Sheng-xian Yuan

Long noncoding RNA high expression in hepatocellular carcinoma facilitates tumor growth through enhancer of zeste homolog 2 in humans

In recent years, long noncoding RNAs (lncRNAs) have been shown to have critical regulatory roles in cancer biology. However, the contributions of lncRNAs to hepatitis B virus (HBV)‐related hepatocellular carcinoma (HCC) remain largely unknown. Differentially expressed lncRNAs between HBV‐related HCC and paired peritumoral tissues were identified by microarray and validated using quantitative real‐time polymerase chain reaction. Liver samples from patients with HBV‐related HCC were analyzed for levels of a specific differentially expressed lncRNA High Expression In HCC (termed lncRNA‐HEIH); data were compared with survival data using the Kaplan‐Meier method and compared between groups by the log‐rank test. The effects of lncRNA‐HEIH were assessed by silencing and overexpressing the lncRNA in vitro and in vivo. The expression level of lncRNA‐HEIH in HBV‐related HCC is significantly associated with recurrence and is an independent prognostic factor for survival. We also found that lncRNA‐HEIH plays a key role in G0/G1 arrest, and further demonstrated that lncRNA‐HEIH was associated with enhancer of zeste homolog 2 (EZH2) and that this association was required for the repression of EZH2 target genes. Conclusions: Together, these results indicate that lncRNA‐HEIH is an oncogenic lncRNA that promotes tumor progression and leads us to propose that lncRNAs may serve as key regulatory hubs in HCC progression. (HEPATOLOGY 2011

Long noncoding RNA associated with microvascular invasion in hepatocellular carcinoma promotes angiogenesis and serves as a predictor for hepatocellular carcinoma patients' poor recurrence‐free survival after hepatectomy

Survival of patients with hepatocellular carcinoma (HCC) remains poor, which is largely attributed to active angiogenesis. However, the mechanisms underlying angiogenesis in HCC remain to be discovered. In this study, we found that long noncoding RNA associated with microvascular invasion in HCC (lncRNA MVIH) (lncRNA associated with microvascular invasion in HCC) was generally overexpressed in HCC. In a cohort of 215 HCC patients, the overexpression of MVIH was associated with frequent microvascular invasion (P = 0.016) and a higher tumor node metastasis stage (P = 0.009) as well as decreased recurrence‐free survival (RFS) (P < 0.001) and overall survival (P = 0.007). Moreover, the up‐regulation of MVIH served as an independent risk factor to predict poor RFS. We also found that MVIH could promote tumor growth and intrahepatic metastasis by activating angiogenesis in mouse models. Subsequent investigations indicated that MVIH could activate tumor‐inducing angiogenesis by inhibiting the secretion of phosphoglycerate kinase 1 (PGK1). Additionally, in 65 HCC samples, MVIH expression was inversely correlated with the serum level of PGK1 and positively correlated with the microvessel density. Conclusion: Deregulation of lncRNA MVIH is a predictor for poor RFS of HCC patients after hepatectomy and could be utilized as a potential target for new adjuvant therapies against active angiogenesis. (HEPATOLOGY 2012;56:2142–2153)

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

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)

Hepatitis B virus X protein (HBx)‐related long noncoding RNA (lncRNA) down‐regulated expression by HBx (Dreh) inhibits hepatocellular carcinoma metastasis by targeting the intermediate filament protein vimentin

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)

Long noncoding RNA DANCR increases stemness features of hepatocellular carcinoma by derepression of CTNNB1.

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)

Antiviral therapy improves postoperative survival in patients with hepatocellular carcinoma: a randomized controlled trial.

OBJECTIVE A randomized controlled trial was conducted to find out whether antiviral therapy in patients with hepatitis B-related hepatocellular carcinoma (HCC) improves long-term survival after hepatic resection. BACKGROUND Despite advances in surgery and in multidisciplinary treatment, there is still no effective adjuvant treatment to prevent HCC recurrence after R0 resection for HCC. Whether antiviral therapy is useful in reducing postoperative HCC recurrence is unclear. METHODS Between May 2007 and April 2008, patients who received R0 hepatic resection for HBV-related HCC were randomly assigned to receive no treatment (the control group, n = 100) or antiviral therapy (adefovir 10 mg/d, the antiviral group, n = 100). RESULTS The baseline clinical, laboratory, and tumor characteristics of the 2 groups were comparable. The 1-, 3-, and 5-year recurrence-free survival rates for the antiviral group and the control group were 85.0%, 50.3%, 46.1% and 84.0%, 37.9%, 27.1%, respectively. The corresponding overall survival rates for the 2 groups were 96.0%, 77.6%, 63.1% and 94.0%, 67.4%, 41.5%, respectively. The recurrence-free survival and overall survival for the antiviral group were significantly better than the control group (P = 0.026, P = 0.001). After adjusting for the confounding prognostic factors in a Cox model, the relative risks of recurrence and death for antiviral treatment were 0.651 [95% confidence interval (CI): 0.451-0.938; P = 0.021] and 0.420 (95% CI: 0.271-0.651; P < 0.001). Antiviral therapy was an independent protective factor of late tumor recurrence (HR = 0.348, 95% CI: 0.177-0.687; P = 0.002) but not of early tumor recurrence [hazard ratio (HR) = 0.949, 95% CI: 0.617-1.459; P = 0.810]. CONCLUSIONS In patients with hepatitis B-related HCC, adefovir antiviral therapy reduced late HCC recurrence and significantly improved overall survival after R0 hepatic resection.

Antisense long non-coding RNA PCNA-AS1 promotes tumor growth by regulating proliferating cell nuclear antigen in hepatocellular carcinoma

About 61-72% of transcribed regions possess long noncoding RNAs in antisense orientation (Antisense long noncoding RNAs, aslncRNAs). However, the function of aslncRNAs in HCC remains unclear. We found numerous aslncRNAs were deregulated and might be involved in regulatory gene-net of HCC. The PCNA-AS1, antisense to PCNA, is significantly up-regulated in HCC and could promote tumor growth in vitro and in vivo. The effects of PCNA-AS1 rely on regulation of PCNA via forming RNA hybridization to increase PCNA mRNA stability. We concluded that aslncRNAs might act as upstream regulators in HCC and PCNA-AS1 could serve as a novel therapeutic target.

Double-negative feedback loop between microRNA-422a and forkhead box (FOX)G1/Q1/E1 regulates hepatocellular carcinoma tumor growth and metastasis.

Growing evidence indicates that the aberrant expression of microRNAs (miRNAs) contributes to tumor development; however, the function of miRNAs in human hepatocellular carcinoma (HCC) remains largely undefined. In this study, we report that microRNA‐422a (miR‐422a) is significantly down‐regulated in HCC tumor samples and cell lines compared with normal controls, and its expression level is negatively correlated with pathological grading, recurrence, and metastasis. The restoration of miR‐422a expression in HCC tumor cells significantly inhibited cell proliferation and migration in vitro. At the same time, the overexpression of miR‐422a in HCC tumor cells significantly inhibits tumor growth and liver metastasis in xenograft tumor models. A mechanistic study identified three genes, forkhead box G1 (FOXG1), FOXQ1, and FOXE1, as miR‐422a targets in the regulation of HCC development. We also investigated the function of the three targets themselves in HCC tumorigenesis using RNAi manipulation and demonstrated that the knockdown of these targets led to significant inhibition of tumor cell proliferation and migration both in vitro and in vivo. More interestingly, a potential miR‐422a promoter region was identified. Both the promoter activity and miR‐422a expression were negatively regulated by the three targets, indicating that a double‐negative feedback loop exists between miR‐422a and its targets. Moreover, we explored the therapeutic potential of miR‐422a in HCC treatment and found that the therapeutic delivery of miR‐422a significantly inhibited tumor development in a xenograft tumor model and a diethylnitrosamine‐induced primary HCC model. Conclusion: Our findings show the critical roles of miR‐422a and its targets—FOXG1, FOXQ1, and FOXE1—in the regulation of HCC development and provide new potential candidates for HCC therapy. (Hepatology 2015;61:561‐573)

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

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. (Hepatology 2016;63:850‐863)

Genomic and oncogenic preference of HBV integration in hepatocellular carcinoma

Hepatitis B virus (HBV) can integrate into the human genome, contributing to genomic instability and hepatocarcinogenesis. Here by conducting high-throughput viral integration detection and RNA sequencing, we identify 4,225 HBV integration events in tumour and adjacent non-tumour samples from 426 patients with HCC. We show that HBV is prone to integrate into rare fragile sites and functional genomic regions including CpG islands. We observe a distinct pattern in the preferential sites of HBV integration between tumour and non-tumour tissues. HBV insertional sites are significantly enriched in the proximity of telomeres in tumours. Recurrent HBV target genes are identified with few that overlap. The overall HBV integration frequency is much higher in tumour genomes of males than in females, with a significant enrichment of integration into chromosome 17. Furthermore, a cirrhosis-dependent HBV integration pattern is observed, affecting distinct targeted genes. Our data suggest that HBV integration has a high potential to drive oncogenic transformation.