Aberrant Upregulation of 14-3-3σ and EZH2 Expression Serves as an Inferior Prognostic Biomarker for Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common malignancy in the world. It is of important significance to find biomarkers for the prognostic monitoring of HCC. The 14-3-3σ and EZH2 proteins are involved in cell cycle regulation and epigenetic silencing. We herein examined the significance of 14-3-3 σ and EZH2 in HCC (n = 167) by immunohistochemistry, RT-PCR and qRT-PCR. The correlation between 14-3-3σ and EZH2 expression and patients'' clinicopathologic features were examined, as was the correlation between 14-3-3σ and EZH2 expression and the prognosis of HCC patients. We found that 14-3-3σ and EZH2 were highly expressed in HCC (71% and 90%), the expression of EZH2, but not 14-3-3σ, is associated with vascular invasion and tumor differentiation (p<0.01). The coexistence of 14-3-3σ and EZH2 overexpression is associated with a relatively unfavorable prognosis (p<0.01), suggesting that aberrant upregulation of 14-3-3σ and EZH2 expression serves as an inferior prognostic biomarker for HCC.     

Proteomic analysis of EZH2 downstream target proteins in hepatocellular carcinoma

Enhancer of zeste homolog 2 (EZH2) is suggested to be a potential therapeutic target and a diagnostic marker for cancer. Our previous study also showed the critical role of EZH2 in hepatocellular carcinoma (HCC) tumorigenesis. The present study is aimed at revealing the comprehensive downstream pathways of EZH2 by functional proteomic profiling. Lentivirus mediated RNA interference (RNAi) was employed to knockdown EZH2 in HCC cells. The 2-DE was employed to compare the expression profile difference between parental and EZH2-knockdown HCC cells. In total, 28 spots were differentially expressed during EZH2 inhibition. Among all, 18 proteins were identified by PMF with MALDI-TOF MS. Western blotting further validated upregulation of 60S acidic ribosomal protein P0 (L10E), and downregulation of two proteins with EZH2 inhibition: stathmin1 and probable protein disulfide isomerase (PDI) ER-60 precursor (ERp57). Moreover, L10E was downregulated with overexpression of EZH2 in hepatocytes, and L10E reversed the effect of EZH2 on cell proliferation, suggesting it a downstream target of EZH2. The comprehensive and comparative analyses of proteins associated with EZH2 could further our understanding on the downstream signal cascade of EZH2 leading to tumorigenesis.     

Tumor Suppressive Function of p21-activated Kinase 6 in Hepatocellular Carcinoma

Our previous studies identified the oncogenic role of p21-activated kinase 1 (PAK1) in hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC). Contrarily, PAK6 was found to predict a favorable prognosis in RCC patients. Nevertheless, the ambiguous tumor suppressive function of PAK6 in hepatocarcinogenesis remains obscure. Herein, decreased PAK6 expression was found to be associated with tumor node metastasis stage progression and unfavorable overall survival in HCC patients. Additionally, overexpression and silence of PAK6 experiments showed that PAK6 inhibited xenografted tumor growth in vivo, and restricted cell proliferation, colony formation, migration, and invasion and promoted cell apoptosis and anoikis in vitro. Moreover, overexpression of kinase dead and nuclear localization signal deletion mutants of PAK6 experiments indicated the tumor suppressive function of PAK6 was partially dependent on its kinase activity and nuclear translocation. Furthermore, gain or loss of function in polycomb repressive complex 2 (PRC2) components, including EZH2, SUZ12, and EED, elucidated epigenetic control of H3K27me3-arbitrated PAK6 down-regulation in hepatoma cells. More importantly, negative correlation between PAK6 and EZH2 expression was observed in hepatoma tissues from HCC patients. These data identified the tumor suppressive role and potential underlying mechanism of PAK6 in hepatocarcinogenesis.     

MiR-214 Targets β-Catenin Pathway to Suppress Invasion, Stem-Like Traits and Recurrence of Human Hepatocellular Carcinoma

The down-regulation of miR-214 has previously been observed in human hepatocellular carcinoma (HCC). Here, we demonstrated the down-regulation of miR-214 is associated with cell invasion, stem-like traits and early recurrence of HCC. Firstly, we validated the suppression of miR-214 in human HCC by real-time quantitative RT-PCR (qRT-PCR) in 20 paired tumor and non-tumor liver tissues of HCC patients and 10 histologically normal liver tissues from colorectal cancer patients with liver metastases. Further qRT-PCR analysis of 50 HCC tissues from an independent cohort of HCC patients of whom 29 with early recurrent disease (<2 years) and 21 with late recurrent disease demonstrated that the suppression of miR-214 was significantly more suppressed in samples from HCC patients with early recurrent disease compared those from patients with no recurrence. Re-expression of miR-214 significantly suppressed the growth of HCC cells in vitro and reduced their tumorigenicity in vivo. The enhancer of zeste homologue 2 (EZH2) and β-catenin (CTNNB1) was identified as two potential direct downstream targets of miR-214 through bioinformatics analysis and experimentally validated the miRNA-target interactions with a dual-firefly luciferase reporter assay. In corroborate with this, both EZH2 and CTNNB1 are found to be significantly overexpressed in human HCC biopsies. Since EZH2 can regulate CTNNB1, CTNNB1 can also be an indirect target of miR-214 through EZH2. Silencing EZH2 or CTNNB1 expression suppressed the growth and invasion of HCC cells and induced E-cadherin (CDH1), known to inhibit cell invasion and metastasis. Furthermore, the silencing of miR-214 or overexpression of EZH2 increased EpCAM(+) stem-like cells through the activation of CTNNB1. Interestingly, the up-regulation of EZH2, CTNNB1 and the down-regulation of CDH1 in HCC patients correlated with early recurrent disease and can be an independent predictor of poor survival. Therefore, miR-214 can directly or indirectly target CTNNB1 to modulate the β-catenin signaling pathway in HCC.     

LncRNA HOTAIR regulates the expression of E-cadherin to affect nasopharyngeal carcinoma progression by recruiting histone methylase EZH2 to mediate H3K27 trimethylation

Background/AimThere has been increasing evidence for the function of long non-coding RNA (lncRNA) in nasopharyngeal carcinoma (NPC). We aim to delve into the position of lncRNA HOX antisense intergenic RNA (HOTAIR), together with enhancer of zeste homolog 2 (EZH2), E-cadherin and trimethylation of lysine 27 on histone H3 (H3K27me3) in NPC.MethodsHOTAIR, EZH2, and E-cadherin expression in NPC tissues and cells were tested. NPC cell biological functions were examined through gain-of and loss-of function assays. The mechanism of lncRNA HOTAIR/E-cadherin/EZH2/H3K27 axis in NPC was decoded.ResultsLncRNA HOTAIR and EZH2 were highly expressed in NPC, and E-cadherin was lowly expressed. Down-regulation of HOTAIR or EZH2 inhibited NPC cell progression and tumor growth. HOTAIR recruited histone methylase EZH2 to mediate trimethylation of H3K27 and regulated E-cadherin expression.ConclusionHOTAIR inhibits E-cadherin by stimulating the trimethylation of H3K27 to promote NPC cell progression through recruiting histone methylase EZH2.     

P53 suppresses the progression of hepatocellular carcinoma via miR-15a by decreasing OGT expression and EZH2 stabilization

Existing literature has highlighted the tumour suppressive capacity of microRNA-15a (miR-15a); however, its role in hepatocellular carcinoma (HCC) remains relatively unknown. This study aimed to investigate the role of miR-15a in HCC and the associated underlying mechanism. Initially, RT-qPCR was performed to detect the expression of miR-15a in HCC tissues and cells. Bioinformatics analysis, Pearson correlation coefficient, dual-luciferase reporter assay, and molecular approaches were all conducted to ascertain the interaction between miR-15a and O-linked N-acetylglucosamine (GlcNAc) transferase (OGT). PUGNAc treatment and cycloheximide (CHX) assay were performed to evaluate O-GlcNAc and the stabilization of the enhancer of zeste homolog 2 (EZH2). Finally, gain- and loss-of-function studies were employed to elucidate the role of P53 and the miR-15a/OGT/EZH2 axis in the progression of HCC, followed by in vivo experiments based on tumour-bearing nude mice. Our results demonstrated that the miR-15a expression was decreased in the HCC tissues and cells. P53 upregulated miR-15a expression, which inhibited the proliferation, migration and invasion of HCC cells, while inducing apoptosis and triggering a G0/G1 cell cycle phase arrest. OGT stabilized EZH2 via catalysing O-GlcNAc, which reversed the effect of P53 and miR-15a. The results of our in vivo study provided evidence demonstrating that P53 could suppress the development of HCC via the miR-15a/OGT/EZH2 axis. P53 was found to inhibit the OGT expression by promoting the expression of miR-15a, which destabilized EZH2 and suppressed the development of HCC. The key findings of our study highlight a promising novel therapeutic strategy for the treatment of HCC.     

EZH2-Mediated H3K27me3 Is Involved in Epigenetic Repression of Deleted in Liver Cancer 1 in Human Cancers

Enhancer of zeste homolog 2 (EZH2), the histone methyltransferase of the Polycomb Repressive complex 2 catalyzing histone H3 lysine 27 tri-methylation (H3K27me3), is frequently up-regulated in human cancers. In this study, we identified the tumor suppressor Deleted in liver cancer 1 (DLC1) as a target of repression by EZH2-mediated H3K27me3. DLC1 is a GTPase-activating protein for Rho family proteins. Inactivation of DLC1 results in hyper-activated Rho/ROCK signaling and is implicated in actin cytoskeleton reorganization to promote cancer metastasis. By chromatin immunoprecipitation assay, we demonstrated that H3K27me3 was significantly enriched at the DLC1 promoter region of a DLC1-nonexpressing HCC cell line, MHCC97L. Depletion of EZH2 in MHCC97L by shRNA reduced H3K27me3 level at DLC1 promoter and induced DLC1 gene re-expression. Conversely, transient overexpression of GFP-EZH2 in DLC1-expressing Huh7 cells reduced DLC1 mRNA level with a concomitant enrichment of EZH2 on DLC1 promoter. An inverse relation between EZH2 and DLC1 expression was observed in the liver, lung, breast, prostate, and ovarian cancer tissues. Treating cancer cells with the EZH2 small molecular inhibitor, 3-Deazaneplanocin A (DZNep), restored DLC1 expression in different cancer cell lines, indicating that EZH2-mediated H3K27me3 epigenetic regulation of DLC1 was a common mechanism in human cancers. Importantly, we found that DZNep treatment inhibited HCC cell migration through disrupting actin cytoskeleton network, suggesting the therapeutic potential of DZNep in targeting cancer metastasis. Taken together, our study has shed mechanistic insight into EZH2-H3K27me3 epigenetic repression of DLC1 and advocated the significant pro-metastatic role of EZH2 via repressing tumor and metastasis suppressors.     

LncRNA MAGI2-AS3 inhibits hepatocellular carcinoma cell proliferation and migration by targeting the miR-374b-5p/SMG1 signaling pathway

Long noncoding RNAs (lncRNAs) have been proven to play critical roles in cancer progression. Recently, lncRNA MAGI2-AS3 has been revealed to be a tumor suppressor and inhibit cell growth by targeting the Fas/FasL signalling pathway in breast cancer. However, the role and underlying mechanism of MAGI2-AS3 in hepatocellular carcinoma (HCC) remain largely unknown. In the current study, we found that MAGI2-AS3 expression is downregulated in HCC tissues and closely associated with some clinical characteristics (tumor size, lymph node metastasis, and TNM stage) and poor overall survival. Overexpression of MAGI2-AS3 inhibits HCC cell proliferation and migration in vitro, while impedes tumor growth in vivo accordantly. In addition, our data suggest that MAGI2-AS3 could function as an endogenous sponge of miR-374b-5p by directly binding to it and suppressing its expression. Furthermore, miR-374b-5p upregulation could restore the inhibitory effect of MAGI2-AS3 on HCC cells processes. Moreover, suppressor with morphogenetic effect on genitalia family member 1 (SMG1) is positively regulated by MAGI2-AS3 via absorbing miR-374b-5p in HCC cells. More important, SMG1 knockdown reverses the suppressive function of MAGI2-AS3 in HCC cell processes. Taken together, we reveal a functional MAGI2-AS3/miR-374b-5p/SMG1 axis that suppresses HCC progression, potently suggesting a new road for HCC treatment.     

LncRNA CASC11 promoted gastric cancer cell proliferation,migration and invasion in vitro by regulating cell cycle pathway

In this study, we aimed to investigate the effects of lncRNA CASC11 on gastric cancer (GC) cell progression through regulating miR-340-5p and cell cycle pathway. Expressions of lncRNA CASC11 in gastric cancer tissues and cell lines were determined by qRT-PCR. Differentially expressed lncRNAs, mRNAs and miRNAs were screened through microarray analysis. The relationship among CASC11, CDK1 and miR-340-5p was predicted by TargetScan and validated through dual luciferase reporter assay. Western blot assay examined the protein level of CDK1 and several cell cycle regulatory proteins. GO functional analysis and KEGG pathway analysis were used to predict the association between functions and related pathways. Cell proliferation was determined by CCK-8 assays. Cell apoptosis and cell cycle were detected by flow cytometry assay. CASC11 was highly expressed in GC tissues and cell lines. Knockdown of CASC11 inhibited GC cell proliferation, promoted cell apoptosis and blocked cell cycle. KEGG further indicated an enriched cell cycle pathway involving CDK1. QRT-PCR showed that miR-340-5p was down-regulated in GC cells tissues, while CDK1 was up-regulated. Furthermore, CASC11 acted as a sponge of miR-340-5p which directly targeted CDK1. Meanwhile, miR-340-5p overexpression promoted GC cell apoptosis and induced cell cycle arrest, while CDK1 overexpression inhibited cell apoptosis and accelerated cell cycle. Our study revealed the mechanism of CASC11/miR-340-5p/CDK1 network in GC cell line, and suggested that CASC11 was a novel facilitator that exerted a biological effect by activating the cell cycle signaling pathway. This finding provides a potential therapeutic target for GC.     

Loss of let-7 up-regulates EZH2 in prostate cancer consistent with the acquisition of cancer stem cell signatures that are attenuated by BR-DIM

The emergence of castrate-resistant prostate cancer (CRPC) contributes to the high mortality of patients diagnosed with prostate cancer (PCa), which in part could be attributed to the existence and the emergence of cancer stem cells (CSCs). Recent studies have shown that deregulated expression of microRNAs (miRNAs) contributes to the initiation and progression of PCa. Among several known miRNAs, let-7 family appears to play a key role in the recurrence and progression of PCa by regulating CSCs; however, the mechanism by which let-7 family contributes to PCa aggressiveness is unclear. Enhancer of Zeste homolog 2 (EZH2), a putative target of let-7 family, was demonstrated to control stem cell function. In this study, we found loss of let-7 family with corresponding over-expression of EZH2 in human PCa tissue specimens, especially in higher Gleason grade tumors. Overexpression of let-7 by transfection of let-7 precursors decreased EZH2 expression and repressed clonogenic ability and sphere-forming capacity of PCa cells, which was consistent with inhibition of EZH2 3'UTR luciferase activity. We also found that the treatment of PCa cells with BR-DIM (formulated DIM: 3,3'-diindolylmethane by Bio Response, Boulder, CO, abbreviated as BR-DIM) up-regulated let-7 and down-regulated EZH2 expression, consistent with inhibition of self-renewal and clonogenic capacity. Moreover, BR-DIM intervention in our on-going phase II clinical trial in patients prior to radical prostatectomy showed upregulation of let-7 consistent with down-regulation of EZH2 expression in PCa tissue specimens after BR-DIM intervention. These results suggest that the loss of let-7 mediated increased expression of EZH2 contributes to PCa aggressiveness, which could be attenuated by BR-DIM treatment, and thus BR-DIM is likely to have clinical impact.     

MER-ERK信号通路调控H3K27me3甲基化酶和去甲基化酶基因表达的研究

在人的某些癌症细胞中,组蛋白H3K27me3甲基化酶EZH2基因存在过表达的现象,很多研究已经证明,这可能是受MEK ERK信号通路调控的.为了确定这种调控模式在小鼠细胞系中是否同样存在,以及MEK ERK信号通路是否同时调控H3K27me3甲基化酶EZH1基因和去甲基化酶UTX、JMJD3基因的表达,用RT PCR和Western印迹方法检测不同浓度的MEK ERK抑制剂U0126（0、10、20、40 μmol/L）对C2C12、C127、NIH3T3三种小鼠细胞系处理后,EZH1、EZH2基因和UTX、JMJD3基因表达变化.结果显示：MEK-ERK抑制剂处理后,3种细胞中EZH1和EZH2基因的表达与对照相比都有不同程度的降低,其中EZH2基因表达变化在C2C12、NIH3T3两种细胞达到显著水平(P<0.05). H3K27me3去甲基化酶UTX、JMJD3基因在3种细胞中表达均有升高,JMJD3升高达到显著水平（P<0.05).因此,在小鼠细胞系MEK ERK信号通路可能参与调控EZH2、JMJD3基因的表达,但对EZH1、UTX基因的表达调控作用不明显.
关键词MEK ERK信号通路;     

UNC5B‐AS1 promoted ovarian cancer progression by regulating the H3K27me on NDRG2 via EZH2

The role of long non‐coding RNAs (lncRNAs) in tumorigenesis and development of ovarian cancer (OC) has caught the attention of scientists. UNC5B antisense RNA 1 (UNC5B‐AS1) is a newly identified carcinogenic lncRNA in thyroid papillary carcinoma, but its role in OC remains unclear. This study is proposed to investigate the function and mechanism of UNC5B‐AS1 in OC. UNC5B‐AS1 expression in OC samples was obtained from gene expression profiling interactive analysis (GEPIA) based on The Cancer Genome Atlas data. Gene expressions were detected by quantitative real‐time polymerase chain reaction (RT‐qPCR) and western blot. Biological functions of UNC5B‐AS1 were assessed by cell counting kit‐8, colony formation, and caspase‐3 analysis. GEPIA revealed the UNC5B‐AS1 upregulation in OC samples. RT‐qPCR assay confirmed the upregulation of UNC5B‐AS1 in OC cells. Functionally, depletion of UCN5B‐AS1 hindered proliferation and prompted apoptosis in OC cells. Mechanistically, we found that UNC5B‐AS1 interacted with zeste 2 polycomb repressive complex 2 subunit (EZH2) to trigger trimethylation of histone H3 at lysine 27 (H3K27me3) on N‐myc downstream regulated gene‐2 (NDRG2) promoter and epigenetically repressed NDRG2. Rescue assay indicated the participation of NDRG2 in the regulation of UNC5B‐AS1 on OC progression. Together, we first illustrated that UNC5B‐AS1 promoted OC progression by regulating the H3K27me on NDRG2 via EZH2, indicating UNC5B‐AS1 as a potential molecular target for OC treatment.     

MicroRNA‐137 is negatively associated with clinical outcome and regulates tumor development through EZH2 in cervical cancer

We intend to evaluate the expression, clinical relevance, and functional role of microRNA‐137 (miR‐137) in human cervical cancer (CC). MiR‐137 expressions were assessed by qPCR in CC cell lines and human CC tumors. The correlation between endogenous miR‐137 expression and CC patients’ postoperative overall survival was examined statistically. CC cell lines, Ca‐Ski, and SiHa cells were transduced with lentivirus to ectopically upregulate endogenous miR‐137 expressions. Possible inhibitory effects of miR‐137 upregulation on CC in vitro proliferation and migration, as well as in vivo transplantation were evaluated. Targeting of enhancer of zeste homolog 2 (EZH2) gene by miR‐137 in CC was assessed by dual‐luciferase activity assay and qPCR. In CC cells with upregulated miR‐137, EZH2 was overexpressed to assess its direct function in miR‐137 mediated CC proliferation and migration. MiR‐137 was downregulated in both CC cells and human CC tumors. Downregulation of endogenous miR‐137 was significantly correlated with CC patients’ short overall survival. In CC cells, miR‐137 upregulation is tumor‐suppressive by inhibiting proliferation and migration in vitro, and transplantation in vivo. EZH2 was a direct downstream target gene of miR‐137 in CC. Forced overexpression of EZH2 in miR‐137‐upregulated CC cells reversed the tumor‐suppression induced by miR‐137. MiR‐137 is lowly expressed in CC and possibly acting as a negative biomarker for CC patients’ clinical outcome. MiR‐137 upregulation may suppress CC, very likely by inversely regulating EZH2.     

PCI-24781 down-regulates EZH2 expression and then promotes glioma apoptosis by suppressing the PIK3K/Akt/mTOR pathway

PCI-24781 is a novel histone deacetylase inhibitor that inhibits tumor proliferation and promotes cell apoptosis. However, it is unclear whether PCI-24781 inhibits Enhancer of Zeste 2 (EZH2) expression in malignant gliomas. In this work, three glioma cell lines were incubated with various concentrations of PCI-24781 (0, 0.25, 0.5, 1, 2.5 and 5 μM) and analyzed for cell proliferation by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay and colony formation, and cell cycle and apoptosis were assessed by flow cytometry. The expression of EZH2 and apoptosis-related proteins was assessed by western blotting. Malignant glioma cells were also transfected with EZH2 siRNA to examine how PCI-24781 suppresses tumor cells. EZH2 was highly expressed in the three glioma cell lines. Incubation with PCI-24781 reduced cell proliferation and increased cell apoptosis by down-regulating EZH2 in a concentration-dependent manner. These effects were simulated by EZH2 siRNA. In addition, PCI-24781 or EZH2 siRNA accelerated cell apoptosis by down-regulating the expression of AKT, mTOR, p70 ribosomal protein S6 kinase (p70s6k), glycogen synthase kinase 3A and B (GSK3a/b) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). These data suggest that PCI-24781 may be a promising therapeutic agent for treating gliomas by down-regulating EZH2 which promotes cell apoptosis by suppressing the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway.     

Downregulation of MEG3 and upregulation of EZH2 cooperatively promote neuroblastoma progression

Neuroblastoma (NB), an embryonic tumour originating from sympathetic crest cells, is the most common extracranial solid tumour type in children with poor overall prognosis. Accumulating evidence has demonstrated the involvement of long non‐coding RNA (lncRNA) in numerous biological processes and their associations with embryonic development and multiple diseases. Ectopic lncRNA expression is linked to malignant tumours. Previous studies by our team indicate that MEG3 attenuates NB autophagy through inhibition of FOXO1 and epithelial‐mesenchymal transition via the mTOR pathway in vitro. Moreover, MEG3 and EZH2 negatively regulate each other. In present study, we first collected 60 NB tissues and 20 adjacent tissues for Quantitative real‐time polymerase chain reaction (Q‐PCR) experiments and performed clinical correlation analysis of the results. At the same time, nude mice were used for subcutaneous tumour formation to detect the effect of MEG3 in vivo. Two NB cell lines, SK‐N‐AS and SK‐N‐BE(2)C, were overexpressed MEG3 and rescued with EZH2 and then were subjected to proliferation, migration, invasion, apoptosis and autophagy experiments. RNA‐binding protein immunoprecipitation (RIP) and Co‐Immunoprecipitation (Co‐IP) experiments were performed to explore the molecular mechanism of MEG3 and EZH2 interaction. Q‐PCR revealed that MEG3 expression was negatively correlated with INSS stage and risk grade of NB. Moreover, MEG3 overexpression was associated with inhibition of NB growth in vivo. MEG3 exerted an anti‐cancer effect via stimulatory effects on EZH2 ubiquitination leading to its degradation. Conversely, EZH2 interacted with DNMT1 and HDAC1 to induce silencing of MEG3. The EZH2 inhibitor, DZNep, and HDAC inhibitor, SAHA, displayed synergistic activity against NB. Combined treatment with DZNep and SAHA inhibited proliferation, migration and invasion of NB through suppression of the PI3K/AKT/mTOR/FOXO1 pathway. In conclusion, downregulation of MEG3 and upregulation of EZH2 forms a feedback loop that concertedly promotes the development of NB. Combined blockage of EZH2 and HDAC1 with the appropriate inhibitors may therefore present an effective treatment strategy for NB cases with low MEG3 and high EZH2 expression.