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The effects and mechanisms of folic acid (FA) as a chemopreventive agent for tumorigenesis of hepatocellular carcinoma (HCC) remain unclear. In this study, the QSG-7701, a human normal liver cell line, was cultured in different FA levels (High, Normal or No) for 6 months. Then, the biological characteristics, the expression of main stem cell-like genes or epithelial-mesenchymal transition (EMT) related genes and the tumorigenicity in vivo of cells cultured in different treatment groups were detected. Our results showed that No FA improved the malignant transformation of cells but High FA depressed the malignant transformation. Meanwhile, cells in different treatment groups were mapped by transcriptome sequencing. Then the relativity of increased LCN2 and decreased FA level was identified and confirmed in vitro and vivo. We also revealed that intracellular control of LCN2 would recover the effects of FA on cell proliferation, cell cycle and tumor formation in vitro and vivo. Finally, our studies displayed that increased FA level induced the down-regulation of LCN2 not by DNA hypermethylation of LCN2 promoter but by promoting the level of histone H3 lysine 9 di-methylation (H3K9Me2) in LCN2 promoter. In conclusion, our studies disclosed the chemopreventive effect of FA supplementation on hepatocarcinogenesis, which partial attributed to the inhibition of LCN2 by regulating histone methylation in promoter. Our results provide a potential mechanism of the chemoprevention of FA supplementation on tumorigenesis of HCC and may be helpful in developing treatment target against HCC.

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).

// Jie Cai 1, * , Sheng-Xian Yuan 1, * , Fu Yang 2, * , Qi-Fei Tao 1, * , Yuan Yang 1 , Qing-Guo Xu 1 , Zhen-Guang Wang 1 , Jian Yu 1 , Kong-Ying Lin 1 , Zong-Yan Wang 1 , Jin-Zhao Ma 2 , Chuan-Chuan Zhou 2 , Fang Wang 2 , Shu-Han Sun 2 , Wei-Ping Zhou 1 1 The Third Department of Hepatic Surgery, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China 2 The Department of Medical Genetics, Second Military Medical University, Shanghai, China * These authors contributed equally to this work Correspondence to: Wei-Ping Zhou, email: ehphwp3@126.com Shu-Han Sun, email: shsun@vip.sina.com Keywords: paraoxonase 3, hepatocellular carcinoma, prognostic predictor, cell proliferation, cDNA microarray Received: February 26, 2016 Accepted: September 02, 2016 Published: September 20, 2016 ABSTRACT Paraoxonase 3 (PON3) exerts prominent anti-inflammation and anti-oxidation properties mainly at the cellular level, and is primarily expressed in the liver. However, its role in HCC remains unexplored. Here, we investigated the expression pattern, clinical significance, and function of PON3 in HCC. PON3 mRNA and protein levels were respectively determined in two large cohorts using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) of tissue microarray. We found that PON3 was downregulated in most HCCs. Kaplan-Meier and log-rank test showed that PON3 downregulation predicted shorter recurrence-free survival (RFS) and overall survival (OS) time in all HCC patients, especially early-stage HCC patients. Cox regression analysis revealed that the PON3 downregulation was an independent risk factor for RFS and OS. Gain- and loss-of-function experiments revealed that PON3 suppressed cell proliferation in vivo and in vitro , which was attributed to its cell-cycle arrest effect. In addition, microarray analysis showed that some pro-proliferative genes were elevated when PON3 was knockdown, and these genes possibly involved in the underlying mechanisms. In conclusion, our studies reveal the cell proliferation inhibitory function of PON3 and offer a potential prognostic predictor and therapeutic target for HCC.

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)

In this study, we found that H19 is the most strongly differentially expressed long noncoding RNA (lncRNA) during liver development. H19 may inhibit the proliferation of fetal liver cells by blocking the interaction between heterogeneous nuclear ribonucleoprotein (hnRNP) U and actin, which results in gene transcriptional repression. Based on ChIP-seq analysis, we found that genes involved in the Wnt signaling pathway are enriched among hnRNP U-binding genes. Further investigation demonstrated that hnRNP U has opposing effects on cell proliferation and Wnt/β-catenin signaling pathway activity compared to H19 and that hnRNP U is very important in this process.