contributes to the progression of prostate cancer via modulating AR signaling, DNA damage repair and immune infiltration.

Metastatic castration-resistant prostate cancer (mCRPC) is a lethal form of prostate cancer, and the molecular mechanism driving mCRPC progression has not yet been fully elucidated. Immunotherapies such as chimeric antigen receptor, T-cell therapy and immune checkpoint blockade have exerted promising antitumor effects in hematological and solid tumor malignancies; however, no encouraging responses have been observed against mCRPC. The deubiquitinase USP13 functions as a tumor suppressor in many human cancers, as it sustains the protein stability of PTEN and TP53; however, its role in prostate cancer (PCa) and involvement in DNA damage and AR signaling remain unclear. In the current study, we explored the prognostic value of USP13 in PCa based on the TCGA database, and we analyzed the expression of USP13 in PCa tissues and adjacent normal tissues based on TCGA and our cohort. The results suggested that USP13 is overexpressed in PCa tumors and has the potential to be an independent biomarker for the overall survival of PCa patients. Additionally, enrichment analysis indicated that USP13 may participate in the AR pathway and PI3k/Wnt signaling, which are closely related to PCa progression. We also observed a significant correlation between the expression of USP13 and AR-related genes, DDR genes and mismatch repair genes based on the TCGA_PRAD dataset, which further supported the critical role of USP13 in AR activation and the DNA damage response of PCa. USP13 was also found to be enriched in protein neddylation, and expression of USP13 was significantly associated with infiltration of immune cells and expression of immunomodulators. Taken together, our study revealed a key role of USP13 in contributing to PCa progression by participating in multiple oncogenic signaling pathways, the DNA damage response and the immunosuppressive tumor microenvironment. Targeting USP13 may inhibit tumor growth and provide additional benefits in cooperation with DDR inhibitors and immunotherapy.

Study on the Interaction between Wheel Polygon and Rail Corrugation in High-Speed Railways.

The wheel polygonization and rail corrugation are typical wheel-rail periodic wear problems, which seriously affect the safe operation of high-speed railways. In the present paper, the interaction between the wheel polygon and the rail corrugation in the long-slope section of high-speed railways is mainly studied based on theory of friction coupling vibration. Firstly, the simulation model of the wheel-rail contact model is established, as well as the polygonal wear of the wheel and the corrugated wear of the rail. Then, the stability analyses of the wheel-rail system with periodic wear are studied, in which the four working conditions of smooth rail-smooth wheel, polygonal wheel-smooth rail, smooth wheel-corrugated rail and polygonal wheel-corrugated rail are compared. Finally, the competition mechanisms between the wheel polygon and rail corrugation under different parameters are discussed, including the wheel-rail friction coefficient and the depth of periodic wear of the wheel-rail system. The numerical results show that both the periodic wear of the wheel and rail with certain relevance will increase the friction coupling vibration of the wheel-rail system, which may aggravate the subsequent relevant wheel polygonal and rail corrugation wear. With the increase of the friction coefficient between wheel and rail, as well as the depth of the wheel polygon and rail corrugation, the vibration trend of the friction coupling vibration of the wheel-rail system increases gradually. Moreover, the proportion of the wheel polygon's influence on the friction coupling vibration of the wheel-rail system is greater than that of rail corrugation.

Integrative Analysis and Experimental Validation Indicated That SNHG17 Is a Prognostic Marker in Prostate Cancer and a Modulator of the Tumor Microenvironment via a Competitive Endogenous RNA Regulatory Network.

The incidence of prostate cancer (PC) is growing rapidly worldwide, and studies uncovering the molecular mechanisms driving the progression and modulating the immune infiltration and antitumor immunity of PC are urgently needed. The long noncoding RNA SNHG family has been recognized as a prognostic marker in cancers and contributes to the progression of multiple cancers, including PC. In this study, we aimed to clarify the prognostic values and underlying mechanisms of SNHGs in promoting the progression and modulating the tumor microenvironment of PC through data mining based on The Cancer Genome Atlas (TCGA) database. We identified that within the SNHG family, SNHG17 was most correlated with the overall survival of PC patients and could act as an independent predictor. Moreover, we constructed a competitive endogenous RNA (ceRNA) network by which SNHG17 promotes progression and potentially inhibits the immune infiltration and immune response of prostate cancer. By interacting with miR-23a-3p/23b-3p/23c, SNHG17 upregulates the expression of UBE2M and OTUB1, which have been demonstrated to play critical roles in the tumorigenesis of human cancers, more importantly promoting cancer cell immunosuppression and resistance to cytotoxic stimulation. Finally, we examined the correlation between SNHG17 expression and the clinical progression of PC patients based on our cohort of 52 PC patients. We also verified the SNHG17/miR-23a/OTUB1 axis in RV-1 and PC-3 cells by dual luciferase and RIP assays, and we further identified that SNHG17 promoted cellular invasive capacity by modulating OTUB1. In summary, the current study conducted a ceRNA-based SNHG17-UBE2M/OTUB1 axis and indicated that SNHG17 might be a novel prognostic factor associated with the progression, immunosuppression, and cytotoxic resistance of PC.

p300-Mediated Acetylation of Histone Demethylase JMJD1A Prevents Its Degradation by Ubiquitin Ligase STUB1 and Enhances Its Activity in Prostate Cancer.

The androgen receptor (AR) pathway plays a central role in the development of castration-resistant prostate cancer (CRPC). The histone demethylase JMJD1A has been shown to regulate activities of AR and c-Myc transcription factors and promote prostate cancer progression. Here, we report that JMJD1A protein stability is controlled by the ubiquitin ligase STUB1. High levels of JMJD1A were strongly correlated with low STUB1 levels in human CRPC specimens. STUB1 inhibited AR activity, AR-V7 levels, and prostate cancer cell growth partly through degradation of JMJD1A. Furthermore, the acetyltransferase p300 acetylated JMJD1A at lysine (K) 421, a modification that recruits the BET family member BRD4 to block JMJD1A degradation and promote JMJD1A recruitment to AR targets. Increased levels of both total and K421-acetylated JMJD1A were observed in prostate cancer cells as they developed resistance to the AR antagonist enzalutamide. Treatment of prostate cancer cells with either p300 or BET inhibitors destabilized JMJD1A, and enzalutamide-resistant prostate cancer cells were more sensitive than parental cells to these inhibitors. Together, our findings identify a critical role for acetylation of JMJD1A in regulating JMJD1A stability and AR activity in CRPC. These newly identified mechanisms controlling JMJD1A protein stability provide potential druggable targets to encourage the development of additional therapies for advanced prostate cancer. SIGNIFICANCE: Identification of mechanisms regulating JMJD1A protein stability reveals new strategies to destabilize JMJD1A and concomitantly inhibit AR activities as potential prostate cancer therapy.

Histone demethylase JMJD1A promotes expression of DNA repair factors and radio-resistance of prostate cancer cells.

The DNA damage response (DDR) pathway is a promising target for anticancer therapies. The androgen receptor and myeloblastosis transcription factors have been reported to regulate expression of an overlapping set of DDR genes in prostate cancer cells. Here, we found that histone demethylase JMJD1A regulates expression of a different set of DDR genes largely through c-Myc. Inhibition of JMJD1A delayed the resolution of γ-H2AX foci, reduced the formation of foci containing ubiquitin, 53BP1, BRCA1 or Rad51, and inhibited the reporter activity of double-strand break (DSB) repair. Mechanistically, JMJD1A regulated expression of DDR genes by increasing not only the level but also the chromatin recruitment of c-Myc through H3K9 demethylation. Further, we found that ubiquitin ligase HUWE1 induced the K27-/K29-linked noncanonical ubiquitination of JMJD1A at lysine-918. Ablation of the JMJD1A noncanonical ubiquitination lowered DDR gene expression, impaired DSB repair, and sensitized response of prostate cells to irradiation, topoisomerase inhibitors or PARP inhibitors. Thus, development of agents that target JMJD1A or its noncanonical ubiquitination may sensitize the response of prostate cancer to radiotherapy and possibly also genotoxic therapy.

ZNFX1 anti-sense RNA 1 promotes the tumorigenesis of prostate cancer by regulating c-Myc expression via a regulatory network of competing endogenous RNAs.

ZNFX1 anti-sense RNA 1 (ZFAS1) has been indicated in the tumorigenesis of various human cancers. However, the role of ZFAS1 in prostate cancer (PCa) progression and the underlying mechanisms remain incompletely understood. In the present study, we discovered that ZFAS1 is upregulated in PCa and that ZFAS1 overexpression predicted poor clinical outcomes. ZFAS1 overexpression notably promoted the proliferation, invasion, and epithelial-mesenchymal transition of PCa cells. Furthermore, we not only discovered that miR-27a/15a/16 are targeted by ZFAS1, which binds to their miRNA-response elements, but also revealed their tumor suppressor roles in PCa. We also identified that the Hippo pathway transducer YAP1, as well as its cooperator, TEAD1, are common downstream targets of miR-27a/15a/16. In addition, H3K9 demethylase KDM3A was found to be another target gene of miR-27a. Importantly, YAP1, TEAD1, and KDM3A all act as strong c-Myc inducers in an androgen-independent manner. Taken together, we suggest a regulatory network in which ZFAS1 is capable of enhancing c-Myc expression by inducing the expression of YAP1, TEAD1, and KDM3A through crosstalk with their upstream miRNAs, thereby globally promoting prostate cancer tumorigenesis.

USP13 functions as a tumor suppressor by blocking the NF-kB-mediated PTEN downregulation in human bladder cancer.

BACKGROUND: USP13 has been reported to be involved in the tumorigenesis of human cancers, however, its functional role and regulatory mechanisms in bladder cancer (BC) remain unclear. METHODS: q-RT-PCR was performed to examine the expression of miR-130b-3p, miR-301b-3p and USP13 in BC tissue samples. Western blot, q-RT-PCR, bioinformatic analysis and dual-luciferase reporter assay were conducted to identify the regulatory function of miR-130b-3p/301b-3p for USP13. Co-immunoprecipitation assay was performed to assess the interaction between USP13 and PTEN protein. Cell-counting-kit 8, colony formation assay and transwell assay were performed to value the proliferative, migrative and invasive capacities of BC cells in vitro. Mouse xenograft model of BC cells was established to verify the function of USP13 in vivo. Immunohistochemistry was performed to identify the protein expression of USP13, NF-kB p65 or PTEN in clinical/xenograft tumor tissues. RESULTS: Our present study reveals that USP13 functions as a tumor suppressor by interacting with PTEN protein and increasing its expression in bladder cancer. We found that loss of USP13 led to the downregulation of PTEN and promoted proliferative, invasive and migrative capacities of bladder cancer cells. Furthermore, we discovered that USP13 was a common target of miR-130b-3p and miR-301b-3p, and the miR-130b/301b cluster, which could be transcriptionally upregulated by NF-kB. Our data demonstrated that NF-kB activation decreased expression level of USP13 and PTEN, and promoted the tumorigenesis phenotypes of BC cells. In addition, reintroduction of USP13 partially rescued PTEN expression as well as the oncogenesis trend caused by NF-kB. CONCLUSION: We reported a potential regulatory loop that the NF-kB-induced miR-130b/301b overexpression decreased USP13 expression and subsequently resulted in the downregulation of PTEN protein and promoted tumorigenesis of bladder cancer. Moreover, NF-kB-mediated PTEN downregulation is very likely to facilitate the full activation of NF-kB.

Inhibition of stearoyl CoA desaturase-1 activity suppresses tumour progression and improves prognosis in human bladder cancer.

Urinary bladder neoplasm is one of the most common cancers worldwide. Cancer stem cells (CSCs) have been proven to be an important cause of cancer progression and poor prognosis. In the present study, we established bladder CSCs and identified the crucial differentially expressed genes (DEGs) between these cells and parental bladder cancer cells. Analyses of bioinformatics data and clinical samples from local hospitals showed that stearoyl CoA desaturase-1 (SCD) was the key factor among the DEGs. A significant correlation between SCD gene expression and poor prognosis among patients with bladder cancer was observed in our data. Loss-of-function experiments further revealed that the SCD inhibitor A939572 and SCD gene interference reduced cell proliferation and invasion. The above data suggest that SCD may serve as a novel marker for the prediction of tumour progression and poor prognosis in patients with bladder cancer.

Histone demethylase JMJD1A promotes alternative splicing of AR variant 7 (AR-V7) in prostate cancer cells.

Formation of the androgen receptor splicing variant 7 (AR-V7) is one of the major mechanisms by which resistance of prostate cancer to androgen deprivation therapy occurs. The histone demethylase JMJD1A (Jumonji domain containing 1A) functions as a key coactivator for AR by epigenetic regulation of H3K9 methylation marks. Here, we describe a role for JMJD1A in AR-V7 expression. While JMJD1A knockdown had no effect on full-length AR (AR-FL), it reduced AR-V7 levels in prostate cancer cells. Reexpression of AR-V7 in the JMJD1A-knockdown cells elevated expression of select AR targets and partially rescued prostate cancer cell growth in vitro and in vivo. The AR-V7 protein level correlated positively with JMJD1A in a subset of human prostate cancer specimens. Mechanistically, we found that JMJD1A promoted alternative splicing of AR-V7 through heterogeneous nuclear ribonucleoprotein F (HNRNPF), a splicing factor known to regulate exon inclusion. Knockdown of JMJD1A or HNRNPF inhibited splicing of AR-V7, but not AR-FL, in a minigene reporter assay. JMJD1A was found to interact with and promote the recruitment of HNRNPF to a cryptic exon 3b on AR pre-mRNA for the generation of AR-V7. Taken together, the role of JMJD1A in AR-FL coactivation and AR-V7 alternative splicing highlights JMJD1A as a potentially promising target for prostate cancer therapy.

Curcumin attenuates hypoxic-ischemic brain injury in neonatal rats through induction of nuclear factor erythroid-2-related factor 2 and heme oxygenase-1.

Curcumin has previously demonstrated anti-inflammatory, anti-infective and immuno-suppressive effects. In the present study, whether the attenuating effects of curcumin against hypoxic-ischemic brain injury in neonatal rats are mediated via nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was investigated. A model of hypoxic-ischemic brain injury was created using 1-week-old Sprague Dawley rats (weight, 52±1 g). The model rats were treated with 150 mg/kg curcumin by gavage for 3 days. Malondialdehyde levels, and superoxide dismutase and caspase-3 activities were assayed using commercial kits and western blot analysis was used to measure inducible nitric oxide synthase (iNOS), Nrf2 and HO-1 expression levels. Treatment with curcumin effectively reduced the brain injury score, increased myelin basic protein (MBP) expression and increased the quantity of neuronal cells in neonatal rats with hypoxic-ischemic brain injury. Furthermore, treatment with curcumin significantly attenuated the changes in SOD activity and MDA levels and suppressed the iNOS protein expression induced in neonatal rats by hypoxic-ischemic brain injury. Treatment with curcumin significantly increased Nrf2 and HO-1 expression in the neonatal rats with hypoxic-ischemic brain injury. The present study indicated that curcumin attenuates hypoxic-ischemic brain injury in neonatal rats through the induction of Nrf2 and HO-1.

Protein kinase C-α (PKCα) modulates cell apoptosis by stimulating nuclear translocation of NF-kappa-B p65 in urothelial cell carcinoma of the bladder.

BACKGROUND: The protein kinase C (PKC) family comprises central regulators of multiple signal transduction processes and is involved in the progression of many cancers. Nuclear factor Kappa-B (NF-κB) is constitutively expressed in cancer tissues and stimulates the transcription of various tumor-related genes. The present study aims to investigate the clinical significance of PKCα and NF-κB p65 in bladder cancer tissues and the mechanism underlying PKCα induction of bladder cancer cell apoptotic resistance through stimulation of p65 nuclear translocation. METHODS: Expression of PKCα and NF-κB subunit p65 was detected in seven bladder cancer cell lines by western blot and in 30 bladder cancer tissue specimens by immunostaining. Immunofluorescence was performed to evaluate p65 nuclear translocation induced by Phorbol 12-myristate 13-acetate (PMA). PKCα/ß selective inhibitor Gö6976, PKC pan-inhibitor sotrastaurin, and the PKC siRNA were employed to conduct PKC inhibition/knockdown in bladder cancer cells. Luciferase reporter assays were performed to measure the activity of NF-κB. Flow cytometry and TUNEL analysis were used to assess cell apoptosis. RESULTS: Expression of PKCα and NF-κB was found to positively correlate with tumor progression in 30 tumor tissue specimens. Furthermore, a Pearson's correlation coefficient analysis revealed a positive correlation between PKCα and NF-κB expression. Among the PKC inhibitors, the PKCα/ß selective inhibitor Gö6976 yielded the most significant block of PKCα and NF-κB activation by PMA. Knockdown of NF-κB p65 remarkably induced cell apoptosis, but PMA restored p65 expression and significantly suppressed cell apoptosis that was otherwise induced by the p65 knockdown alone. CONCLUSION: Our study showed that PKCα modulated cell resistance to apoptosis by stimulating NF-κB activation and thus promoted the tumorigenesis of bladder cancer.

NF-κB suppresses apoptosis and promotes bladder cancer cell proliferation by upregulating survivin expression in vitro and in vivo.

Nuclear factor kappa-B (NF-κB) activation is a common phenomenon in cancers, which results in the aberrant expression of NF-κB target genes and leads to malignant transformation, metastatic dissemination, abnormal cell proliferation or resistance to cell death. Survivin is a unique member of the IAP family, a well-known cancer-specific molecule and a molecular marker of poor clinical outcome in several cancer types, including bladder cancer. YM-155, a potent survivin suppressor, has been shown to have anti-tumor activity in preclinical cell lines, xenograft models and phase I/II studies. In the present study, we investigated the function of the NF-κB/survivin pathway in bladder cancer. We found that NF-κB can promote cell cycle progression and reduce apoptosis by upregulating survivin expression, thereby increasing cellular proliferation. We further confirmed the tumorigenic function of the NF-κB/survivin pathway in vivo using a xenograft tumor model of stable NF-κB-overexpressing 5637 cells. Moreover, we found that YM-155 significantly induced apoptosis and decreased cellular proliferation as well as tumor growth in mice. Our results demonstrate the carcinogenic function of the NF-κB/survivin pathway in bladder cancer and the role of YM-155 as a promising agent for the strategic treatment of bladder cancer.

miR-130b, an onco-miRNA in bladder cancer, is directly regulated by NF-κB and sustains NF-κB activation by decreasing Cylindromatosis expression.

Persistent activation of NF-κB signaling is closely related to chronic inflammation and tumorigenesis. Commonly, NF-κB signaling is tightly controlled by multiple feedback loops and regulators, such as the deubiquitinases (DUBs). However, in cancer cells, NF-κB may override these feedbacks through special pathways and lead to the sustained activation. In the present study, we demonstrate that in transitional cell carcinoma (TCC) of bladder, miR-130b plays an oncogenesis role, it enhanced proliferation, invasion and migration of TCC cell, and was highly correlated with tumor progression. On the other hand, NF-κB directly regulated the transcription of miR-130b by binding with its promoter region. Importantly, we verify that, through deceasing the expression of Cylindromatosis (CYLD), a K63-specific DUB and endogenous blocker of NF-κB signaling, miR-130b can in return sustain the persistent activation of NF-κB, which may promote the malignant progression of TCC. Thus, the present study uncovers a potential signaling transduction in which NF-κB is continuously activated, and may provide a novel therapeutic approach for the clinical management of TCC.

Effects of injection of anti-corticotropin release hormone serum in the lateral ventricles and electroacupuncture analgesia on pain threshold in rats with adjuvant arthritis.

Rat models of adjuvant arthritis were established, and anti-corticotropin release hormone serum injection in the lateral ventricles and electroacupuncture at right Jiaji (EX-B2) were performed. The pain threshold was decreased at 45 and 60 minutes after injection of the anti-corticotropin release hormone serum. Electroacupuncture at Jiaji can resist this effect. Immunohistochemical staining results showed that the expression of corticotropin release hormone in the hypothalamic paraventricular nucleus was greater in the electroacupuncture + anti-corticotropin release hormone serum group compared with the anti-corticotropin release hormone serum group. The expression of corticotropin release hormone was correlated with the pain threshold. The effect of endogenous corticotropin release hormone in pain modulation can be obstructed by anti-corticotropin release hormone serum. The analgesia of electroacupuncture can partially resist the depressed pain threshold caused by injection of anti-corticotropin release hormone serum. The analgesic effect of electroacupuncture is associated with the corticotropin release hormone content in the hypothalamus.