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// Xiu-Yan Huang 1, * , Zi-Li Huang 2, * , Tao Niu 3, * , Zhen-Qian Wu 1, * , Bin Xu 4, * , Yong-Hua Xu 2 , Xin-Yu Huang 1 , Qi Zheng 1 , Jian Zhou 5 , Zi Chen 6 and Zhao-You Tang 5 1 Department of General Surgery, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai, P.R. China 2 Department of Radiology, Xuhui Central Hospital, Shanghai, P.R. China 3 Department of General Surgery, People’s Hospital of Menghai County, Yunnan Province, P.R. China 4 Department of General Surgery, The Tenth People’s Hospital of Tongji University, Shanghai, P.R. China 5 Liver Cancer Institute and Zhongshan Hospital, Fudan University, Shanghai, P.R. China 6 Thayer School of Engineering, Norris Cotton Cancer Center, Dartmouth College, Hanover, NH, USA * These authors have contributed equally to this work Correspondence to: Xiu-Yan Huang, email: xyhuang1119@163.com Keywords: hepatocellular carcinoma; missing in metastasis B; caveolin-1; epidermal growth factor receptor; metastasis Received: December 14, 2016 Accepted: August 04, 2017 Published: September 08, 2017 ABSTRACT Background: Increasing amounts of evidence indicate that Missing in metastasis B (MIM-B) promotes cancer metastasis. Here, we sought to better understand the mechanism through which MIM-B promotes tumor metastasis in hepatocellular carcinoma (HCC). Methods: We performed confocal microscopy analysis to determine the distributions of MIM-B and caveolin-1 and conducted co-immunoprecipitation assays to detect the interactions between MIM-B and caveolin-1 in vitro . We performed transwell assays to analyze the invasive ability of HCC cells. Changes in the expression levels of key genes and some molecular makers were detected by immunohistochemistry and western blotting in HCC tissue samples. Results: We found that MIM-B co-localizes with caveolin-1 and demonstrated that MIM-B and caveolin-1 interact in vitro . Repressing MIM-B and caveolin-1 expression inhibited the epidermal growth factor receptor signaling pathway. We overexpressed MIM-B and caveolin-1 in Hep3B cells, which enhanced Hep3B cell invasiveness. Furthermore, MHCC97H cell invasiveness was significantly decreased in cells in which MIM-B and caveolin-1 expression was inhibited. Additionally, we found that MIM-B and caveolin-1 were expressed at higher levels in HCC tissues than in paired normal tissues. Moreover, HCC patients with MIM-B and caveolin-1 up-regulation experienced significantly worse outcomes than controls ( P < 0.001), and HCC patients with high MIM-B and caveolin-1 expression levels often developed pulmonary metastasis ( P < 0.001). Conclusions: MIM-B combined with caveolin-1 promotes metastasis of HCC, and elevated MIM-B and caveolin-1 expression levels are associated with a poor prognosis in HCC patients; therefore, MIM-B and caveolin-1 may represent novel targets for the diagnosis and treatment of HCC.

ARG1, which encodes Arginase1, is expressed in the liver cytoplasm and plays a major role in the hepatic urea cycle. The past research works shed light on the fact that ARG1 participates in anti-inflammation, tumor immunity, and immunosuppression-related diseases. Nevertheless, the concrete role and clinical significance of ARG1 in the progression of hepatocellular carcinoma (HCC) remain unclear. Herein, we aimed at examining the expression and clinicopathological significance of ARG1 in HCC, together with determining the effect of ARG1 on the progression and metastasis of HCC. In the current study, evaluation of the expression of ARG1 and clinicopathological significance of ARG1 was carried out in the human HCC tissues microarray, and the ARG1 overexpression vector and shRNA-ARG1 plasmids were constructed for the assessment of the concrete effect of ARG1 on cellular behaviors of Huh7 cells. As our data revealed, ARG1 was significantly downregulated in HCC, and the higher expression of ARG1 was positively correlated with more aggressive tumor growth, size, ALT, and GGT level. Significantly, we found that the high expression of ARG1 was correlated with poor DFS of HCC patients. Besides, in vitro study revealed that overexpression of ARG1 could enhance arginase activity, cell viability, migration, and invasion of Huh7 cells, and loss-of-function of ARG1 by shRNA interference could inhibit these cellular behaviors. Additionally, overexpression of ARG1 led to a significant increase in the expression of Vimentin, N-cadherin, and

Backgroud It has been shown that aberrant expression of microRNAs (miRNAs) and transcriptional factors (TFs) is tightly associated with the development of HCC. Therefore, in order to further understand the pathogenesis of HCC, it is necessary to systematically study the relationship between the expression of miRNAs, TF and genes. In this study, we aim to identify the potential transcriptomic markers of HCC through analyzing common microarray datasets, and further establish the differential co-expression network of miRNAs–TF–mRNA to screen for key miRNAs as candidate diagnostic markers for HCC. Method We first downloaded the mRNA and miRNA expression profiles of liver cancer from the GEO database. After pretreatment, we used a linear model to screen for differentially expressed genes (DEGs) and miRNAs. Further, we used weighed gene co-expression network analysis (WGCNA) to construct the differential gene co-expression network for these DEGs. Next, we identified mRNA modules significantly related to tumorigenesis in this network, and evaluated the relationship between mRNAs and TFs by TFBtools. Finally, the key miRNA was screened out in the mRNA–TF–miRNA ternary network constructed based on the target TF of differentially expressed miRNAs, and was further verified with external data set. Results A total of 465 DEGs and 215 differentially expressed miRNAs were identified through differential genes expression analysis, and WGCNA was used to establish a co-expression network of DEGs. One module that closely related to tumorigenesis was obtained, including 33 genes. Next, a ternary network was constructed by selecting 256 pairs of mRNA–TF pairs and 100 pairs of miRNA–TF pairs. Network mining revealed that there were significant interactions between 18 mRNAs and 25 miRNAs. Finally, we used another independent data set to verify that miRNA hsa-mir-106b and hsa-mir-195 are good classifiers of HCC and might play key roles in the progression of HCC. Conclusion Our data indicated that two miRNAs—hsa-mir-106b and hsa-mir-195—are identified as good classifiers of HCC.

Numerous studies have investigated the association between patatin-like phospholipase domain-containing protein-3 (PNPLA3) rs738409 C G polymorphism and the risk of hepatocellular carcinoma (HCC). However, the results are conflicting and inconclusive among different populations. Thus, a meta-analysis was performed to resolve this inconsistency.Potentially related studies were investigated in PubMed, Cochrane Library, EMBASE, and Chinese Biomedical Database (CBM) up to June 12, 2018. The odds ratio (OR) with a 95% confidence interval (CI) was used to explore the strength of the associations. Subgroup analysis was performed according to ethnicity and etiology of cases. Publication bias detection was conducted using Egger's test.Fourteen case-control studies were included in this meta-analysis, reporting a total of 3527 HCC patients and 7184 controls. Overall results revealed that PNPLA3 rs738409 C G polymorphism was associated with an increased risk of HCC in the populations studied with various types of etiology under allelic model (OR = 1.59, 95%CI: 1.20-2.10, P = 0.001), dominant model (OR = 1.55, 95%CI: 1.13-2.13, P = 0.007), homozygous model (OR = 2.76, 95%CI: 1.52-5.01, P = 0.001), heterozygous model (OR = 1.31, 95%CI: 1.01-1.69, P = 0.039), and recessive model (OR = 2.42, 95%CI: 1.51-3.87, P 0.001). A significant increased risk was observed in patients with HCC related to alcoholic cirrhosis under all genetic models (C vs. G: OR = 3.35, 95%CI: 2.14-5.24, P 0.001; CC vs.GG: OR = 11.02, 95%CI: 4.35-27.88, P 0.001; CC vs. GC: OR = 2.75, 95%CI: 1.72-4.39, P 0.001; GG vs. CC + CG: OR = 5.82, 95%CI: 2.93-11.57, P 0.001; CG + GG vs. CC: OR = 4.08, 95%CI: 2.33-7.13, P 0.001), with respect to specific etiology of HCC. A significant increased risk was also revealed in patients with HCC due to virus related cirrhosis under allelic model (OR = 1.19, 95%CI: 1.07-1.32, P = 0.001), dominant model (OR = 1.17, 95%CI: 1.02-1.35, P = 0.03), homozygous model (OR = 1.47, 95%CI: 1.17-1.85, P = 0.001), and recessive model (OR = 1.43, 95%CI: 1.15-1.76, P = 0.001). Subgroup analysis on ethnicity revealed that the polymorphism was associated with increased risk of HCC in Caucasians under allelic model (OR = 1.65, 95%CI: 1.12-2.45, P = 0.012), dominant model (OR = 1.63, 95%CI: 1.04-4.25, P = 0.035), homozygous model (OR = 2.88, 95%CI: 1.27-6.55, P = 0.012), and recessive model (OR = 2.48, 95%CI: 1.32-4.65, P = 0.005).Our study suggests a significant increased association between PNPLA3 rs738409 C G polymorphism and HCC risk in the entire populations studied, especially in Caucasians. Therefore, PNPLA3 rs738409 C G polymorphism may be a risk factor for virus and alcoholic-related HCC.

Circular RNAs (circRNAs) represent a class of endogenous noncoding RNAs that have recently been recognized as important regulators of gene expression and pathological networks. However, their transcriptional activities and functional mechanisms in cancer remain largely unknown. Here, we present results from a global circRNA expression and functional analysis of patients with hepatocellular carcinoma (HCC). Using a circRNA microarray, we identified 226 differentially expressed circRNAs, of which 189 were significantly upregulated and 37 were downregulated. High expression of circRNA_100338, one of the upregulated circRNAs in HCC, is closely correlated with a low cumulative survival rate and metastatic progression in HCC patients with hepatitis B. Furthermore, our in silico and experimental analyses identified miR-141-3p as a direct target of circRNA_100338. Thus, circRNA_100338 functions as an endogenous sponge for miR-141-3p in HCC. In addition, we identified the crucial antagonistic roles of circRNA_100338 and miR-141-3p in the regulation of invasive potential in liver cancer cells. Overall, the differential expression of multiple circRNAs in HCC tissues and their clinical significance in hepatitis B-related HCC patients as revealed by our study suggests that circRNA_100338 is a potentially valuable biomarker for HCC diagnosis and target for HCC therapeutics.

The present study aimed to quantitatively determine the aberrant methylation signal of the adenomatous polyposis coli (APC) gene in hepatocellular carcinoma (HCC), and to evaluate whether hypermethylation of the APC promoter could be a prognostic biomarker for HCC. Taqman probe-based quantitative methylation-specific polymerase chain reaction was performed to identify the APC promoter methylation levels in 57 HCC and corresponding non-tumorous liver tissues. In the present study, the methylation level of the APC promoter was upregulated by 4.51-fold in the HCC tissues compared with the non-cancerous tissues (P=0.0003). With regard to the clinicopathological data, the methylation level of the APC promoter in the HCC samples was higher in the patients with larger tumors when the cut-off was set at 4 cm (P=0.0008), and in the older patients when the cut-off was set at 60 years old (P=0.0438). However, the methylation status in the HCC samples appeared not to affect the overall patient survival rate (P=0.1684). The findings of the present study showed that APC promoter hypermethylation accumulates during the development of HCC, but that it may not be a promising prognostic biomarker for HCC.

Background Hepatectomy generally offers the best chance of long-term survival for patients with hepatocellular carcinoma (HCC). Many studies have shown that hepatectomy accelerates tumor metastasis, but the mechanism remains unclear. Methods An orthotopic nude mice model with palliative HCC hepatectomy was performed in this study. Metastasis-related genes in tumor following resection were screened; HCC invasion, metastasis, and some molecular alterations were examined in vivo and in vitro. Clinical significance of key gene mRNA expression was also analyzed. Results Metastasis suppressor 1 (MTSS1) located in the central position of gene function net of residual HCC. MTSS1 was up-regulated in residual tumor after palliative resection. In hepatitis B-related HCC patients undergone palliative hepatectomy, those with higher MTSS1 mRNA expression accompanied by activation of matrix metalloproteinase 2 (MMP2) in residual HCC, had earlier residual HCC detection after hepatectomy and poorer survival when compared to those with lower MTSS1. In different cell lines, the levels of MTSS1 mRNA increased in parallel with metastatic potential. MTSS1 down regulation via siRNA decreased MMP2 activity, reduced invasive potentials of HCC by 28.9 % in vitro, and averted the deteriorated lung metastatic extent in vivo. Conclusions The poor prognosis of hepatitis B-related HCC patients following palliative hepatectomy associates with elevated MTSS1 mRNA expression; therefore, MTSS1 may provide a new research field for HCC diagnosis and treatment. Electronic supplementary material The online version of this article (doi:10.1186/s13046-016-0361-8) contains supplementary material, which is available to authorized users.

Objective: Although signal transducer and activator of transcription 1 (STAT1), a transcription factor, plays a critical role in carcinogenesis and has been implicated as a tumor suppressor, few studies have investigated the associations between polymorphisms of this gene and the risk of cancer development. The aim of this study was to examine whether STAT1 gene polymorphisms are associated with the risk of hepatocellular carcinoma (HCC). Methods: Ten single nucleotide polymorphisms in the STAT1 gene were genotyped by TaqMan assays in 469 HCC cases and 558 age-, sex- and HBsAg-matched controls in a Chinese population. Results: Minor allele homozygous genotypes at rs867637 (9,046 bp 3′ of STP A>G), rs3771300 (IVS24–153T>G), and rs2280235 (IVS20–103G>A), compared with their homozygote genotypes of common alleles, were associated with 1.6- (95% CI 1.1–2.3), 1.6- (95% CI 1.1–2.4), and 1.4-fold (95% CI 0.95–1.9) increased risk of HCC, respectively. The GGA haplotype, comprised of risk alleles at rs867637, rs3771300 and rs2280235, conferred a 1.2-fold (95% CI 1.0–1.5) increased risk of HCC, as compared to the most common haplotype of ATG. Diplotype GGA/GGA conferred a 1.6-fold (95% CI 1.0–2.5) increased risk of HCC compared with diplotype ATG/ATG. Conclusion: Our results demonstrate for the first time that polymorphisms in the STAT1 gene are associated with HCC susceptibility.

Objective: To study the relevance of uPA, uPAR and PAI-1 to hepatoceilular carcinoma (HCC). Methods: The expression at protein level of uPA, uPAR and PAI-1 was determined in 48 cases of HCC and 12 cases of benign tumors of liver (as control) by immunohistochemistry. Results: When compared to cancer-adjacent liver tissue and the control, positive rate of immune staining for uPA, uPAR and PAI-1 on cell membrane were significantly higher in HCC cells (P

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide and is caused by the accumulation of genetic and epigenetic alterations in regulatory genes. In this study, we used methylight to detect the methylation status of the RASSF1A promoter in 87 paired HCC samples and analysed the relationship between methylation status and clinicopathological parameters, including prognosis after surgery. We found that the methylation level of the RASSF1A promoter in HCC tissues was significantly higher than that in the corresponding non-tumorous tissues (p

Global DNA hypomethylation has been associated with increased risk for cancers of the colorectum, bladder, breast, head and neck, and testicular germ cells. The aim of this study was to examine whether global hypomethylation in blood leukocyte DNA is associated with the risk of hepatocellular carcinoma (HCC). A total of 315 HCC cases and 356 age-, sex- and HBsAg status-matched controls were included. Global methylation in blood leukocyte DNA was estimated by analyzing long interspersed element-1 (LINE-1) repeats using bisulfite-polymerase chain reaction (PCR) and pyrosequencing. We observed that the median methylation level in HCC cases (percentage of 5-methylcytosine (5mC)=77.7%) was significantly lower than that in controls (79.5% 5mC) (P=0.004, Wilcoxon rank-sum test). The odds ratios (ORs) of HCC for individuals in the third, second, and first (lowest) quartiles of LINE-1 methylation were 1.1 (95% confidence interval (CI) 0.7–1.8), 1.4 (95% CI 0.8–2.2), and 2.6 (95% CI 1.7–4.1) (P for trend median) LINE-1 methylation. Our results demonstrate for the first time that individuals with global hypomethylation measured in LINE-1 repeats in blood leukocyte DNA have an increased risk for HCC. Our data provide the evidence that global hypomethylation detected in the easily obtainable DNA source of blood leukocytes may help identify individuals at risk of HCC.

Background Liver resection is a widely accepted treatment for hepatocellular carcinoma (HCC). Our previous clinical study showed that the rate of palliative resection was 34.0% (1958-2008, 2754 of 8107). However, the influence of palliative resection on tumor metastasis remains controversial. The present study was conducted to evaluate the effect of palliative resection on residual HCC and to explore interventional approaches. Methods Palliative resection was done in an orthotopic nude mice model of HCC (MHCC97H) with high metastatic potential. Tumor growth, invasion, metastasis, lifespan, and some molecular alterations were examined in vivo and in vitro. Mice that underwent palliative resection were treated with the Chinese herbal compound "Songyou Yin," interferon-alfa-1b (IFN-α), or their combination to assess their effects. Results In the palliative resection group, the number of lung metastatic nodules increased markedly as compared to the sham operation group (14.3 ± 4.7 versus 8.7 ± 3.6, P < 0.05); tumor matrix metalloproteinase 2 (MMP2) activity was elevated by 1.4-fold, with up-regulation of vascular endothelial growth factor (VEGF) and down-regulation of tissue inhibitor of metalloproteinase 2 (TIMP2). The sera of mice undergoing palliative resection significantly enhanced cell invasiveness by 1.3-fold. After treatment, tumor volume was 1205.2 ± 581.3 mm3, 724.9 ± 337.6 mm3, 507.6 ± 367.0 mm3, and 245.3 ± 181.2 mm3 in the control, "Songyou Yin," IFN-α, and combination groups, respectively. The combined therapy noticeably decreased the MMP2/TIMP2 ratio and prolonged the lifespan by 42.2%. Moreover, a significant (P < 0.001) reduction of microvessel density was found: 43.6 ± 8.5, 34.5 ± 5.9, 23.5 ± 5.6, and 18.2 ± 8.0 in the control and treatment groups, respectively. Conclusion Palliative resection-stimulated HCC metastasis may occur, in part, by up-regulation of VEGF and MMP2/TIMP2. "Songyou Yin" reinforced the ability of IFN-α to inhibit the metastasis-enhancing potential induced by palliative resection, which indicated its potential postoperative use in patients with HCC.