Normal Liver Tissue Samples Research Articles

Abstract 5336: Methylation patterns in hepatoblastoma: A report from the Children's Oncology Group

Abstract Introduction. Hepatoblastoma (HB) accounts for over 80% of all liver cancer diagnosed in children under 15 years of age in the United States. Aberrant DNA methylation has been implicated in the pathogenesis of multiple types of cancer, including HB. Global hypomethylation and DNA methylation in the promoter region of selected tumor suppressor genes, including RASSF1A, p16, SOCS1, CASP8, and MT1G, has been observed in previous studies of HB. Methods. A total of 47 flash-frozen samples of HB tumors were available for analysis. These tumors include 26 tumors from cases enrolled in Children's Oncology Group study AEPI04C1 (the HOPE study) and 21 tumors obtained as part of the Children's Oncology Group P9346 HB biology protocol. The Cooperative Human Tissue Network Pediatric Division (CHTN; Columbus, OH) provided 23 normal liver tissue samples from children ages 0—6 to serve as controls in this analysis. Initial multi-dimensional scaling (MDS) plots showed that 8 samples, 2 control and 6 patients, did not cluster with the other samples of the same group and were considered outliers. These 8 samples were removed from the study leaving a total of 62 samples, 21 control and 41 patients, remaining. Differentially methylated regions (DMR) were identified between control and patient samples using the bumphunter algorithm included in the minfi package. We used Ingenuity Pathway Analysis (IPA) to identify pathways that were enriched in the group of CpG with a change in methylation beta value of ≥39% between patients and controls. Results. We found 4833 DMR, which are associated with 3543 genes, when comparing HB patients and controls. Pathway analysis revealed 76 pathways enriched among the DMR. Conclusions. Identification of methylation patterns may elucidate the developmental stage at which HB arose and the at-risk period when environmental exposures could be most harmful. Further, understanding the relevant genetic pathways could lead to the development of new targets for therapy. Citation Format: Erin Marcotte, Logan G. Spector, Heather Nelson, Gail Tomlinson, Mark Krailo, Lauren Mills, Jenny Poynter. Methylation patterns in hepatoblastoma: A report from the Children's Oncology Group [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5336.

Deletion of Smad4 reduces hepatic inflammation and fibrogenesis during nonalcoholic steatohepatitis progression

To explore the effects of mothers against decapentaplegic homolog family member 4 (Smad4) deletion on inflammation and fibrogenesis in nonalcoholic steatohepatitis (NASH). Biopsied liver samples from NASH patients and normal liver tissue samples from patients who had received liver resection for trauma were collected. Smad4Co/Co and wild-type (WT) mice were used to construct the NASH model using a high-fat diet (HFD) or methionine- and choline-deficient diet (MCD). HE staining and TUNEL assay were used to observe the pathological changes and cell apoptosis, respectively. Quantitative real-time polymerase chain reaction was used to detect the expression of inflammatory, fibrogenesis and apoptosis-related genes, and immunohistochemistry to determine the protein expression of SMAD4, MCP-1 and α-SMA. SMAD4 protein expression significantly increased in NASH patients than in the control group. Compared with WT mice, HFD- and MCD-fed Smad4Co/Co mice showed decreased hepatic steatosis, inflammation, liver cell apoptosis and nonalcoholic fatty liver activity score, reduced plasma glucose, triglyceride, free fatty acids, alanine aminotransferase and aspartate aminotransferase levels but increased adiponectin. Moreover, Smad4Co/Co decreased the expression of inflammatory markers (TNF-α, MCP-1, IFN-γ), fibrogenetic markers (COL1A1, α-SMA and TGF-β1), lipogenic (Srebp1c, Fas and Acc) and proapoptotic genes (Bax and caspase-3), but increased the expression of β-oxidation (Ppar-α, Cpt1 and Aco) and antiapoptotic genes (Bcl-2). Smad4 deletion may inhibit lipogenesis, stimulate β-oxidation, improve lipid metabolism and liver function, alleviate inflammation and fibrosis, and reduce cell apoptosis in NASH.

MicroRNA-33a downregulation is associated with tumorigenesis and poor prognosis in patients with hepatocellular carcinoma.

In order to examine the prognostic significance of miR-33a in patients with hepatocellular carcinoma (HCC), total RNA was extracted from 149 HCC biopsies, 36 of which were paired with para-carcinoma tissues, and miR-33a expression was measured by reverse transcription-quantitative polymerase chain reaction. The results demonstrated that miR-33a expression was decreased in HCC biopsies compared with normal liver tissue samples. It was also demonstrated that miR-33a expression was significantly associated with tumor foci number. Furthermore, overall and progression-free survival time was decreased in patients expressing low miR-33a with multiple tumor foci. Taken together, the low expression of miR-33a may be a potential risk factor for HCC.

The Abnormal Expression of MicroRNA-542-3p in Hepatocellular Carcinoma and Its Clinical Significance.

Aim To evaluate the expression of miRNA-542-3p in hepatocellular carcinoma, establish its function, and evaluate whether it could serve as a biomarker for diagnosis and prognosis of HCC patients. Methods qRT-PCR analysis was performed to determine the expression level of miRNA-542-3p in normal liver cells and HCC cell lines. Additionally, samples from TCGA consortium and from our patients were analyzed using biostatistical methods to ascertain whether miR-542-3p could be a good biomarker for HCC diagnosis and prognosis. The effects of miRNA-542-3p on HCC were investigated in HCCLM9 cells. Results The expression of miRNA-542-3p in HCC cells was significantly downregulated compared with normal liver cells. A lower level of expression of miRNA-542-3p was found in HCC tissue samples than in adjacent normal liver tissue samples from TCGA cases and our patients. Further evaluation revealed that the downregulation was clearly related to aggressive clinicopathological characteristics and affected the prognosis, as low-expressing patients tended to have shorter overall survival. Moreover, cell assays revealed that miR-542-3p overexpression inhibited HCC cell growth and induced apoptosis. Conclusion We demonstrated for the first time that miRNA-542-3p appears to function as a novel tumor suppressor in HCC and may serve as a promising prognostic biomarker in HCC patients.

Correlation between PPAR-α methylation level in peripheral blood and inflammatory factors of NAFLD patients with DM.

The correlation between the methylation levels of peroxisome proliferator-activated receptor-α (PPAR-α) in the peripheral blood and the inflammatory factors associated with non-alcoholic fatty liver disease (NAFLD) patients with diabetes mellitus (DM) was investigated. Thirty-two samples of normal liver tissues (group N) and 35 samples of liver tissues from NAFLD patients with DM (group M) were used for the present study. The levels of alanine transaminase (ALT) and aspartate transaminase (AST) were measured using commercially available kits. The accumulation of lipid droplets and glycogen in the two groups was determined through Oil Red O staining and Sudan III staining. mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-lβ (IL-1β) and IL-6 in liver tissues of groups N and M were detected using reverse transcription-polymerase chain reaction (RT-PCR). In addition, western blotting was used to detect the protein expression of PPAR-α in liver tissues of both groups. The Statistical Product and Service Solutions (SPSS) 17.0 statistical software was used to analyze the expression difference of PPAR-α in liver tissues in the groups. The high levels of ALT and AST indicated severe liver injury in group M. Oil Red O staining and Sudan III staining showed a large number of lipid droplets and glycogen accumulation in the liver of group M patients. RT-PCR showed that the expression of inflammatory factors was extremely high and that the inflammatory injury was severe in the liver of group M patients. Western blotting showed that the expression of PPAR-α in group N was significantly higher than that in group M. ANOVA results showed that the expression of PPAR-α in liver tissues of groups N and M patients were statistically significantly different (P<0.01). Therefore, the abnormal expression of PPAR-α is closely associated with the occurrence and development of NAFLD complicated with DM, and that the abnormal expression of PPAR-α is closely related to inflammatory factors. Results from the present study suggest PPAR-α has important value in the study on NAFLD complicated with DM. The expression of PPAR-α can be used as a new basis for the diagnosis and treatment of NAFLD complicated with DM.

Silencing HOXD10 by promoter region hypermethylation activates ERK signaling in hepatocellular carcinoma

BackgroundHepatocellular carcinoma is the fifth most common malignancy and the third leading cause of cancer-related death worldwide. Dysregulation of HomeoboxD10 (HOXD10) was found to suppress or promote cancer progression in different cancer types. The function and regulation of HOXD10 remain unclear in human hepatocellular carcinoma (HCC).MethodsPrimary HCC samples (117), normal liver tissue samples (15), and 13 HCC cell lines (SNU182, SNU449, HBXF344, SMMC7721, Huh7, HepG2, LM3, PLC/PRF/5, BEL7402, SNU387, SNU475, QGY7703, and Huh1) were included in this study. Methylation-specific PCR, flow cytometry, western blot, transwell, siRNA, and chromatin immunoprecipitation assays were employed.ResultsHOXD10 was methylated in 76.9% (90/117) of human primary HCC samples. HOXD10 methylation was significantly associated with vessel cancerous embolus, tumor cell differentiation, and the 3-year overall survival rate (all P < 0.05). The expression of HOXD10 was regulated by promoter region methylation. HOXD10 suppressed colony formation, cell proliferation, cell invasion and migration, and induced G2/M phase arrest and apoptosis in HCC cells. HOXD10 suppressed HCC cell xenograft growth in mice. HOXD10 suppresses HCC growth by inhibiting ERK signaling.ConclusionHOXD10 is frequently methylated in human HCC, and the expression of HOXD10 is regulated by promoter region methylation. HOXD10 suppresses HCC cell growth both in vitro and in vivo. HOXD10 suppresses human HCC by inhibiting ERK signaling.

The expression, induction and pharmacological activity of CYP1A2 are post-transcriptionally regulated by microRNA hsa-miR-132-5p

Cytochrome P450 1A2 (CYP1A2) is one of the most abundant and important drug metabolizing enzymes in human liver. However, little is known about the post-transcriptional regulation of CYP1A2, especially the mechanisms involving microRNAs (miRNAs). This study applied a systematic approach to investigate the post-transcriptional regulation of CYP1A2 by miRNAs. Candidate miRNAs targeting the 3′-untranslated region (3′-UTR) of CYP1A2 were screened in silico, resulting in the selection of sixty-two potential miRNAs for further analysis. The levels of two miRNAs, hsa-miR-132-5p and hsa-miR-221-5p, were inversely correlated with the expression of CYP1A2 mRNA transcripts in normal human liver tissue samples represented in The Cancer Genome Atlas (TCGA) dataset. The interactions between these miRNAs and cognate CYP1A2 mRNA sequences were evaluated using luciferase reporter gene studies and electrophoretic mobility shift assays, by which a direct interaction was confirmed involving hsa-miR-132-5p and a cognate binding site present in the CYP1A2 3′-UTR. Experiments by which hsa-miR-132-5p or random miRNA controls were introduced into HepG2, Huh-7 and HepaRG hepatic cell lines showed that only hsa-miR-132-5p suppressed the endogenous and lansoprazole-induced expression of CYP1A2, at biological activity, protein production, and mRNA transcript levels. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays showed that hsa-miR-132-5p attenuates CYP1A2-mediated, lansoprazole-enhanced, flutamide-induced hepatic cell toxicity. Results from multilayer experiments demonstrate that hsa-miR-132-5p suppresses the expression of CYP1A2 and that this suppression is able to decrease the extent of an adverse drug-drug interaction involving lansoprazole and flutamide.

Candidate genes investigation for severe nonalcoholic fatty liver disease based on bioinformatics analysis.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver condition worldwide. However, its etiology and fundamental pathophysiology for the disease process are poorly understood. In this study, we thus used bioinformatics to identify candidate genes potentially causative of severe NAFLD. Gene expression profile data GSE49541 were downloaded from the Gene Expression Omnibus database. Tissues samples from 32 severe and 40 mild NAFLD patients were evaluated to identify differentially expressed genes (DEGs) between the 2 groups, followed by analyses of Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes pathways. Then, a weighted protein-protein interaction (PPI) network was constructed, and subnetworks and candidate genes were screened. Moreover, the GSE48452 data (14 normal liver tissue samples and 18 nonalcoholic steatohepatitis samples) were used to verify the results obtained from the above analyses. A total of 100 upregulated genes and 24 downregulated ones were identified in severe NAFLD. Functional enrichment and pathway analyses showed that these DEGs were mainly associated with cell adhesion, inflammatory response, and chemokine activity. The top 5 subnetworks were selected based on the PPI network. A total of 5 hub genes, including ubiquilin 4 (UBQLN4), amyloid-beta precursor protein (APP), sex hormone-binding globulin (SHBG), cadherin-associated protein beta 1 (CTNNB1) and collagen type I alpha 1 (COL1A1), were considered to be candidate genes for NAFLD. In addition, the verification data confirmed the status of COL1A1, SHBG, and APP as candidate genes. UBQLN4, APP, CTNNB1, SHBG, and COL1A1 might be involved in the development of NAFLD, and are proposed as the potential markers for predicting the development of this condition.

Identification of Potential Gene Network Associated with HCV-Related Hepatocellular Carcinoma Using Microarray Analysis.

In order to identify potential specific gene networks of Hepatitis C virus (HCV) related hepatocellular carcinoma (HCC), weighted gene co-expression network analysis (WGCNA) was performed, which may provide an insight into the potential mechanism of the HCC development. HCV-related HCC and normal sample data were downloaded from GEO, T test of limma package was used to screen different expression genes (DEGs); KEGG pathway was used to analyze related biochemical pathways, and WGCNA was used to construct clustering trees and screen hub genes in the HCC-specific modules. A total of 1151 DEGs were authenticated between the HCC and normal liver tissue samples, including 433 upregulated and 718 downregulated genes. Among these genes, three specific modules of HCC were constructed, including Tan, Yellow and Cyan, but only Yellow module had a significant enrichment score in substance combination module with three hub genes: SLA2547, EFNA4 and MME. Although Tan and Cyan separately had four and three hub genes, but the bio-functions of them did not have significant enrichment scores (score<2). SLA2547, EFNA4 and MME may play important roles in the substance combination of HCV-related HCC, so studying the function of this gene network may provide us a deeper understanding of HCV-related HCC.

Expression of ATAD2 in different liver lesions and its clinical significance

Objective: To examine the expression of ATAD2 in different liver lesions and its clinical significance. Methods: ATAD2 expression in 60 hepatocellular carcinoma (HCC) surgical specimens (49 of which have concurrent liver cirrhosis), 43 HCC biopsy specimens, 2 high-grade liver dysplastic nodule specimens, 3 low-grade liver dysplastic nodule specimens, 50 liver cirrhosis tissue samples, and 20 normal liver tissue samples were measured using immunohistochemistry. The F-test, q-test, t-test, and chi-square test were used for statistical analysis of data. Results: ATAD2 was expressed in 56 HCC surgical specimens (93.33%), 35 HCC biopsy specimens (81.40%), and 2 high-grade liver dysplastic nodule specimens (2/2), but not in the low-grade liver dysplastic nodule, liver cirrhosis tissue, and normal liver tissue samples. The mean expression of ATAD2 was significantly higher in HCC tissues than in high-grade and low-grade liver dysplastic nodule tissues, liver cirrhosis tissue, and normal liver tissue (F = 22.96, q = 3.138, 3.972, 12.272, and 9.101, respectively, all P < 0.01). There were no significant differences in the mean expression and positive expression rate of ATAD2 between HCC surgical and biopsy specimens (t = 1.40, P > 0.05; χ² = 3.47, P >0.05). Of the 35 HCC biopsy specimens that expressed ATAD2, the mean ATAD2 expression was ≥1% in 35 specimens (100%), ≥3% in 27 specimens (77.14%), and ≥5 % in 23 specimens (65.71%). In addition, among the pathological grade I-II HCC biopsy specimens, the mean ATAD2 expression was ≥1% in 28 specimens (100%), ≥3% in 22 specimens (62.86%), and ≥5% in 19 specimens (54.29%). Moreover, ATAD2 expression in HCC was associated with serum alpha-fetoprotein level, presence of hepatitis B virus surface antigen (HBsAg), and presence of concurrent liver cirrhosis (t = 2.09, 2.30, and 2.18, respectively, all P < 0.05). Conclusion: ATAD2 may play an important role in HCC tumorigenesis, and may be involved in malignant transformation of cells. ATAD2 expression can be a valuable marker for differentiating the nature of lesions in liver biopsy tissues during clinical practice.

Immunofluorescence identifies distinct subsets of endothelial cells in the human liver

As well as systemic vascular endothelial cells, the liver has specialised sinusoidal endothelial cells (LSEC). LSEC dysfunction has been documented in many diseased states yet their phenotype in normal human liver has not been comprehensively assessed. Our aim was to improve characterisation of subsets of endothelial cells and associated pericytes in the human liver. Immunofluorescence microscopy was performed on normal human liver tissue samples to assess endothelial and structural proteins in a minimum of three donors. LSEC are distributed in an acinar pattern and universally express CD36, but two distinctive subsets of LSEC can be identified in different acinar zones. Type 1 LSEC are CD36hiCD32−CD14−LYVE-1− and are located in acinar zone 1 of the lobule, while Type 2 LSEC are LYVE-1+CD32hiCD14+CD54+CD36mid-lo and are located in acinar zones 2 and 3 of the lobule. Portal tracts and central veins can be identified using markers for systemic vascular endothelia and pericytes, none of which are expressed by LSEC. In areas of low hydrostatic pressure LSEC are lined by stellate cells that express the pericyte marker CD146. Our findings identify distinctive populations of LSEC and distinguish these cells from adjacent stellate cells, systemic vasculature and pericytes in different zones of the liver acinus.

Genome-wide DNA methylation analysis during non-alcoholic steatohepatitis-related multistage hepatocarcinogenesis: comparison with hepatitis virus-related carcinogenesis.

The aim of this study was to clarify the significance of DNA methylation alterations during non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis. Single-CpG-resolution genome-wide DNA methylation analysis was performed on 264 liver tissue samples using the Illumina Infinium HumanMethylation450 BeadChip. After Bonferroni correction, 3331 probes showed significant DNA methylation alterations in 113 samples of non-cancerous liver tissue showing NASH (NASH-N) as compared with 55 samples of normal liver tissue (NLT). Principal component analysis using the 3331 probes revealed distinct DNA methylation profiles of NASH-N samples that were different from those of NLT samples and 37 samples of non-cancerous liver tissue showing chronic hepatitis or cirrhosis associated with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection (viral-N). Receiver operating characteristic curve analysis identified 194 probes that were able to discriminate NASH-N samples from viral-N samples with area under the curve values of more than 0.95. Jonckheere-Terptsra trend test revealed that DNA methylation alterations in NASH-N samples from patients without hepatocellular carcinoma (HCC) were inherited by or strengthened in NASH-N samples from patients with HCC, and then inherited by or further strengthened in 22 samples of NASH-related HCC (NASH-T) themselves. NASH- and NASH-related HCC-specific DNA methylation alterations, which were not evident in viral-N samples and 37 samples of HCC associated with HBV or HCV infection, were observed in tumor-related genes, such as WHSC1, and were frequently associated with mRNA expression abnormalities. These data suggested that NASH-specific DNA methylation alterations may participate in NASH-related multistage hepatocarcinogenesis.

Aberrant KLK4 gene promoter hypomethylation in pediatric hepatoblastomas

DNA methylation has a crucial role in cancer biology and has been recognized as an activator of oncogenes and inactivator of tumor suppressor genes, both of which are mechanisms for tumorigenesis. Kallikrein-related peptidase 4 (KLK4), has been suggested to be an oncogene in various types of cancer. The aim of the present study was to assess the DNA methylation patterns of the KLK4 gene in cancerous samples harvested from patients with hepatoblastoma (HB). KLK4 mRNA expression levels were detected using reverse transcription-quantitative polymerase chain reaction and assessed its DNA methylation patterns using high-throughput mass spectrometry on a matrix-assisted laser desorption/ionization time-of-flight mass array. A total of 10 HB and 10 normal liver tissue samples were obtained from patients with HB. The results of the present study showed that a significantly higher level of KLK4 mRNA expression levels were detected in HB tissues, as compared with the matched controls. Furthermore, the KLK4 gene promoter region was distinctively less methylated in the HB samples compared with the controls and negatively correlated with KLK4 mRNA expression levels. These findings indicate that aberrant methylation of KLK4 may contribute to its upregulated mRNA expression in HB.

Gut and liver T-cells of common clonal origin in primary sclerosing cholangitis-inflammatory bowel disease

Recruitment of gut-derived memory T-cells to the liver is believed to drive hepatic inflammation in primary sclerosing cholangitis (PSC). However, whether gut-infiltrating and liver-infiltrating T-cells share T cell receptors (TCRs) and antigenic specificities is unknown. We used paired gut and liver samples from PSC patients with concurrent inflammatory bowel disease (PSC-IBD), and normal tissue samples from colon cancer controls, to assess potential T cell clonotype overlap between the two compartments. High-throughput sequencing of TCRβ repertoires was applied on matched colon, liver and blood samples from patients with PSC-IBD (n=10), and on paired tumor-adjacent normal gut and liver tissue samples from colon cancer patients (n=10). An average of 9.7% (range: 4.7-19.9%) memory T cell clonotypes overlapped in paired PSC-IBD affected gut and liver samples, after excluding clonotypes present at similar frequencies in blood. Shared clonotypes constituted on average 16.0% (range: 8.7-32.6%) and 15.0% (range: 5.9-26.3%) of the liver and gut memory T-cells, respectively. A significantly higher overlap was observed between paired PSC-IBD affected samples (8.7%, p=0.0007) compared to paired normal gut and liver samples (3.6%), after downsampling to equal number of reads. Memory T-cells of common clonal origin were detected in paired gut and liver samples of patients with PSC-IBD. Our data indicate that this is related to PSC-IBD pathogenesis, suggesting that memory T-cells driven by shared antigens are present in the gut and liver of PSC-IBD patients. Our findings support efforts to therapeutically target memory T cell recruitment in PSC-IBD. Primary sclerosing cholangitis (PSC) is a devastating liver disease strongly associated with inflammatory bowel disease (IBD). The cause of PSC is unknown, but it has been suggested that the immune reactions in the gut and the liver are connected. Our data demonstrate for the first time that a proportion of the T-cells in the gut and the liver react to similar triggers, and that this proportion is particularly high in patients with PSC and IBD.

Hepatitis B virus X protein mediates yes-associated protein 1 upregulation in hepatocellular carcinoma.

Hepatitis B virus (HBV) X protein (HBx) is implicated in the development of hepatocellular carcinoma (HCC). Yes-associated protein 1 (YAP) is an important proto-oncogene, which is a downstream effector molecule in the Hippo signaling pathway. The aim of the present study was to investigate the association between HBx expression in HCC samples and YAP expression in the Hippo pathway. A total of 20 pathologically confirmed HCC samples, 20 corresponding adjacent non-tumor liver tissues and 5 normal liver tissue samples were collected. The expression of HBx and YAP in the tissues was analyzed by quantitative reverse transcription-polymerase chain reaction and western blot analysis. The intensity and location of YAP expression were analyzed by immunohistochemistry. YAP mRNA and protein expression levels in HCC samples infected with HBV were significantly higher than those of normal liver tissues. Furthermore, YAP expression was positively correlated with HBx expression in HBV-positive HCC samples. Immunohistochemical staining revealed that YAP was predominantly expressed in the nuclei in HBV-positive HCC tissues. YAP expression was significantly decreased in the normal liver tissue and corresponding adjacent liver tissue when compared with the HCC tissues and by contrast to HCC tissues, YAP was predominantly located in the cytoplasm. In conclusion, these results indicate that the YAP gene is a key driver of HBx-induced liver cancer. Therefore, YAP may present a novel target in the treatment of HBV-associated HCC.

Expression of sal-like 4 in primary hepatocellular carcinoma and its association with epithelial-mesenchymal transition

Objective: To investigate the expression of sal-like 4 (SALL4) in tissues of primary hepatocellular carcinoma (HCC) and its association with epithelial-mesenchymal transition (EMT) and clinicopathological characteristics. Methods: Immunohistochemistry and nucleic acid in situ hybridization were used to measure the mRNA expression of SALL4, epithelial cadherin (E-cadherin), vimentin, and Snail in 72 HCC samples, 2 fetal liver samples, and 2 normal adult liver tissue samples. Results: Strong expression of SALL4 was observed in hepatoblasts in fetal liver, but SALL4 expression was not observed in primitive hematopoietic cells and normal adult hepatocytes or biliary epithelial cells. In the HCC samples, the positive rate of SALL4 was 47.2% (34/72), showing focal positive nuclear staining. The HCC patients with microscopic microvascular tumor thrombus and portal vein tumor thrombus, a serum alpha-fetoprotein level of≥ 350 ng/ml, International Union Against Cancer (UICC) stage III+IV, and an age of < 46 years showed higher positive expression of SALL4 than those with no microscopic microvascular tumor thrombus or portal vein tumor thrombus, a serum alpha-fetoprotein level of < 350 ng/ml, UICC stage I+II, and an age of≥46 years. The HCC patients with positive SALL4 showed lower postoperative disease-free survival rate and overall survival rate than those with negative SALL4 (P < 0.05). Sixty percent of the patients with microvascular tumor thrombus (21/35) showed positive expression of SALL4. The positive rate of SALL4 was negatively correlated with E-cadherin (r = -0.434, P < 0.01), but positively correlated with vimentin and Snail (vimentin: r = 0.516, P < 0.01; Snail: r = 0.571, P < 0.01). Conclusion: In patients with primary HCC, the expression of SALL4 greatly affects EMT, which helps with the research on invasion and metastasis of liver cancer and prognostic evaluation.

Genome-wide analysis of DNA methylation in hepatoblastoma tissues.

DNA methylation has a crucial role in cancer biology. In the present study, a genome-wide analysis of DNA methylation in hepatoblastoma (HB) tissues was performed to verify differential methylation levels between HB and normal tissues. As alpha-fetoprotein (AFP) has a critical role in HB, AFP methylation levels were also detected using pyrosequencing. Normal and HB liver tissue samples (frozen tissue) were obtained from patients with HB. Genome-wide analysis of DNA methylation in these tissues was performed using an Infinium HumanMethylation450 BeadChip, and the results were confirmed with reverse transcription-quantitative polymerase chain reaction. The Infinium HumanMethylation450 BeadChip demonstrated distinctively less methylation in HB tissues than in non-tumor tissues. In addition, methylation enrichment was observed in positions near the transcription start site of AFP, which exhibited lower methylation levels in HB tissues than in non-tumor liver tissues. Lastly, a significant negative correlation was observed between AFP messenger RNA expression and DNA methylation percentage, using linear Pearson's R correlation coefficients. The present results demonstrate differential methylation levels between HB and normal tissues, and imply that aberrant methylation of AFP in HB could reflect HB development. Expansion of these findings could provide useful insight into HB biology.

Role of microRNA miR-34a in liver cancer stem cells

&lt;p class="Abstract"&gt;The present study focuses on the role of microRNA miR-34a in liver cancer stem cells. Liver tissue samples were collected from the control and liver cancer patients. Immunohistochemistry experiment with CD90 antibodies suggests that the liver cancer stem cells were present in the liver cancer tissue samples. Interestingly, flow cytometry analysis followed by qRT-PCR confirmed that the CD90&lt;sup&gt;+&lt;/sup&gt; cells also called as liver cancer stem cells shows expression of miR-34a at the levels of 30-80%, when compared with that of normal liver tissue samples. The present study concludes that the liver cancer stem cells shows high expression of miR-34a, which is the important target unique to liver cancer stem cells in order to design liver cancer stem cells-specific therapies.&lt;/p&gt;&lt;p&gt; &lt;/p&gt;

Long non-coding RNA HOTAIR is a marker for hepatocellular carcinoma progression and tumor recurrence.

The present study aimed to investigate the expression level of HOX transcript antisense RNA (HOTAIR) in hepatocellular carcinoma (HCC) and its association with various clinicopathological characteristics, and to further explore the molecular mechanisms of HOTAIR function in HCC. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression level of HOTAIR in 60 paired fresh HCC samples and adjacent normal liver tissue samples. The association between HOTAIR expression and clinicopathological parameters was analyzed. Lentivirus-mediated HOTAIR-specific small hairpin RNA vectors were transfected into HepG2 cells. Cell proliferation and invasion in vitro were examined by MTT and Transwell assays, respectively. A xenograft model was used to analyze the tumorigenesis of liver cancer cells in vivo. In addition, semi-quantitative RT-PCR was used to detect the expression level of Wnt/β-catenin signaling molecules under the condition of HOTAIR inhibition. The results revealed that the expression level of HOTAIR in HCC tissues was higher than that in adjacent non-cancerous tissues. HOTAIR expression was significantly associated with poor tumor differentiation (P=0.002), metastasis (P=0.002) and early recurrence (P=0.001). In vitro, the inhibition of HOTAIR in liver cancer cells resulted in the suppression of cell proliferation and invasion. HOTAIR depletion significantly inhibited the rate of growth of liver cancer cells in vivo. Furthermore, the expression levels of Wnt and β-catenin were downregulated when HOTAIR expression was suppressed. In conclusion, HOTAIR is important in the progression and recurrence of HCC, partly through the regulation of the Wnt/β-catenin signaling pathway. Targeting HOTAIR may be a novel therapeutic strategy for HCC.

Correlation between the expression of hTERT gene and the clinicopathological characteristics of hepatocellular carcinoma.

The aim of the present study was to detect the expression levels of the human telomerase reverse transcriptase (hTERT) gene in hepatocellular carcinoma (HCC), and investigate its significance in the incidence and development of the cancer. HCC tissues and corresponding para-carcinoma liver tissues were surgically collected from 78 patients with HCC who presented to Shandong Provincial Hospital (Jinan, Shandong, China). hTERT expression at the protein and mRNA levels were detected by immunohistochemistry (streptavidin peroxidase method) and reverse transcription polymerase chain reaction, respectively, in the HCC tissues and corresponding para-carcinoma liver tissues of 78 HCC patients and in 12 samples of normal liver tissue. The data were analyzed using SPSS 17.0 statistical software, and employing χ2 tests and t-tests. hTERT protein was mainly expressed in the HCC cell cytoplasm, but was occasionally observed in the cell nucleus. The positive rates of hTERT protein and mRNA expression in the HCC patients were 84.62% (66/78) and 78.21% (61/78), respectively, which was significantly higher compared with the rates of 10.26% (8/78) and 8.97% (7/78) in the paired para-carcinoma liver tissues (P<0.01). hTERT protein and mRNA were not expressed in the normal liver tissues (0/12). χ2 test and t-test analysis revealed that hTERT gene expression was correlated with tumor grade, the presence/absence of a portal vein tumor thrombus, hepatitis B surface antigen positivity and a high α-fetoprotein level (P<0.05) rather than patient age, gender or tumor size. Expression of the hTERT gene may play a pivotal role in the incidence and development of HCC. The hTERT gene potentially serves as an important molecular and biological index for diagnosing and predicating the biological behavior of HCC.