B cell expression of E3 ubiquitin ligase Cul4b promotes chronic gammaherpesvirus infection in vivo.

IMPORTANCE: The human gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are etiologic agents of numerous B cell lymphomas. A hallmark of gammaherpesvirus infection is their ability to establish lifelong latency in B cells. However, the specific mechanisms that mediate chronic infection in B cells in vivo remain elusive. Cellular E3 ubiquitin ligases regulate numerous biological processes by catalyzing ubiquitylation and modifying protein location, function, or half-life. Many viruses hijack host ubiquitin ligases to evade antiviral host defense and promote viral fitness. Here, we used the murine gammaherpesvirus 68 in vivo system to demonstrate that the E3 ligase Cul4b is essential for this virus to establish latency in germinal center B cells. These findings highlight an essential role for this E3 ligase in promoting chronic gammaherpesvirus infection in vivo and suggest that targeted inhibition of E3 ligases may provide a novel and effective intervention strategy against gammaherpesvirus-associated diseases.

Proteomic analysis of DZIP3 interactome and its role in proliferation and metastasis in gastric cancer cells.

Gastric cancer is a serious malignant tumor in the world, accounting for the third cause of cancer death worldwide. The pathogenesis of gastric cancer is very complex, in which epigenetic inheritance plays an important role. In our study, we found that DZIP3 was significantly up-regulated in gastric cancer tissues as compared to adjacent normal tissue, which suggested it may be play a crucial part in gastric cancer. To clarify the mechanism of it, we further analyzed the interacting proteome and transcriptome of DZIP3. An association between DZIP3 and some epigenetic regulators, such as CUL4B complex, was verified. We also present the first proteomic characterization of the protein-protein interaction (PPI) network of DZIP3. Then, the transcriptome analysis of DZIP3 demonstrated that knockdown DZIP3 increased a cohort of genes, including SETD7 and ZBTB4, which have essential role in tumors. We also revealed that DZIP3 promotes proliferation and metastasis of gastric cancer cells. And the higher expression of DZIP3 is positively associated with the poor prognosis of several cancers. In summary, our study revealed a mechanistic role of DZIP3 in promoting proliferation and metastasis in gastric cancer, supporting the pursuit of DZIP3 as a potential target for gastric cancer therapy.

CUL4B enhances the malignant phenotype of esophageal squamous cell carcinoma by suppressing TGFBR3 expression.

Cullin 4B (CUL4B), which acts as a scaffold protein in CUL4B-RING ubiquitin ligase complexes (CRL4B), is frequently overexpressed in cancer and represses tumor suppressors through epigenetic mechanisms. However, the expression and function of CUL4B in esophageal squamous cell carcinoma (ESCC) have not been well illustrated. In this study, we show that upregulation of CUL4B in ESCC cells enhances proliferation, invasion and cisplatin (CDDP)-resistance, while knockdown of CUL4B significantly represses the malignant activities. Mechanistically, we demonstrate that CUL4B promotes proliferation and migration of ESCC cells through inhibiting expression of transforming growth factor beta receptor III (TGFBR3). CRL4B complex binds to the promoter of TGFBR3, and represses its transcription by catalyzing monoubiquitination at H2AK119 and coordinating with PRC2 and HDAC complexes. Taken together, our findings establish a critical role for the CUL4B/TGFBR3 axis in the regulation of ESCC malignancy.

Knockdown of circMFN2 inhibits cell progression and glycolysis by miR-198/CUL4B pathway in ovarian cancer.

Circular RNA (circRNA) regulates malignant tumors, including ovarian cancer (OC). The present research study aimed to reveal the biological mechanism of circRNA mitofusin 2 (circMFN2) in OC. Cell biological behaviors were investigated using clonogenicity assay, EdU assay, transwell assay, and flow cytometry analysis. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analysis were implemented to detect the levels of circMFN2, miR-198, Cullin 4B (CUL4B), and apoptosis-related proteins. Glycolysis was assessed by glucose assay kit, lactate assay kit, and ATP level detection kit. The relationships among miR-198, circMFN2, and CUL4B were verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. The xenograft mice model was used to analyze tumor growth in vivo. The expression of circMFN2 and CUL4B was increased, while miR-330-5p was decreased in OC tissues or cells. The absence of CircMFN2 hindered cell proliferation, migration, invasion, and glycolysis and promoted apoptosis in OC cells. We found that circMFN2 promoted CUL4B expression via sponging miR-198. MiR-198 depletion reversed circMFN2 knockdown-induced effects in OC cells. Furthermore, CUL4B overexpression overturned the inhibitory effect of miR-198 in OC cells. And the absence of circMFN2 inhibited tumor growth in vivo. CircMFN2 repressed OC progression by regulating the miR-198/CUL4B axis.

Exosomal circKDM4A Induces CUL4B to Promote Prostate Cancer Cell Malignancy in a miR-338-3p-Dependent Manner.

Circular RNA lysine demethylase 4A (circKDM4A) is also named circ_0012098 and its abnormal expression has been confirmed in serum exosomes of prostate cancer (PC) patients. However, whether PC progression involves the exosomal circ_0012098 remains unknown. RNA expression of circKDM4A, microRNA-338-3p (miR-338-3p) and cullin 4B (CUL4B) was detected by quantitative real-time polymerase chain reaction. Protein expression was checked by Western blot. The positive expression rate of nuclear proliferation marker (ki-67) was analyzed by immunohistochemistry assay. Dual-luciferase reporter assay and RNA immunoprecipitation assay were used to identify the interaction between miR-338-3p and circKDM4A or CUL4B. Mouse model assay was performed to determine the effect of exosomal circKDM4A on tumorigenesis in vivo. CircKDM4A expression was significantly upregulated in the serum exosomes from PC patients compared with the exosomes from healthy volunteers. Exosomes treatment promoted the proliferation, migration and invasion of PC cells but inhibited apoptosis; however, these effects were attenuated after circKDM4A knockdown. Meanwhile, circKDM4A depletion restored exosome-increased circKDM4A expression. Additionally, circKDM4A acted as a miR-338-3p sponge, and miR-338-3p bound to CUL4B in PC cells. CircKDM4A regulated the effect of exosome-induced PC cell malignancy by interacting with miR-338-3p and CUL4B. Moreover, circKDM4A silencing relieved exosome-induced tumor growth in vivo. Exosomal circKDM4A promoted PC malignant progression by the miR-338-3p/CUL4B axis, providing a therapeutic target for PC.

CUL4B increases platinum-based drug resistance in colorectal cancer through EMT: A study in its mechanism.

Platinum-based chemotherapy drugs play a very important role in the treatment of patients with advanced colorectal cancer, but the drug resistance of platinum-based chemotherapy drugs is an important topic that puzzles us. If we can find mechanisms of resistance, it will be revolutionary for us. We analysed the differential genes, core genes and their enrichment pathways in platinum-resistant and non-resistant patients through a public database. Platinum-resistant cell lines were cultured in vitro for in vitro colony and Transwell analysis. Tumorigenesis analysis of nude mice in vivo. Verify the function of core genes. Through differential gene and enrichment analysis, we found that CUL4B was the main factor affecting platinum drug resistance and EMT. Our hypothesis was further verified by in vitro drug-resistant and wild-type cell lines and in vivo tumorigenesis analysis of nude mice. CUL4B leads to platinum drug resistance in colorectal cancer by affecting tumour EMT.

Roles of the ubiquitin ligase CUL4B and ADP-ribosyltransferase TiPARP in TCDD-induced nuclear export and proteasomal degradation of the transcription factor AHR.

The aryl hydrocarbon receptor (AHR) is a transcription factor activated by exogenous halogenated polycyclic aromatic hydrocarbon compounds, including the environmental toxin TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and naturally occurring dietary and endogenous compounds. The activated AHR enhances transcription of specific genes including phase I and phase II metabolism enzymes and other targets genes such as the TCDD-inducible poly(ADP-ribose) polymerase (TiPARP). The regulation of AHR activation is a dynamic process: immediately after transcriptional activation of the AHR by TCDD, the AHR is exported from the nucleus to the cytoplasm where it is subjected to proteasomal degradation. However, the mechanisms regulating AHR degradation are not well understood. Here, we studied the role of two enzymes reported to enhance AHR breakdown: the cullin 4B (CUL4B)AHR complex, an E3 ubiquitin ligase that targets the AHR and other proteins for ubiquitination, and TiPARP, which targets proteins for ADP-ribosylation, a posttranslational modification that can increase susceptibility to degradation. Using a WT mouse embryonic fibroblast (MEF) cell line and an MEF cell line in which CUL4B has been deleted (MEFCul4b-null), we discovered that loss of CUL4B partially prevented AHR degradation after TCDD exposure, while knocking down TiPARP in MEFCul4b-null cells completely abolished AHR degradation upon TCDD treatment. Increased TCDD-activated AHR protein levels in MEFCul4b-null and MEFCul4b-null cells in which TiPARP was knocked down led to enhanced AHR transcriptional activity, indicating that CUL4B and TiPARP restrain AHR action. This study reveals a novel function of TiPARP in controlling TCDD-activated AHR nuclear export and subsequent proteasomal degradation.

Insulin alleviates LPS-induced ARDS via inhibiting CUL4B-mediated proteasomal degradation and restoring expression level of Na,K-ATPase α1 subunit through elevating HCF-1.

Acute respiratory distress syndrome (ARDS) is a critical disease with a high mortality rate, characterized by obstinate hypoxemia caused by accumulation of alveolar fluid and excessive uncontrolled inflammation. Na,K-ATPase α1 (ATP1A1) subunit is an important component of Na,K-ATPase that transports Na+ and K+ and scavenges alveolar fluid. The function of Na,K-ATPase is always impaired during ARDS and results in more severe symptoms of ARDS. However, the regulatory mechanism of Na,K-ATPase after ARDS remains unclear. Here, we revealed ATP1A1 was downregulated post-transcriptionally by an E3 ligase component CUL4B mediated proteasomal degradation. Moreover, we found insulin could inhibit the upregulation of CUL4B in an insulin receptor cofactor HCF-1-dependent manner. Our study resolved the molecular mechanism underlying the clearance impairment of alveolar fluid and provided a clue for the usage of insulin as a potential therapeutic medicine for ARDS.

MiR-674-5p Suppresses the Proliferation and Migration of Glioma Cells by Targeting Cul4b.

Glioma multiforme (GBM) is the most common malignant primary brain tumors. Despite the considerable advances in GBM treatment, it is still one of the most lethal forms of brain tumor. New clinical biomarkers and therapeutic targets are immediately required. MicroRNAs (miRNAs) are a class of small, evolutionarily conserved noncoding RNAs and have emerged as the key regulators of many cancers. Here in this study, we showed that miR-674-5p was probably an important regulator of glioma cell growth. After the transfection with miR-674-5p mimic or inhibitor, we found that the expression level of miR-674-5p was negatively related with cell proliferation and migration in C6 cells. Based on the prediction of the target genes of miR-674-5p on the website, we chose Cullin 4B (Cul4b), a gene upregulated in GBM, and proved that it was a target of miR-674-5p. In addition, we explored the role of miR-674-5p in glioma growth in vivo. Taken together, the present study indicated that miR-674-5p suppressed glioma cell proliferation and migration by targeting Cul4b.

CUL4B renders breast cancer cells tamoxifen-resistant via miR-32-5p/ER-α36 axis.

Tamoxifen (TAM) resistance is a significant clinical challenge in endocrine therapies for estrogen receptor (ER)-positive breast cancer patients. Cullin 4B (CUL4B), which acts as a scaffold protein in CUL4B-RING ubiquitin ligase complexes (CRL4B), is frequently overexpressed in cancer and represses tumor suppressors through diverse epigenetic mechanisms. However, the role and the underlying mechanisms of CUL4B in regulating drug resistance remain unknown. Here, we showed that CUL4B promotes TAM resistance in breast cancer cells through a miR-32-5p/ER-α36 axis. We found that upregulation of CUL4B correlated with decreased TAM sensitivity of breast cancer cells, and knockdown of CUL4B or expression of a dominant-negative CUL4B mutant restored the response to TAM in TAM-resistant MCF7-TAMR and T47D-TAMR cells. Mechanistically, we demonstrated that CUL4B renders breast cancer cells TAM-resistant by upregulating ER-α36 expression, which was mediated by downregulation of miR-32-5p. We further showed that CRL4B epigenetically represses the transcription of miR-32-5p by catalyzing monoubiquitination at H2AK119 and coordinating with PRC2 and HDAC complexes to promote trimethylation at H3K27 at the promoter of miR-32-5p. Pharmacologic or genetic inhibition of CRL4B/PRC2/HDAC complexes significantly increased TAM sensitivity in breast cancer cells in vitro and in vivo. Taken together, our findings thus establish a critical role for the CUL4B-miR-32-5p-ER-α36 axis in the regulation of TAM resistance and have important therapeutic implications for combined application of TAM and the inhibitors of CRL4B/PRC2/HDAC complex in breast cancer treatment. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Calcifying pseudoneoplasms of the neuraxis (CAPNON). A case report.

Calcifying pseudoneoplasms of the neuraxis (CAPNON) are rare, slow-growing, benign lesions occurring throughout the neuroaxis that are frequently misdiagnosed and overlooked by clinicians. Here, we report a case of a 56-year-old woman who presented with a history of recurrent headache for the previous six years. Magnetic resonance imaging (MRI) revealed a 2.3-cm-sized solid mass in the right frontal lobe that was surrounded by marked edematous areas. The lesion demonstrated dense calcification and avid enhancement. The lesion was initially diagnosed as oligodendroglioma, and then found to be CAPNON based on histopathology of a surgically resected tissue. Genetic analysis revealed a nonsense mutation in the CUL4B gene. The patient's condition appeared to reflect a reactive, rather than neoplastic, process. Clinicians should be prepared to detect such pseudotumors histopathologically in order to avoid unnecessary differential tests of neoplastic or infectious diseases, as well as potentially harmful therapies.

Cul4b Promotes Progression of Malignant Cutaneous Melanoma Patients by Regulating CDKN2A.

Although several molecular targeted therapy and immunotherapy have been developed, cutaneous melanoma prognosis is still not satisfying. Cul4b promotes the progression of several malignant tumors by regulating cell proliferation. However, its prognostic role in malignant cutaneous melanoma has not been evaluated. In this study, immunohistochemistry was performed to assess the expression of Cul4b in a consecutive patient cohort. The prognostic role of Cul4b was estimated with univariate and multivariate analysis. Cul4b was knocked down in melanoma cell line to evaluate its role in promoting cell proliferation. The results revealed that Cul4b was highly expressed in some of the cutaneous malignant melanoma patients and high expression of Cul4b was associated with poor melanoma-specific overall survival and poor disease-free survival. Cul4b expression was associated with Breslow categories, Clark level, and Ki67 expression. Univariate and multivariate analysis revealed that Cul4b is an independent prognosis risk factor of cutaneous melanoma. Downregulation of Cul4b inhibited the proliferation ability of melanoma cells and downregulated the expression of CDKN2A. These results suggest that Cul4b plays an essential role in cutaneous melanoma progression and may serve as a promising treatment target.

[CRL4B complex promotes the development of pancreatic cancer by inhibiting secreted frizzled related protein 1].

Objective: To investigate the role of CUL4B-RING E3 ubiquitin ligase (CRL4B) complex in pancreatic tumorigenesis and the molecular mechanism. Methods: Pancreatic cells were divided into control group (transfected with negative control lentivirus), shCUL4B group (transfected with CUL4B lentivirus), shDDB1 group [transfected with DNA damage binding protein 1 (DDB1) lentivirus], and shCUL4B+ siSFRP1 group (transfected with CUL4B lentivirus and SFRP1-siRNA). RNA-seq was performed in pancreatic cancer cell lines with CUL4B and DDB1 knocked down respectively, to identify the target genes regulated by CRL4B complex. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression levels of target genes. Chromatin immunoprecipitation (ChIP) assay was used to identify the target genes directly regulated by CUL4B and DDB1. Western blot was used to detect the protein expression levels of the epithelial-mesenchymal transition (EMT) markers. The EdU cell proliferation test was used to detect cell proliferation ability. The scratch repair test and Transwell cell invasion test were used to detect cell migration and invasion ability. Finally, the sequencing data of pancreatic cancer-related tumor samples and normal samples in GEO, TCGA and GTEx databases were used to analyze the expression correlations of CUL4B, DDB1 and their downstream target genes. Results: RNA-seq results showed that target genes regulated by CRL4B complex involved in a number of malignant tumor-related signaling pathways. qRT-PCR results verified that the mRNA expression levels of the target genes of CUL4B or DDB1 knockdown groups were higher than those of the control group, and the difference was statistically significant (P<0.05). ChIP-PCR results showed that CRL4B complex directly bound to the promoter regions of the target genes, NME1 and SFRP1, and the enrichment of monoubiquitination of lysine at 119 of histone H2A (H2AK119ub1) in the promoter region of target gene was reduced after CUL4B knockdown. The proliferation rate in PANC-1 cell line of the control group was (32.10±3.58)%, higher than (13.95±1.66)% in the shCUL4B group and (22.38±0.77)% in the shCUL4B+ siSFRP1 group (P<0.05). The proliferation rate in AsPC-1 cell line of the control group was (35.47±7.80)%, higher than (19.60±3.58)% in the shCUL4B group and (30.09±0.81)% in the shCUL4B+ siSFRP1 group (P<0.05). The scratch repair experiment showed that the migration rate of PANC-1 cell line control group was (53.18±3.70)%, higher than that (17.46±2.62)% in the shCUL4B group and (44.99±9.18)% in the shCUL4B + siSFRP1 group (P<0.05). Western blot showed the expression levels of epithelial markers including α-catenin and γ-catenin in the control group were 1.00±0.03 and 1.01±0.11, respectively, lower than 1.44±0.01 and 1.21±0.06 in the shCUL4B group (P<0.05). The expression levels of mesenchymal markers including fibronectin and vimentin in the control group were 1.01±0.14 and 1.02±0.18, respectively, higher than 1.53±0.13 and 1.22±0.07 in the shCUL4B+ siSFRP1 group (P<0.05). The cell metastasis rate of the control group was (100.00±3.96)%, higher than the (35.49±0.34)% in the shCUL4B group and (107.06±2.77)% in the shCUL4B+ siSFRP1 group, the difference was statistically significant (P<0.05). The expressions of CUL4B and DDB1 were significantly upregulated in the pancreatic cancer tissues, and were negatively correlated with the expression of SFRP1 (r=-0.342 and r=-0.264, respectively). Conclusions: CRL4B complex inhibits the transcription of target gene SFRP1 and promotes the development of pancreatic cancer. Moreover, CRL4B complex is upregulated in pancreatic cancer, which provide a potential of therapeutic target for pancreatic cancer.

Circ_0015756 promotes the progression of ovarian cancer by regulating miR-942-5p/CUL4B pathway.

BACKGROUND: Ovarian cancer (OC) is the gynecologic cancer with the highest mortality. Circular RNAs (circRNAs) play a vital role in the development and progression of cancer. This study aimed to explore the potential role of circ_0015756 in OC and its molecular mechanism. METHODS: The levels of circ_0015756, microRNA-942-5p (miR-942-5p) and Cullin 4B (CUL4B) were determined by quantitative real-time PCR (qRT-PCR) or Western blot assay. Cell proliferation, apoptosis, migration and invasion were assessed by Cell Counting Kit-8 (CCK-8), colony formation assay, flow cytometry and transwell assay. The levels of proliferation-related and metastasis-related proteins were measured by Western blot assay. The relationship between miR-942-5p and circ_0015756 or CUL4B was verified by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay. Xenograft assay was used to analyze tumor growth in vivo. RESULTS: Circ_0015756 and CUL4B levels were increased, while miR-942-5p level was decreased in OC tissues and cells. Depletion of circ_0015756 suppressed proliferation, migration and invasion and promoted apoptosis in OC cells. Down-regulation of circ_0015756 hindered OC cell progression via modulating miR-942-5p. Also, up-regulation of miR-942-5p impeded OC cell development by targeting CUL4B. Mechanistically, circ_0015756 up-regulated CUL4B via sponging miR-942-5p. Moreover, circ_0015756 silencing inhibited tumor growth in vivo. CONCLUSION: Knockdown of circ_0015756 suppressed OC progression via regulating miR-942-5p/CUL4B axis, suggesting that circ_0015756 might be a potential therapeutic target for ovarian cancer.

Inflammation-dependent overexpression of c-Myc enhances CRL4DCAF4 E3 ligase activity and promotes ubiquitination of ST7 in colitis-associated cancer.

Inflammation is well known as an important driver of the initiation of colitis-associated cancer (CAC). Some cytokines, such as IL-6 and TNF-α can activate expression of the oncogene c-Myc (MYC) and regulate its downstream effects. Cullin-RING E3 Ligases (CRLs) are emerging as master regulators controlling tumorigenesis. Here, we demonstrate that two cullin genes, CUL4A and CUL4B, but not other members, are specifically overexpressed in CAC tumour samples and positively correlate with levels of the proinflammatory cytokines IL-1ß and IL-6. In vitro experiments revealed that the transcription factor c-Myc can specifically activate the expression of CUL4A and CUL4B by binding to a conserved site (CACGTG) located in their promoters. Additionally, we found that both CUL4A and CUL4B can form an E3 complex with DNA damage-binding protein 1 (DDB1) and DDB1-CUL4-associated factor 4 (DCAF4). In vitro and in vivo ubiquitination analyses indicate that CRL4DCAF4 E3 ligase specifically directs degradation of ST7 (suppression of tumorigenicity 7). Overexpression of c-Myc in human colon epithelial cells resulted in the accumulation of CUL4A, CUL4B and DCAF4, but degradation of ST7. In contrast, knockdown of c-Myc, CUL4A or CUL4B in the colon adenocarcinoma cell line HT29 caused accumulation of ST7 and inhibition of cell proliferation, colony formation ability and in vivo tumour growth. Collectively, our results provide in vitro and in vivo evidence that c-Myc regulates CRL4DCAF4 E3 ligase activity to mediate ubiquitination of ST7, whose presence is physiologically essential for CAC tumorigenesis. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

NCBP1 promotes the development of lung adenocarcinoma through up-regulation of CUL4B.

Lung cancer is the most frequent cancer type and is the leading cause of tumour-associated deaths worldwide. Nuclear cap-binding protein 1 (NCBP1) is necessary for capped RNA processing and intracellular localization. It has been reported that silencing of NCBP1 resulted in cell growth reduction in HeLa cells. Nevertheless, its clinical significance and underlying molecular mechanisms in non-small-cell lung cancer remain unclear. In this study, we found that NCBP1 was significantly overexpressed in lung cancer tissues and several lung cancer cell lines. Through knockdown and overexpression experiments, we showed that NCBP1 promoted lung cancer cell growth, wound healing ability, migration and epithelial-mesenchymal transition. Mechanistically, we found that cullin 4B (CUL4B) was a downstream target gene of NCBP1 in NSCLC. NCBP1 up-regulated CUL4B expression via interaction with nuclear cap-binding protein 3 (NCBP3). CUL4B silencing significantly reversed NCBP1-induced tumorigenesis in vitro. Based on these findings, we propose a model involving the NCBP1-NCBP3-CUL4B oncoprotein axis, providing novel insight into how CUL4B is activated and contributes to LUAD progression.

Inflammation-dependent downregulation of miR-194-5p contributes to human intervertebral disc degeneration by targeting CUL4A and CUL4B.

Inflammation is one of the major causes of intervertebral disc degeneration (IDD). Emerging evidence has revealed that increase in the levels of pro-inflammatory cytokines, such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α), can activate a variety of signaling pathways, eventually resulting in IDD. Here, we show that the two cullin family genes, CUL4A and CUL4B, but not other cullins, are specifically overexpressed in IDD samples compared with healthy controls, and the CUL4A and CUL4B levels are positively correlated with the severity of IDD. In vitro analyses in human osteoblast cells (hFOB1.19), nucleus pulposus cells (hNPCs), and annulus fibrosus cells (hAFCs) indicated that treatment with IL-6 and TNF-α can increase CUL4A and CUL4B levels. By performing a microRNA-based microarray analysis, we found a set of microRNAs (miRNAs) that were differentially expressed in IDD samples compared with samples from healthy controls. Of these miRNAs, miR-194-5p, was significantly downregulated in IDD samples and could bind to the three prime untranslated regions (3'-UTRs) of both CUL4A and CUL4B, thereby downregulating their expression. The in vitro overexpression or downregulation of miR-194-5p, with a miR-194-5p-mimic or with anti-miR-194-5p, can cause the repression or induction of both CUL4A and CUL4B, respectively. Interestingly, treatment with IL-6 and TNF-α inhibitors in primary hNPCs and hAFCs that were isolated from patients with IDD led to the downregulation of CUL4A and CUL4B. Together, these findings provide insight into how the inflammation-dependent downregulation of miR-194-5p contributes to the pathogenesis of IDD, which may aid in the development of new therapeutic approaches for IDD by directly targeting miR-194-5p or CUL4A and CUL4B.

CUL4B/miR-33b/C-MYC axis promotes prostate cancer progression.

BACKGROUND: Cullin 4B (CUL4B), a scaffold protein that assembles CRL4B ubiquitin ligase complexes, is overexpressed in many types of solid tumors and contributes to epigenetic silencing of tumor suppressors. However, its clinical significance and underlying molecular mechanisms in prostate cancer (PCa) remain unknown. METHODS: The clinical significance of CUL4B in PCa was characterized by in silico method. RT-qPCR and Western blot were used to study the transcript and protein expression levels of CUL4B and C-MYC. Bioinformatics tools, chromatin immunoprecipitation (ChIP) and luciferase reporter assay were utilized to identify and characterize the microRNAs (miRNAs) regulated by CUL4B. The biological function of CUL4B and miR-33b-5p was evaluated by MTS, transwell, and wound healing assays, accordingly. RESULTS: CUL4B is significantly overexpressed in PCa tissues compared with benign prostatic tissues and its overexpression is correlated with poor prognosis. CUL4B promotes proliferation and aggressiveness of PCa cells in vitro. Mechanistically, we demonstrate that CUL4B upregulates the expression of C-MYC at post-transcriptional level through epigenetic silencing of miR-33b-5p. Importantly, CUL4B-induced oncogenic activity in PCa by targeting C-MYC is repressed by miR-33b-5p. CONCLUSIONS: Our results suggested a novel CUL4B/miR-33b/C-MYC axis implicated in PCa cell growth and progression. This might provide novel insight into how CUL4B contributed to PCa aggressiveness and progression.

CUL4B regulates cancer stem-like traits of prostate cancer cells by targeting BMI1 via miR200b/c.

BACKGROUND: Cancer stem-like traits contribute to prostate cancer (PCa) progression and metastasis. Cullin 4B (CUL4B) is a member of the ubiquitin E3 ligase family and overexpressed in several solid malignancies including PCa. CUL4B has been suggested to be an oncogene through epigenetic repression of tumor suppressors. However, the link between CUL4B expression and cancer stem-like phenotype remains unclear. METHODS: Western blot analysis, sphere formation, and colony formation assays were used to examine the effect of CUL4B on cancer stem-like traits in PCa cells. Mechanically, bioinformatic analysis was utilized to evaluate whether BMI1 was a target of CUL4B. Moreover, real-time polymerase chain reaction, chromatin immunoprecipitation, and luciferase reporter assays were performed to identify microRNAs regulated by CUL4B. Finally, Western blot assay was used to validate the regulation of CUL4B, miR200b, and miR200c (miR200b/c) on the stem-like characteristics of PCa cells. RESULTS: CUL4B promotes PCa pluripotency-associated markers expression, sphere formation, and anchorage-independent growth ability in vitro. Mechanically, CUL4B upregulates BMI1 expression via epigenetically repressing miR200b/c expression. In addition, miR200b/c could partially reverse CUL4B-induced BMI1 and pluripotency-associated marker expression. CONCLUSIONS: Our study revealed that CUL4B regulates cancer stem-like traits of prostate cancer cells by targeting BMI1 via miR200b/c, which might give novel insight into how CUL4B promotes PCa progression through regulating cancer stem-like traits.

CUL4B promotes metastasis and proliferation in pancreatic cancer cells by inducing epithelial-mesenchymal transition via the Wnt/ß-catenin signaling pathway.

This study determines whether cullin 4B (CUL4B) promotes pancreatic cancer (PC) metastasis by inducing epithelial-mesenchymal transition (EMT) via the Wnt/ß-catenin signaling pathway. A total of 64 PC patients were enrolled in this study. Human PC cell lines were distributed into blank, negative control, shCUL4B, PLOC, PLOC-CUL4B, and PLOC-CUL4B + siRNA-ß-catenin groups. The expressions of CUL4B, Wnt/ß-catenin signaling pathway-related proteins, and EMT-related proteins were determined using RT-qPCR and Western blotting. The positive expressions of CUL4B and ß-catenin protein in tissues were detected by immunohistochemistry. MTT assay and flow cytometry was performed for cell proliferation and cell cycle, scratch test, and transwell assay for cell migration and invasion ability. CUL4B and ß-catenin were expressed at a higher level in PC tissues than in paracancerous tissues though paracancerous tissues had higher expressions of CUL4B and ß-catenin than normal tissues. The PLOC-CUL4B group showed increased CUL4B, Wnt, ß-catenin, LEF-1, c-Jun, Cyclin D1, N-cadherin, Vimentin, Snail, and ZEB1 expression; decreased E-cadherin expression; accelerated cell proliferation; increased S-phase cell percentages; increased cell migration ability; more liver metastases; and enlarged tumor than the PLOC and PLOC-CUL4B + siRNA-ß-catenin groups. The shCUL4B group showed decreased CUL4B, Wnt, ß-catenin, LEF-1, c-Jun, Cyclin D1, N-cadherin, Vimentin, Snail, and ZEB1 expression; increased E-cadherin expression; decelerated cell proliferation; decreased S-phase cell percentages; reduced cell migration ability; less liver metastases; and decreased tumor weight than the blank and negative control groups. We demonstrate that CUL4B promotes PC metastasis by inducing EMT via the Wnt/ß-catenin signaling pathway. Therefore, CUL4B might be the clinical target for treating PC.