N6‐Methyladenosine (m6A) modification has been implicated in many biological processes. However, its role in cancer has not been well studied. Here, we demonstrate that m6A modifications are ...decreased in hepatocellular carcinoma, especially in metastatic hepatocellular carcinoma, and that methyltransferase‐like 14 (METTL14) is the main factor involved in aberrant m6A 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 m6A‐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 m6A modification in tumor progression. (Hepatology 2017;65:529‐543).
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•Circular RNAs in human HCC were identified using RNA-sequencing.•Circular RNA cSMARCA5 was downregulated in HCC and associated with poor prognosis.•Downregulation of cSMARCA5 in HCC ...was attributed to the upregulation of DHX9.•cSMARCA5 inhibited HCC growth and metastasis both in vitro and in vivo.•cSMARCA5 acted as the sponge of miR-17-3p and miR-181b-5p to upregulate TIMP3.
In recent years, circular RNAs (circRNAs) have been shown to have critical regulatory roles in cancer biology. However, the contributions of circRNAs to hepatocellular carcinoma (HCC) remain largely unknown.
cSMARCA5 (a circRNA derived from exons 15 and 16 of the SMARCA5 gene, hsa_circ_0001445) was identified by RNA-sequencing and validated by quantitative reverse transcription PCR. The role of cSMARCA5 in HCC progression was assessed both in vitro and in vivo. circRNAs in vivo precipitation, luciferase reporter assay, biotin-coupled microRNA capture and fluorescence in situ hybridization were conducted to evaluate the interaction between cSMARCA5 and miR-17-3p/miR-181b-5p.
The expression of cSMARCA5 was lower in HCC tissues, because of the regulation of DExH-Box Helicase 9, an abundant nuclear RNA helicase. The downregulation of cSMARCA5 in HCC was significantly correlated with aggressive characteristics and served as an independent risk factor for overall survival and recurrence-free survival in patients with HCC after hepatectomy. Our in vivo and in vitro data indicated that cSMARCA5 inhibits the proliferation and migration of HCC cells. Mechanistically, we found that cSMARCA5 could promote the expression of TIMP3, a well-known tumor suppressor, by sponging miR-17-3p and miR-181b-5p.
These results reveal an important role of cSMARCA5 in the growth and metastasis of HCC and provide a fresh perspective on circRNAs in HCC progression.
Herein, we studied the role of cSMARCA5, a circular RNA, in hepatocellular carcinoma. Our in vitro and in vivo data showed that cSMARCA5 inhibits the growth and migration of hepatocellular carcinoma cells, making it a potential therapeutic target.
Tumor cells with stemness (stem‐cell) features contribute to initiation and progression of hepatocellular carcinoma (HCC), but involvement of long noncoding RNAs (lncRNAs) remains largely unclear. ...Genome‐wide analyses were applied to identify tumor‐associated lncRNA‐DANCR. DANCR expression level and prognostic values of DANCR were assayed in two HCC cohorts (China and Korea, n = 135 and 223). Artificial modulation of DANCR (down‐ and overexpression) was done to explore the role of DANCR in tumorigenesis and colonization, and tumor‐bearing mice were used to determine therapeutic effects. We found that lncRNA‐DANCR is overexpressed in stem‐like HCC cells, and this can serve as a prognostic biomarker for HCC patients. Experiments showed that DANCR markedly increased stemness features of HCC cells to promote tumorigenesis and intra‐/extrahepatic tumor colonization. Conversely, DANCR knockdown attenuated the stem‐cell properties and in vivo interference with DANCR action led to decreased tumor cell vitality, tumor shrinkage, and improved mouse survival. Additionally, we found that the role of DANCR relied largely on an association with, and regulation of, CTNNB1. Association of DANCR with CTNNB1 blocked the repressing effect of microRNA (miR)−214, miR‐320a, and miR‐199a on CTNNB1. This observation was confirmed in vivo, suggesting a novel mechanism of tumorigenesis involving lncRNAs, messenger RNAs, and microRNAs. Conclusions: These studies reveal a significance and mechanism of DANCR action in increasing stemness features and offer a potential prognostic marker and a therapeutic target for HCC. (Hepatology 2016;63:499–511)
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. (Hepatology 2014;60:1278–1290)
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.
The oxygen reduction reaction (ORR) is essential in many life processes and energy conversion systems. It is desirable to design transition metal molecular catalysts inspired by enzymatic oxygen ...activation/reduction processes as an alternative to noble‐metal‐Pt‐based ORR electrocatalysts, especially in view point of fuel cell commercialization. We have fabricated bio‐inspired molecular catalysts electrografted onto multiwalled carbon nanotubes (MWCNTs) in which 5,10,15,20‐tetra(pentafluorophenyl) iron porphyrin (iron porphyrin FeF20TPP) is coordinated with covalently electrografted axial ligands varying from thiophene to imidazole on the MWCNTs’ surface. The catalysts’ electrocatalytic activity varied with the axial coordination environment (i. e., S‐thiophene, N‐imidazole, and O‐carboxylate); the imidazole‐coordinated catalyst MWCNTs‐Im‐FeF20TPP exhibited the highest ORR activity among the prepared catalysts. When MWCNT‐Im‐FeF20TPP was loaded onto the cathode of a zinc−air battery, an open‐cell voltage (OCV) of 1.35 V and a maximum power density (Pmax) of 110 mW cm−2 were achieved; this was higher than those of MWCNTs‐Thi‐FeF20TPP (OCV=1.30 V, Pmax=100 mW cm−2) and MWCNTs‐Ox‐FeF20TPP (OCV=1.28 V, Pmax=86 mW cm−2) and comparable with a commercial Pt/C catalyst (OCV=1.45 V, Pmax=120 mW cm−2) under similar experimental conditions. This study provides a time‐saving method to prepare covalently immobilized molecular electrocatalysts on carbon‐based materials with structure–performance correlation that is also applicable to the design of other electrografted catalysts for energy conversion.
A time‐saving electrografting method (within a couple of minutes) for the covalent functionalization of carbon nanotube surfaces with an imidazole or thiophene ligand that is axially coordinated with iron porphyrin as electrocatalyst for the oxygen reduction reaction (ORR) has been demonstrated. The N‐imidazole‐coordinated catalyst, MWCNT‐Im‐FeF20TPP, exhibited the highest ORR activity followed by the S‐thiophene and O‐carboxylate axially coordinated catalysts.
To explore whether plasma circular RNAs (circRNAs) can diagnose hepatitis B virus (HBV)‐related hepatocellular carcinoma (HCC), microarray and qPCR were used to identify plasma circRNAs that were ...increased in HBV‐related HCC patients compared to controls (including healthy controls, chronic hepatitis B and HBV‐related liver cirrhosis). A logistic regression model was constructed using a training set (n = 313) and then validated using another two independent sets (n = 306 and 526, respectively). Area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy. We identified a plasma circRNA panel (CircPanel) containing three circRNAs (hsa_circ_0000976, hsa_circ_0007750 and hsa_circ_0139897) that could detect HCC. CircPanel showed a higher accuracy than AFP (alpha‐fetoprotein) to distinguish individuals with HCC from controls in all three sets (AUC, 0.863 95% confidence interval, CI: 0.819–0.907 vs. 0.790 0.738–0.842, p = 0.036 in training set; 0.843 0.796–0.890 vs. 0.747 0.691–0.804, p = 0.011 in validation set 1 and 0.864 0.830–0.898 vs. 0.769 0.728–0.810, p < 0.001 in validation set 2). CircPanel also performed well in detecting Small‐HCC (solitary, ≤3 cm), AFP‐negative HCC and AFP‐negative Small‐HCC.
What's new?
To date, one limitation in the treatment of hepatocellular carcinoma (HCC) is the lack of serum biomarkers with satisfactory diagnostic accuracy. Here, the authors explored whether plasma circRNAs can be biomarkers to diagnose hepatitis B virus‐related HCC in 1155 participants from three hospitals in China. They identified a plasma circRNA panel (CircPanel, including hsa_circ_0000976, hsa_circ_0007750, and hsa_circ_0139897) that showed higher accuracy than the clinically‐used serum biomarker AFP in distinguishing individuals with HCC or Small‐HCC from controls and performed well in diagnosing AFP‐negative HCC and AFP‐negative Small‐HCC. Altogether, the findings point to CircPanel as a promising potential biomarker in HCC diagnosis.
There is little evidence that adjuvant therapy after radical surgical resection of hepatocellular carcinoma (HCC) improves recurrence-free survival (RFS) or overall survival (OS). We conducted a ...multicentre, randomised, controlled, phase IV trial evaluating the benefit of an aqueous extract of
Murr (Huaier granule) to address this unmet need.
A total of 1044 patients were randomised in 2:1 ratio to receive either Huaier or no further treatment (controls) for a maximum of 96 weeks. The primary endpoint was RFS. Secondary endpoints included OS and tumour extrahepatic recurrence rate (ERR). The Huaier (n=686) and control groups (n=316) had a mean RFS of 75.5 weeks and 68.5 weeks, respectively (HR 0.67; 95% CI 0.55 to 0.81). The difference in the RFS rate between Huaier and control groups was 62.39% and 49.05% (95% CI 6.74 to 19.94; p=0.0001); this led to an OS rate in the Huaier and control groups of 95.19% and 91.46%, respectively (95% CI 0.26 to 7.21; p=0.0207). The tumour ERR between Huaier and control groups was 8.60% and 13.61% (95% CI -12.59 to -2.50; p=0.0018), respectively.
This is the first nationwide multicentre study, involving 39 centres and 1044 patients, to prove the effectiveness of Huaier granule as adjuvant therapy for HCC after curative liver resection. It demonstrated a significant prolongation of RFS and reduced extrahepatic recurrence in Huaier group.
NCT01770431; Post-results.
Autophagy participates in the progression of hepatocellular carcinoma (HCC) and the resistance of HCC cells to sorafenib. We investigated the feasibility of sensitising HCC cells to sorafenib by ...modulating miR-541-initiated microRNA-autophagy axis.
Gain- and loss-of-function assays were performed to evaluate the effects of miR-541 on the malignant properties and autophagy of human HCC cells. Autophagy was quantified by western blotting of LC3, transmission electron microscopy analyses and confocal microscopy scanning of mRFP-GFP-LC3 reporter construct. Luciferase reporter assays were conducted to confirm the targets of miR-541. HCC xenograft tumours were established to analyse the role of miR-541 in sorafenib-induced lethality.
The expression of miR-541 was downregulated in human HCC tissues and was associated with malignant clinicopathologic phenotypes, recurrence and survival of patients with HCC. miR-541 inhibited the growth, metastasis and autophagy of HCC cells both in vitro and in vivo. Prediction software and luciferase reporter assays identified autophagy-related gene 2A (ATG2A) and Ras-related protein Rab-1B (RAB1B) as the direct targets of miR-541. Consistent with the effects of the miR-541 mimic, inhibition of ATG2A or RAB1B suppressed the malignant phenotypes and autophagy of HCC cells. Furthermore, siATG2A and siRAB1B partially reversed the enhancement of the malignant properties and autophagy in HCC cells mediated by the miR-541 inhibitor. More interestingly, higher miR-541 expression predicted a better response to sorafenib treatment, and the combination of miR-541 and sorafenib further suppressed the growth of HCC cells in vivo compared with the single treatment.
Dysregulation of miR-541-ATG2A/RAB1B axis plays a critical role in patients' responses to sorafenib treatment. Manipulation of this axis might benefit survival of patients with HCC, especially in the context of the highly pursued strategies to eliminate drug resistance.
The hepatitis B virus X protein (HBx) has been implicated as an oncogene in both epigenetic modifications and genetic regulation during hepatocarcinogenesis, but the underlying mechanisms are not ...entirely clear. Long noncoding RNAs (lncRNAs), which regulate gene expression with little or no protein‐coding capacity, are involved in diverse biological processes and in carcinogenesis. We asked whether HBx could promote hepatocellular carcinoma (HCC) by regulating the expression of lncRNAs. In this study we investigated the alteration in expression of lncRNAs induced by HBx using microarrays and real‐time quantitative polymerase chain reaction (PCR). Our results indicate that HBx transgenic mice have a specific profile of liver lncRNAs compared with wildtype mice. We identified an lncRNA, down‐regulated expression by HBx (termed lncRNA‐Dreh), which can inhibit HCC growth and metastasis in vitro and in vivo, act as a tumor suppressor in the development of hepatitis B virus (HBV)‐HCC. LncRNA‐Dreh could combine with the intermediate filament protein vimentin and repress its expression, and thus further change the normal cytoskeleton structure to inhibit tumor metastasis. We also identified a human ortholog RNA of Dreh (hDREH) and found that its expression level was frequently down‐regulated in HBV‐related HCC tissues in comparison with the adjacent noncancerous hepatic tissues, and its decrement significantly correlated with poor survival of HCC patients. Conclusion: These findings support a role of lncRNA‐Dreh in tumor suppression and survival prediction in HCC patients. This discovery contributes to a better understanding of the importance of the deregulated lncRNAs by HBx in HCC and provides a rationale for the potential development of lncRNA‐based targeted approaches for the treatment of HBV‐related HCC. (HEPATOLOGY 2013)
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