METTL14 downregulation drives S100A4+ monocyte-derived macrophages via MyD88/NF-κB pathway to promote MAFLD progression.

Without intervention, a considerable proportion of patients with metabolism-associated fatty liver disease (MAFLD) will progress from simple steatosis to metabolism-associated steatohepatitis (MASH), liver fibrosis, and even hepatocellular carcinoma. However, the molecular mechanisms that control progressive MAFLD have yet to be fully determined. Here, we unraveled that the expression of the N6-methyladenosine (m6A) methyltransferase METTL14 is remarkably downregulated in the livers of both patients and several murine models of MAFLD, whereas hepatocyte-specific depletion of this methyltransferase aggravated lipid accumulation, liver injury, and fibrosis. Conversely, hepatic Mettl14 overexpression alleviated the above pathophysiological changes in mice fed on a high-fat diet (HFD). Notably, in vivo and in vitro mechanistic studies indicated that METTL14 downregulation decreased the level of GLS2 by affecting the translation efficiency mediated by YTHDF1 in an m6A-depedent manner, which might help to form an oxidative stress microenvironment and accordingly recruit Cx3cr1+Ccr2+ monocyte-derived macrophages (Mo-macs). In detail, Cx3cr1+Ccr2+ Mo-macs can be categorized into M1-like macrophages and S100A4-positive macrophages and then further activate hepatic stellate cells (HSCs) to promote liver fibrosis. Further experiments revealed that CX3CR1 can activate the transcription of S100A4 via CX3CR1/MyD88/NF-κB signaling pathway in Cx3cr1+Ccr2+ Mo-macs. Restoration of METTL14 or GLS2, or interfering with this signal transduction pathway such as inhibiting MyD88 could ameliorate liver injuries and fibrosis. Taken together, these findings indicate potential therapies for the treatment of MAFLD progression.

SPHK1/S1P/S1PR pathway promotes the progression of peritoneal fibrosis by mesothelial-mesenchymal transition.

Long-term exposure to non-physiologically compatible dialysate inevitably leads to peritoneal fibrosis (PF) in patients undergoing peritoneal dialysis (PD), and there is no effective prevention or treatment for PF. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid produced after catalysis by sphingosine kinase (SPHK) 1/2 and activates signals through the S1P receptor (S1PR) via autocrine or paracrine. However, the role of SPHK1/S1P/S1PR signaling has never been elucidated in PF. In our research, we investigated S1P levels in peritoneal effluents and demonstrated the role of SPHK1/S1P/S1PR pathway in peritoneal fibrosis. It was found that S1P levels in peritoneal effluents were positively correlated with D/P Cr (r = 0.724, p < .001) and negatively correlated with 4 h ultrafiltration volume (r = -0.457, p < .001). S1PR1 and S1PR3 on peritoneal cells were increased after high glucose exposure in vivo and in vitro. Fingolimod was applied to suppress S1P/S1PR pathway. Fingolimod restored mouse peritoneal function by reducing interstitial hyperplasia, maintaining ultrafiltration volume, reducing peritoneal transport solute rate, and mitigating the protein expression changes of fibronectin, vimentin, α-SMA, and E-cadherin induced by PD and S1P. Fingolimod preserved the morphology of the human peritoneal mesothelial cells, MeT-5A, and moderated the mesothelial-mesenchymal transition (MMT) process. We further delineated that SPHK1 was elevated in peritoneal cells after high glucose exposure and suppression of SPHK1 in MeT-5A cells reduced S1P release. Overexpression of SPHK1 in MeT-5A cells increased S1P levels in the supernatant and fostered the MMT process. PF-543 treatment, targeting SPHK1, alleviated deterioration of mouse peritoneal function. In conclusion, S1P levels in peritoneal effluent were correlated with the deterioration of peritoneal function. SPHK1/S1P/S1PR pathway played an important role in PF.

The ligation between ERMAP, galectin-9 and dectin-2 promotes Kupffer cell phagocytosis and antitumor immunity.

Kupffer cells, the liver tissue resident macrophages, are critical in the detection and clearance of cancer cells. However, the molecular mechanisms underlying their detection and phagocytosis of cancer cells are still unclear. Using in vivo genome-wide CRISPR-Cas9 knockout screening, we found that the cell-surface transmembrane protein ERMAP expressed on various cancer cells signaled to activate phagocytosis in Kupffer cells and to control of liver metastasis. ERMAP interacted with ß-galactoside binding lectin galectin-9 expressed on the surface of Kupffer cells in a manner dependent on glycosylation. Galectin-9 formed a bridging complex with ERMAP and the transmembrane receptor dectin-2, expressed on Kupffer cells, to induce the detection and phagocytosis of cancer cells by Kupffer cells. Patients with low expression of ERMAP on tumors had more liver metastases. Thus, our study identified the ERMAP-galectin-9-dectin-2 axis as an 'eat me' signal for Kupffer cells.

Tamoxifen exerts anti-peritoneal fibrosis effects by inhibiting H19-activated VEGFA transcription.

BACKGROUND: Peritoneal dialysis (PD) remains limited due to dialysis failure caused by peritoneal fibrosis. Tamoxifen (TAM), an inhibitor of estrogen receptor 1 (ESR1), has been reported to treat fibrosis, but the underlying mechanism remains unknown. In this study, we sought to explore whether tamoxifen played an anti-fibrotic role by affecting transcription factor ESR1. METHODS: ESR1 expression was detected in the human peritoneum. Mice were daily intraperitoneally injected with 4.25% glucose PD dialysate containing 40 mM methylglyoxal for 2 weeks to establish PD-induced peritoneal fibrosis. Tamoxifen was administrated by daily gavage, at the dose of 10 mg/kg. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assay were performed to validate ESR1 bound H19 promoter. Gain-of-function and loss-of-function experiments were performed to investigate the biological roles of H19 on the mesothelial-mesenchymal transition (MMT) of human peritoneal mesothelial cells (HPMCs). Intraperitoneal injection of nanomaterial-wrapped 2'-O-Me-modified small interfering RNA was applied to suppress H19 in the mouse peritoneum. RNA immunoprecipitation and RNA pull-down assays demonstrated binding between H19 and p300. Exfoliated peritoneal cells were obtained from peritoneal dialysis effluent to analyze the correlations between ESR1 (or H19) and peritoneal solute transfer rate (PSTR). RESULTS: ESR1 was increased significantly in the peritoneum after long-term exposure to PD dialysate. Tamoxifen treatment ameliorated high glucose-induced MMT of HPMCs, improved ultrafiltration rate, and decreased PSTR of mouse peritoneum. Tamoxifen reduced the H19 level by decreasing the ESR1 transcription of H19. Depletion of H19 reversed the pro-fibrotic effect of high glucose while ectopic expression of H19 exacerbated fibrotic pathological changes. Intraperitoneal injection of nanomaterial-wrapped 2'-O-Me-modified siRNAs targeting H19 mitigated PD-related fibrosis in mice. RNA immunoprecipitation (RIP) and RNA pull-down results delineated that H19 activated VEGFA expression by binding p300 to the VEGFA promoter and inducing histone acetylation of the VEGFA promoter. ESR1 and H19 were promising targets to predict peritoneal function. CONCLUSIONS: High glucose-induced MMT of peritoneal mesothelial cells in peritoneal dialysis via activating ESR1. In peritoneal mesothelial cells, ESR1 transcribed the H19 and H19 binds to transcription cofactor p300 to activate the VEGFA. Targeting ESR1/H19/VEGFA pathway provided new hope for patients undergoing peritoneal dialysis.

Identification of novel factors that affect the onset of idiopathic chronic pancreatitis: The role for microRNA-323b-5p.

BACKGROUND: The c.194+2 T>C variant of serine protease inhibitor Kazal type 1 (SPINK1) is a known genetic risk factor found in Chinese patients with idiopathic chronic pancreatitis (ICP), but the early-onset mechanisms of ICP are still unclear. METHODS: Complementary experimental approaches were used to pursue other potential pathologies in the present study. The serum level of SPINK1 of ICP patients in the Han population in China was detected and verified by an enzyme-linked immunosorbent assay. Next, differentially expressed proteins and microRNAs from plasma samples of early-onset and late-onset ICP patients were screened by proteomic analysis and microarray, respectively. RESULTS: Combined with these advanced methods, the data strongly suggest that the regulatory effects of microRNAs were involved in the early-onset mechanism of the ICP by in vitro experiments. There was no significant difference in the plasma SPINK1 expression between the early-onset ICP and the late-onset patients. However, the expression of plasma glutathione peroxidase (GPx3) in early-onset ICP patients was markedly lower than that in late-onset ICP patients, although the level of hsa-miR-323b-5p was lower in late-onset patients compared to the early-onset ICP group. In vitro experiments confirmed that hsa-miR-323b-5p could increase apoptosis in caerulein-treated pancreatic acinar cells and inhibit the expression of GPx3. CONCLUSIONS: The up-regulated hsa-miR-323b-5p might play a crucial role in the early-onset mechanisms of ICP by diminishing the antioxidant activity through the down-regulation of GPx3.

METTL16 promotes hepatocellular carcinoma progression through downregulating RAB11B-AS1 in an m6A-dependent manner.

BACKGROUND: The molecular mechanisms driving hepatocellular carcinoma (HCC) remain largely unclear. As one of the major epitranscriptomic modifications, N6-methyladenosine (m6A) plays key roles in HCC. The aim of this study was to investigate the expression, roles, and mechanisms of action of the RNA methyltransferase methyltransferase-like protein 16 (METTL16) in HCC. METHODS: The expression of METTL16 and RAB11B-AS1 was determined by RT-qPCR. The regulation of RAB11B-AS1 by METTL16 was investigated by RNA immunoprecipitation (RIP), methylated RIP (MeRIP), and RNA stability assays. In vitro and in vivo gain- and loss-of-function assays were performed to investigate the roles of METTL16 and RAB11B-AS1. RESULTS: METTL16 was upregulated in HCC, and its increased expression was correlated with poor prognosis of HCC patients. METTL16 promoted HCC cellular proliferation, migration, and invasion, repressed HCC cellular apoptosis, and promoted HCC tumoral growth in vivo. METTL16 directly bound long noncoding RNA (lncRNA) RAB11B-AS1, induced m6A modification of RAB11B-AS1, and decreased the stability of RAB11B-AS1 transcript, leading to the downregulation of RAB11B-AS1. Conversely to METTL16, RAB11B-AS1 is downregulated in HCC, and its decreased expression was correlated with poor prognosis of patients with HCC. Furthermore, the expression of RAB11B-AS1 was negatively correlated with METTL16 in HCC tissues. RAB11B-AS1 repressed HCC cellular proliferation, migration, and invasion, promoted HCC cellular apoptosis, and inhibited HCC tumoral growth in vivo. Functional rescue assays revealed that overexpression of RAB11B-AS1 reversed the oncogenic roles of METTL16 in HCC. CONCLUSIONS: This study identified the METTL16/RAB11B-AS1 regulatory axis in HCC, which represented novel targets for HCC prognosis and treatment.

SLC38A4 functions as a tumour suppressor in hepatocellular carcinoma through modulating Wnt/ß-catenin/MYC/HMGCS2 axis.

BACKGROUND: Many molecular alterations are shared by embryonic liver development and hepatocellular carcinoma (HCC). Identifying the common molecular events would provide a novel prognostic biomarker and therapeutic target for HCC. METHODS: Expression levels and clinical relevancies of SLC38A4 and HMGCS2 were investigated by qRT-PCR, western blot, TCGA and GEO datasets. The biological roles of SLC38A4 were investigated by functional assays. The downstream signalling pathway of SLC38A4 was investigated by qRT-PCR, western blot, immunofluorescence, luciferase reporter assay, TCGA and GEO datasets. RESULTS: SLC38A4 silencing was identified as an oncofetal molecular event. DNA hypermethylation contributed to the downregulations of Slc38a4/SLC38A4 in the foetal liver and HCC. Low expression of SLC38A4 was associated with poor prognosis of HCC patients. Functional assays demonstrated that SLC38A4 depletion promoted HCC cellular proliferation, stemness and migration, and inhibited HCC cellular apoptosis in vitro, and further repressed HCC tumorigenesis in vivo. HMGCS2 was identified as a critical downstream target of SLC38A4. SLC38A4 increased HMGCS2 expression via upregulating AXIN1 and repressing Wnt/ß-catenin/MYC axis. Functional rescue assays showed that HMGCS2 overexpression reversed the oncogenic roles of SLC38A4 depletion in HCC. CONCLUSIONS: SLC38A4 downregulation was identified as a novel oncofetal event, and SLC38A4 was identified as a novel tumour suppressor in HCC.

LncRNA HOXA11-AS regulates calcium oxalate crystal-induced renal inflammation via miR-124-3p/MCP-1.

Long noncoding RNA (lncRNA) has been suggested to play an important role in a variety of diseases over the past decade. In a previous study, we identified a novel lncRNA, termed HOXA11-AS, which was significantly up-regulated in calcium oxalate (CaOx) nephrolithiasis. However, the biological function of HOXA11-AS in CaOx nephrolithiasis remains poorly defined. Here, we demonstrated that HOXA11-AS was significantly up-regulated in CaOx nephrolithiasis both in vivo and in vitro. Gain-/loss-of-function studies revealed that HOXA11-AS inhibited proliferation, promoted apoptosis and aggravated cellular damage in HK-2 cells exposed to calcium oxalate monohydrate (COM). Further investigations showed that HOXA11-AS regulated monocyte chemotactic protein 1 (MCP-1) expression in HK-2 cell model of CaOx nephrolithiasis. In addition, online bioinformatics analysis and dual-luciferase reporter assay results showed that miR-124-3p directly bound to HOXA11-AS and the 3'UTR of MCP-1. Furthermore, rescue experiment results revealed that HOXA11-AS functioned as a competing endogenous RNA to regulate MCP-1 expression through sponging miR-124-3p and that overexpression of miR-124-3p restored the inhibitory effect of proliferation, promotion effects of apoptosis and cell damage induced by HOXA11-AS overexpression. Taken together, HOXA11-AS mediated CaOx crystal-induced renal inflammation via the miR-124-3p/MCP-1 axis, and this outcome may provide a good potential therapeutic target for nephrolithiasis.

CTGF secreted by mesenchymal-like hepatocellular carcinoma cells plays a role in the polarization of macrophages in hepatocellular carcinoma progression.

M2 macrophages play critical roles in the progression of hepatocellular carcinoma (HCC), and they are associated with poor outcomes. TGF-ß-induced epithelial-mesenchymal transition (EMT) has been shown to be critically important to cancer cell dissemination in HCC. However, the relationship between stromal-like HCC cells and M2 macrophages formation is not clear. Here, we interrogated the molecular link between mesenchymal-like HCC cells and the formation of M2 macrophages. We demonstrated that mesenchymal-like HCC cells secrete connective tissue growth factor (CTGF) to polarized macrophages. Reciprocally, Chemokine ligand 18 (CCL18) from M2 macrophages promotes HCC progression. Furthermore, CTGF and CCL18 were increased significantly in HCC compared to adjacent normal liver tissues. In summary, our study discovered a positive feedback loop between CTGF and CCL18 in HCC metastasis. Targeting CTGF or CCL18 might provide beneficial effects for the clinical treatment of HCC.

Long noncoding RNA MRCCAT1 promotes metastasis of clear cell renal cell carcinoma via inhibiting NPR3 and activating p38-MAPK signaling.

BACKGROUND: Recent evidences showed that long noncoding RNAs (lncRNAs) are frequently dysregulated and play important roles in various cancers. Clear cell renal cell carcinoma (ccRCC) is one of the leading cause of cancer-related death, largely due to the metastasis of ccRCC. However, the clinical significances and roles of lncRNAs in metastatic ccRCC are still unknown. METHODS: lncRNA expression microarray analysis was performed to search the dysregulated lncRNA in metastatic ccRCC. quantitative real-time PCR was performed to measure the expression of lncRNAs in human ccRCC samples. Gain-of-function and loss-of-function experiments were performed to investigate the biological roles of lncRNAs on ccRCC cell proliferation, migration, invasion and in vivo metastasis. RNA pull-down, RNA immunoprecipitation, chromatin immunoprecipitation, and western blot were performed to explore the molecular mechanisms underlying the functions of lncRNAs. RESULTS: The microarray analysis identified a novel lncRNA termed metastatic renal cell carcinoma-associated transcript 1 (MRCCAT1), which is highly expressed in metastatic ccRCC tissues and associated with the metastatic properties of ccRCC. Multivariate Cox regression analysis revealed that MRCCAT1 is an independent prognostic factor for ccRCC patients. Overexpression of MRCCAT1 promotes ccRCC cells proliferation, migration, and invasion. Depletion of MRCCAT1 inhibites ccRCC cells proliferation, migration, and invasion in vitro, and ccRCC metastasis in vivo. Mechanistically, MRCCAT1 represses NPR3 transcription by recruiting PRC2 to NPR3 promoter, and subsequently activates p38-MAPK signaling pathway. CONCLUSIONS: MRCCAT1 is a critical lncRNA that promotes ccRCC metastasis via inhibiting NPR3 and activating p38-MAPK signaling. Our results imply that MRCCAT1 could serve as a prognostic biomarker and therapeutic target for ccRCC.

The MBNL3 splicing factor promotes hepatocellular carcinoma by increasing PXN expression through the alternative splicing of lncRNA-PXN-AS1.

Understanding the roles of splicing factors and splicing events during tumorigenesis would open new avenues for targeted therapies. Here we identify an oncofetal splicing factor, MBNL3, which promotes tumorigenesis and indicates poor prognosis of hepatocellular carcinoma patients. MBNL3 knockdown almost completely abolishes hepatocellular carcinoma tumorigenesis. Transcriptomic analysis revealed that MBNL3 induces lncRNA-PXN-AS1 exon 4 inclusion. The transcript lacking exon 4 binds to coding sequences of PXN mRNA, causes dissociation of translation elongation factors from PXN mRNA, and thereby inhibits PXN mRNA translation. In contrast, the transcript containing exon 4 preferentially binds to the 3' untranslated region of PXN mRNA, protects PXN mRNA from microRNA-24-AGO2 complex-induced degradation, and thereby increases PXN expression. Through inducing exon 4 inclusion, MBNL3 upregulates PXN, which mediates the pro-tumorigenic roles of MBNL3. Collectively, these data demonstrate detailed mechanistic links between an oncofetal splicing factor, a splicing event and tumorigenesis, and establish splicing factors and splicing events as potential therapeutic targets.

An alternative POLDIP3 transcript promotes hepatocellular carcinoma progression.

Alternative splicing plays critical roles in many pathophysiological processes and splicing dysregulation is a hallmark of cancer. The different isoforms may have significantly different effects on cancers. POLDIP3 is a target of ribosomal protein S6 kinase 1, and regulates DNA replication and mRNA translation. In this study, we measured the expression of an alternative POLDIP3 transcript (POLDIP3-ß), which lacks exon 3 and 29 amine acids, in clinical hepatocellular carcinoma (HCC) tissues. The roles of POLDIP3-ß on HCC cell proliferation, apoptosis, and migration were assessed by Glo cell viability assays, Ethynyl deoxyuridine incorporation assays, colony formation assays, TUNEL assays, Annexin V-propidium iodide staining and flow cytometry, transwell assays, wound healing assays, and in vivo xenograft growth. Our results showed that POLDIP3-ß was significantly upregulated in HCC tissues compared with paired adjacent noncancerous hepatic tissues. In vitro and in vivo functional experiments results demonstrated that overexpression of POLDIP3-ß drastically increased HCC cell proliferation, inhibited HCC cell apoptosis, enhanced HCC cell migration, and promoted xenograft growth. While the effects of normal POLDIP3, which contains exon 3, were much weaker. In conclusion, our study demonstrated that an alternative transcript of POLDIP3 is upregulated and functions as a critical oncogene in HCC. Selectively targeting this isoform of POLDIP3 would be a promising therapeutic strategy for HCC.

METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N6 -methyladenosine-dependent primary MicroRNA processing.

N6 -Methyladenosine (m6 A) modification has been implicated in many biological processes. However, its role in cancer has not been well studied. Here, we demonstrate that m6 A modifications are decreased in hepatocellular carcinoma, especially in metastatic hepatocellular carcinoma, and that methyltransferase-like 14 (METTL14) is the main factor involved in aberrant m6 A modification. Moreover, METTL14 down-regulation acts as an adverse prognosis factor for recurrence-free survival of hepatocellular carcinoma and is significantly associated with tumor metastasis in vitro and in vivo. We confirm that METTL14 interacts with the microprocessor protein DGCR8 and positively modulates the primary microRNA 126 process in an m6 A-dependent manner. Further experiments show that microRNA 126 inhibits the repressing effect of METTL14 in tumor metastasis. CONCLUSION: These studies reveal an important role of METTL14 in tumor metastasis and provide a fresh view on m6 A modification in tumor progression. (Hepatology 2017;65:529-543).

Long noncoding RNA glypican 3 (GPC3) antisense transcript 1 promotes hepatocellular carcinoma progression via epigenetically activating GPC3.

Long noncoding RNA (lncRNA) have critical roles in various pathophysiological processes, and are frequently dysregulated in many diseases, particularly in cancer. The lncRNA glypican 3 antisense transcript 1 (GPC3-AS1) has been reported to be a potential biomarker for hepatocellular carcinoma (HCC) screening. However, the exact biological functions of GPC3-AS1 in HCC, and its roles and regulation mechanisms regarding GPC3 are still unknown. In this study, we observed a significant upregulation of GPC3-AS1 in HCC. Increased expression of GPC3-AS1 was associated with α-fetoprotein, tumor size, microvascular invasion, encapsulation, Barcelona Clinic Liver Cancer stage, and worse prognosis of HCC patients. Furthermore, we found that GPC3-AS1 physically associated with P300/CBP-associated factor and recruited it to the GPC3 gene body region, consequently inducing an increase in euchromatic histone marks and activating GPC3 transcription. GPC3-AS1 expression was strongly correlated with GPC3 in HCC tissues. Gain-of-function and loss-of-function analyses showed that GPC3-AS1 overexpression enhanced HCC cell proliferation and migration in vitro and xenograft tumor growth in vivo. GPC3-AS1 knockdown inhibited HCC cell proliferation and migration. Moreover, the effects of GPC3-AS1 on HCC cell proliferation and migration were dependent on the upregulation of GPC3. Collectively, our studies indicate that GPC3-AS1 significantly promotes HCC progression via epigenetically activating GPC3, and identifies GPC3-AS1 as a potential therapeutic target for HCC.

Systemic genome screening identifies the outcome associated focal loss of long noncoding RNA PRAL in hepatocellular carcinoma.

UNLABELLED: Systemic analyses using large-scale genomic profiles have successfully identified cancer-driving somatic copy number variations (SCNVs) loci. However, functions of vast focal SCNVs in "protein-coding gene desert" regions are largely unknown. The integrative analysis of long noncoding RNA (lncRNA) expression profiles with SCNVs in hepatocellular carcinoma (HCC) led us to identify the recurrent deletion of lncRNA-PRAL (p53 regulation-associated lncRNA) on chromosome 17p13.1, whose genomic alterations were significantly associated with reduced survival of HCC patients. We found that lncRNA-PRAL could inhibit HCC growth and induce apoptosis in vivo and in vitro through p53. Subsequent investigations indicated that the three stem-loop motifs at the 5' end of lncRNA-PRAL facilitated the combination of HSP90 and p53 and thus competitively inhibited MDM2-dependent p53 ubiquitination, resulting in enhanced p53 stability. Additionally, in vivo lncRNA-PRAL delivery efficiently reduced intrinsic tumors, indicating its potential therapeutic application. CONCLUSIONS: lncRNA-PRAL, one of the key cancer-driving SCNVs, is a crucial stimulus for HCC growth and may serve as a potential target for antitumor therapy.

Aldolase B inhibits metastasis through Ten-Eleven Translocation 1 and serves as a prognostic biomarker in hepatocellular carcinoma.

BACKGROUND: Downregulation of Aldolase B (ALDOB) has been reported in hepatocellular carcinoma. However, its clinical significance and its role in pathogenesis of HCC remain largely unknown. METHODS: We analyzed the expression of ALDOB and its clinical features in a large cohort of 313 HCC patients using tissue microarray and immunohistochemistry. Moreover, the function of stably overexpressed ALDOB in HCC cells was explored in vitro and in vivo. Gene expression microarray analysis was performed on ALDOB-overexpressing SMMC7721 cells to elucidate its mechanism of action. RESULTS: ALDOB downregulation in HCC was significantly correlated with aggressive characteristics including absence of encapsulation, increased tumor size (>5 cm) and early recurrence. ALDOB downregulation was indicative of a shorter recurrence-free survival (RFS) and overall survival (OS) for all HCC patients and early-stage HCC patients (BCLC 0-A and TNM I stage patients). Multiple analyses revealed that ALDOB downregulation was an independent risk factor of RFS and OS. Stable expression of ALDOB in HCC cell lines reduced cell migration in vitro and inhibited lung metastasis, intrahepatic metastasis, and reduced circulating tumor cells in vivo. Mechanistically, we found that cells stably expressing ALDOB show elevated Ten-Eleven Translocation 1 (TET1) expression. Moreover, ALDOB expressing cells have higher levels of methylglyoxal than do control cells, which can upregulate TET1 expression. CONCLUSION: The downregulation of ALDOB could indicate a poor prognosis for HCC patients, and therefore, ALDOB might be considered a prognostic biomarker for HCC, especially at the early stage. In addition, ALDOB inhibits the invasive features of cell lines partly through TET1 expression.

Oncofetal long noncoding RNA PVT1 promotes proliferation and stem cell-like property of hepatocellular carcinoma cells by stabilizing NOP2.

UNLABELLED: Many protein-coding oncofetal genes are highly expressed in murine and human fetal liver and silenced in adult liver. The protein products of these hepatic oncofetal genes have been used as clinical markers for the recurrence of hepatocellular carcinoma (HCC) and as therapeutic targets for HCC. Herein we examined the expression profiles of long noncoding RNAs (lncRNAs) found in fetal and adult liver in mice. Many fetal hepatic lncRNAs were identified; one of these, lncRNA-mPvt1, is an oncofetal RNA that was found to promote cell proliferation, cell cycling, and the expression of stem cell-like properties of murine cells. Interestingly, we found that human lncRNA-hPVT1 was up-regulated in HCC tissues and that patients with higher lncRNA-hPVT1 expression had a poor clinical prognosis. The protumorigenic effects of lncRNA-hPVT1 on cell proliferation, cell cycling, and stem cell-like properties of HCC cells were confirmed both in vitro and in vivo by gain-of-function and loss-of-function experiments. Moreover, mRNA expression profile data showed that lncRNA-hPVT1 up-regulated a series of cell cycle genes in SMMC-7721 cells. By RNA pulldown and mass spectrum experiments, we identified NOP2 as an RNA-binding protein that binds to lncRNA-hPVT1. We confirmed that lncRNA-hPVT1 up-regulated NOP2 by enhancing the stability of NOP2 proteins and that lncRNA-hPVT1 function depends on the presence of NOP2. CONCLUSION: Our study demonstrates that the expression of many lncRNAs is up-regulated in early liver development and that the fetal liver can be used to search for new diagnostic markers for HCC. LncRNA-hPVT1 promotes cell proliferation, cell cycling, and the acquisition of stem cell-like properties in HCC cells by stabilizing NOP2 protein. Regulation of the lncRNA-hPVT1/NOP2 pathway may have beneficial effects on the treatment of HCC.

A long noncoding RNA activated by TGF-ß promotes the invasion-metastasis cascade in hepatocellular carcinoma.

The role of TGF-ß-induced epithelial-mesenchymal transition (EMT) in cancer cell dissemination is well established, but the involvement of lncRNAs in TGF-ß signaling is still unknown. In this study, we observed that the lncRNA-activated by TGF-ß (lncRNA-ATB) was upregulated in hepatocellular carcinoma (HCC) metastases and associated with poor prognosis. lncRNA-ATB upregulated ZEB1 and ZEB2 by competitively binding the miR-200 family and then induced EMT and invasion. In addition, lncRNA-ATB promoted organ colonization of disseminated tumor cells by binding IL-11 mRNA, autocrine induction of IL-11, and triggering STAT3 signaling. Globally, lncRNA-ATB promotes the invasion-metastasis cascade. Thus, these findings suggest that lncRNA-ATB, a mediator of TGF-ß signaling, could predispose HCC patients to metastases and may serve as a potential target for antimetastatic therapies.

H19 inhibits RNA polymerase II-mediated transcription by disrupting the hnRNP U-actin complex.

BACKGROUND: H19 was one of the earliest identified, and is the most studied, long noncoding RNAs. It is presumed that H19 is essential for regulating development and disease conditions, and it is associated with carcinogenesis for many types. However the biological function and regulatory mechanism of this conserved RNA, particularly with respect to its effect on transcription, remain largely unknown. METHODS: We performed RNA pulldown, RNA immunoprecipitation and deletion mapping to identify the proteins that are associated with H19. In addition, we employed EU (5-ethynyl uridine) incorporation, immunoprecipitation and Western blotting to investigate the functional aspects of H19. RESULTS: Our research further verifies that H19 is bound to hnRNP U, and this interaction is located within the 5' 882 nt region of H19. Moreover, H19 disrupts the interaction between hnRNP U and actin, which inhibits phosphorylation at Ser5 of the RNA polymerase II (Pol II) C-terminal domain (CTD), consequently preventing RNA Pol II-mediated transcription. We also showed that hnRNP U is essential for H19-mediated transcription repression. CONCLUSIONS: In this study, we demonstrate that H19 inhibits RNA Pol II-mediated transcription by disrupting the hnRNP U-actin complex. GENERAL SIGNIFICANCE: These data suggest that H19 regulates general transcription and exerts wide-ranging effects in organisms.

Long noncoding RNAs associated with liver regeneration 1 accelerates hepatocyte proliferation during liver regeneration by activating Wnt/ß-catenin signaling.

UNLABELLED: In recent years, long noncoding RNAs (lncRNAs) have been investigated as a new class of regulators of biological function. A recent study reported that lncRNAs control cell proliferation in hepatocellular carcinoma (HCC). However, the role of lncRNAs in liver regeneration and the overall mechanisms remain largely unknown. To address this issue, we carried out a genome-wide lncRNA microarray analysis during liver regeneration in mice after 2/3 partial hepatectomy (PH) at various timepoints. The results revealed differential expression of a subset of lncRNAs, notably a specific differentially expressed lncRNA associated with Wnt/ß-catenin signaling during liver regeneration (an lncRNA associated with liver regeneration, termed lncRNA-LALR1). The functions of lncRNA-LALR1 were assessed by silencing and overexpressing this lncRNA in vitro and in vivo. We found that lncRNA-LALR1 enhanced hepatocyte proliferation by promoting progression of the cell cycle in vitro. Furthermore, we showed that lncRNA-LALR1 accelerated mouse hepatocyte proliferation and cell cycle progression during liver regeneration in vivo. Mechanistically, we discovered that lncRNA-LALR1 facilitated cyclin D1 expression through activation of Wnt/ß-catenin signaling by way of suppression of Axin1. In addition, lncRNA-LALR1 inhibited the expression of Axin1 mainly by recruiting CTCF to the AXIN1 promoter region. We also identified a human ortholog RNA of lncRNA-LALR1 (lncRNA-hLALR1) and found that it was expressed in human liver tissues. CONCLUSION: lncRNA-LALR1 promotes cell cycle progression and accelerates hepatocyte proliferation during liver regeneration by activating Wnt/ß-catenin signaling. Pharmacological intervention targeting lncRNA-LALR1 may be therapeutically beneficial in liver failure and liver transplantation by inducing liver regeneration.