Human Primary Hepatocellular Carcinoma Tissues Research Articles

STAT5A modulates CDYL2/SLC7A6 pathway to inhibit the proliferation and invasion of hepatocellular carcinoma by targeting to mTORC1.

Chromodomain Y-like 2 (CDYL2), as a member of CDY family known to be involved in spermatogenesis, has been reported to participate in breast cancer development recently, but its exact biological role in hepatocellular carcinoma (HCC) remains unclear. Here, we observed that CDYL2 was down-regulated in human primary HCC tissues and the low levels of CDYL2 expression were correlated with poor survival. Gain- and loss-of-function experiments showed that CDYL2 inhibited the proliferation and metastasis of HCC cells in vitro and in vivo. Mechanistically, CDYL2 down-regulates solute carrier family 7 member 6 (SLC7A6) by decreasing the enrichment of H3K4me3 on the promoter region of SLC7A6. Additionally, we also found that signal transducer and activator of transcription 5A (STAT5A) could directly and positively regulate the expression of CDYL2. Thus, CDYL2 was regulated by STAT5A, and suppressed the amino acid transportation through down-regulation of SLC7A6, and then inhibits the mTORC1/S6K pathway, a master regulator of cell growth. Consistently, CDYL2 expression correlated significantly with STAT5A and SLC7A6 expression in HCC. Collectively, we propose a model for a STAT5A/CDYL2/SLC7A6 axis that provides novel insight into CDYL2, which may serve as a potential factor for predicting prognosis and a therapeutic target for HCC patients.

Isolation of cancer stem-like cells from hepatocellular carcinoma cell line HepG2 by methods of magnetic-activated cell sorting, spheroid culture, and anti-tumor drug-resistant selection: A primary evaluation

Introduction: Recently reported data have suggested that only a small subset of cancer cells possess the capability to initiate malignancies. These observations were based on investigation of cells within the primary tumors displaying a distinct surface marker pattern. CD133 marker is a putative hematopoietic and neuronal stem cell marker, which is also considered to be a tumorigenic marker in brain, prostate and liver. Recent studies have shown that a small population of CD133-positive cells, indeed, exists in human hepatocellular carcinoma (HCC) cell lines and primary HCC tissues. This study was aimed at isolating the cancer stem-like cells from hepatocellular carcinoma cell line HepG2 using three different methods: magnetic-activated cell sorting (MACS), spheroid culture (SC), and anti-tumor drug (ATD) resistant selection.
 Methods: HepG2 hepatocellular carcinoma cells were expanded to yield enough cells that could be used to isolate cancer stem-like cells by these three methods. For MACS, cancer stem-like cells were sorted using anti-CD133 monoclonal antibody. For the second method, cancer stem-like cells were enriched by selection of anti-tumor drug resistance property. Lastly, for the third method, three-dimensional (3D) culture was used to enrich for the cancer stem-like cells. The cells obtained by the three methods were expanded to obtain an adequate number of cells for confirmation of CD133 expression.
 Results: The expression of CD133+ cells in the three methods was found to be different. In the MACS method, the expanded CD133+ sorted cells cultured through 2 passages only contained 0.40 % CD133+ cells. In the 3D spheroid cell culture, of the population of cells there were 38.39 % that were CD133+ cells. Lastly, in the anti-tumor drug (doxorubicin at 150 nM) resistant selection, 66.22 % were CD133+ cells.
 Conclusion: This study shows that isolation of HepG2 derived CD133+ population by culture with doxorubicin (150 nM) yields the highest efficiency and purity of the 3 methods studied.

Combined Chibby and β-Catenin Predicts Clinical Outcomes in Patients with Hepatocellular Carcinoma.

Chibby is an antagonist of β-catenin and is considered a potential tumor suppressor protein, but the role of Chibby in hepatocellular carcinoma (HCC) has not been characterized. The expression patterns of Chibby and β-catenin in HCC specimens and paired adjacent noncancerous tissues were measured by Western blotting and immunohistochemistry. The correlations between Chibby expression and clinicopathological parameters were analyzed. Then the biological functions of Chibby were analyzed in vitro. The Chibby protein was significantly downexpressed in human primary HCC tissues compared to that in matched adjacent normal liver tissue and is a risk factor for HCC recurrence and shorter survival. Furthermore, we found that in HCC tissues the high expression of β-catenin with low expression of Chibby in the nuclei was an independent predictor for disease-free survival (DFS) (p = 0.012) and overall survival (OS) (p = 0.005). Subsequent genetic manipulation in vitro studies revealed that Chibby knockdown induced the expression of β-catenin and C-myc, cyclin D1 protein, which promoted cell proliferation and invasiveness. In contrast, overexpression of Chibby decreased β-catenin expression and inhibited the cell proliferation and invasiveness. Our results suggest that low expression of Chibby was associated with advanced tumor-node-metastasis (TNM) stage and poor differentiation. Furthermore, the combination of Chibby and β-catenin can predict poor prognosis in patients with HCC. Chibby inhibited HCC progression by blocking β-catenin signaling in vitro. Chibby is a biomarker and may be a potential therapeutic target for HCC.

FOXM1 promotes hepatocellular carcinoma progression by regulating KIF4A expression

BackgroundForkhead box M1 (FOXM1) is a proliferation-associated transcription factor of the forkhead box proteins superfamily, which includes four isoforms FOXM1a, b, c, and d. FOXM1 has been implicated in hepatocellular carcinoma (HCC) progression, but the underlying molecular mechanism remains elusive. In this study, we aim to clarify the molecular basis for FOXM1-mediated HCC progression.MethodsBioinformatic analysis was used to explore the differentially expressed genes predicting HCC proliferation. The expression of FOXM1 and kinesin family member (KIF)4A was confirmed by western blotting and immunohistochemistry in HCC tissues. Kaplan-Meier survival analysis was conducted to analyze the clinical impact of FOXM1 and KIF4A on HCC. The effect of FOXM1 on the regulation of KIF4A expression was studied in cell biology experiments. The interaction between KIF4A and FOXM1 was analyzed by chromatin immunoprecipitation and luciferase experiments. A series of experiments was performed to explore the functions of FOXM1/KIF4A in HCC progression, such as cell proliferation, cell growth, cell viability, and cell cycle. A xenograft mouse model was used to explore the regulatory effect of FOXM1-KIF4A axis on HCC tumor growth.ResultsFOXM1 and KIF4A were overexpressed in human primary HCC tissues compared to that in matched adjacent normal liver tissue and are significant risk factors for HCC recurrence and shorter survival. We found that KIF4A was dominantly regulated by FOXM1c among the four isoforms, and further identified KIF4A as a direct downstream target of FOXM1c. Inhibiting FOXM1 decreased KIF4A expression in HCC cells, whereas its overexpression had the opposite effect. FOXM1-induced HCC cell proliferation was dependent on elevated KIF4A expression as KIF4A knockdown abolished FOXM1-induced proliferation of HCC cells both in vitro and in vivo.ConclusionThe FOXM1–KIF4A axis mediates human HCC progression and is a potential therapeutic target for HCC treatment.

Targeting HSP90 with the small molecule inhibitor AUY922 (luminespib) as a treatment strategy against hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly malignant tumor that responds very poorly to existing therapies, most probably due to its extraordinary inter- and intra-tumor molecular heterogeneity. The modest therapeutic response to molecular targeted agents underlines the need for new therapeutic approaches for HCC. In our study, we took advantage of well-characterized human HCC cell lines, differing in transcriptomic subtypes, DNA mutation and amplification alterations, reflecting the heterogeneity of primary HCCs, to provide a preclinical evaluation of the specific heat shock protein 90 (HSP90) inhibitor AUY922 (luminespib). Indeed, HSP90 is highly expressed in different tumor types, but its role in hepatocarcinogenesis remains unclear. Here, we analyzed HSP90 expression in primary human HCC tissues and evaluated the antitumor effects of AUY922 in vitro as well as in vivo. HSP90 expression was significantly higher in HCC tissues than in cirrhotic peritumoral liver tissues. AUY922 treatment reduced the cell proliferation and viability of HCC cells in a dose-dependent manner, but did not do so for normal human primary hepatocytes. AUY922 treatment led to the upregulation of HSP70 and the simultaneous depletion of HSP90 client proteins. In addition, in a cell type-dependent manner, treatment induced either both caspase-dependent β-catenin cleavage and the upregulation of p53, or Mcl-1 expression, or NUPR1 expression, which contributed to the increased efficacy of, or resistance to, treatment. Finally, in vivo AUY922 inhibited tumor growth in a xenograft model. In conclusion, HSP90 is a promising therapeutic target in HCC, and AUY922 could be a drug candidate for its treatment.

Identification of genes and pathways, including the CXCL2 axis, altered by DNA methylation in hepatocellular carcinoma.

Recent genetic studies have suggested that tumor suppressor genes are often silenced during carcinogenesis via epigenetic modification caused by methylation of promoter CpG islands. Here, we characterized genes inactivated by DNA methylation in human hepatocellular carcinoma (HCC) to identify the genes and pathways involved in DNA methylation in hepatocellular carcinoma. Eight HCC-derived cell lines were treated with a DNA demethylating agent, 5-aza-2'-deoxycytidine. Additionally, 100 pairs of primary HCC and adjacent non-cancerous tissues as well as 15 normal liver tissues were analyzed by comprehensive gene expression analysis using microarrays. Moreover, gene set enrichment analysis identified the major molecular pathways associated with DNA methylation. Validation of gene expression and DNA methylation status was performed by real-time PCR after bisulfite modification. We showed that CXCL2, an immune-related chemokine, expression was significantly downregulated in tumor tissues and also significantly upregulated by DAC treatment in cell lines. Furthermore, we observed a statistically significant difference in methylation status between normal liver tissues and tumor tissues (P < 0.05). In addition, tumors with higher CXCL2 expression included significantly more numbers of multiple tumors than the lower expression group. We identified CXCL2, an immune-related chemokine, decreased in hepatocellular carcinoma and the regulation mechanism may be controlled by methylation. Further studies should be warranted to examine if and to what extent the gene is actually suppressed by methylation and if there is a possibility that the CXCL2 axis plays a role for diagnosis and treatment of hepatocellular carcinoma.

NK3 homeobox 1 (NKX3.1) up-regulates forkhead box O1 expression in hepatocellular carcinoma and thereby suppresses tumor proliferation and invasion

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related mortality in China, and the molecular mechanism of uncontrolled HCC progression remains to be explored. NK3 homeobox 1 (NKX3.1), an androgen-regulated prostate-specific transcription factor, suppresses tumors in prostate cancer, but its role in HCC is unknown, especially in hepatocellular carcinoma. In the present study, the differential expression analyses in HCC tissues and matched adjacent noncancerous liver tissues revealed that NKX3.1 is frequently down-regulated in human primary HCC tissues compared with matched adjacent noncancerous liver tissues. We also noted that NKX3.1 significantly inhibits proliferation and mobility of HCC cells both in vitro and in vivo Furthermore, NKX3.1 overexpression resulted in cell cycle arrest at the G1/S phase via direct binding to the promoter of forkhead box O1 (FOXO1) and up-regulation of expression. Of note, FOXO1 silencing in NKX3.1-overexpressing cells reversed the inhibitory effects of NKX3.1 on HCC cell proliferation and invasion. Consistently, both FOXO1 and NKX3.1 were down-regulated in human HCC tissues, and their expression was significantly and positively correlated with each other. These results suggest that NKX3.1 functions as a tumor suppressor in HCC cells through directly up-regulating FOXO1 expression.

MicroRNA-377 Downregulates Bcl-xL and Increases Apoptosis in Hepatocellular Carcinoma Cells.

Aberrantly expressed microRNAs (miRNAs/miRs) and their role in cancer development have recently gained more attention. However, the potential role of miRNAs in hepatocellular carcinoma (HCC) remains largely unknown. In this study, we demonstrated that miR-377 was markedly downregulated in HCC cell lines and primary human HCC tissues. The decreased expression of miR-377 contributes to the upregulation of Bcl-xL expression by targeting its 3′-untranslated region (3′-UTR). Functionally, knockdown of miR-377 noticeably increased HCC cell growth and colony formation and inhibited apoptosis. In contrast, overexpression of miR-377 suppressed cell proliferation and increased apoptosis. This study provides new insights for the use of miR-377 as a potential molecular target in HCC therapy.

High-throughput flow cytometry screening of human hepatocellular carcinoma reveals CD146 to be a novel marker of tumor-initiating cells

Hepatocellular carcinoma (HCC) remains a common and lethal cancer. Cancer stem cells, or tumor-initiating cells (TICs), are thought to contribute to the pathogenesis of HCC, but remain to be fully characterized. Unbiased screens of primary human HCC cells for the identification of novel HCC TIC markers have not been reported. We conducted high-throughput flow cytometry (HT-FC) profiling to characterize the expression of 375 CD antigens on tumor cells from 10 different human HCC samples. We selected 91 of these for further analysis based on HT-FC data that showed consistent expression in discrete, rare, sortable populations of HCC cells. Nine of these CD antigens demonstrated significantly increased expression in the EpCAM+ stem/progenitor fraction of a human HCC cell line and were further evaluated in primary human HCC tissues from 30 different patients. Of the nine tested, only CD146 demonstrated significantly increased expression in HCC tumor tissue as compared with matched adjacent non-tumor liver tissue. CD146+CD31−CD45− cells purified from HCC tumors and cell lines demonstrated a unique phenotype distinct from mesenchymal stem cells. As compared with other tumor cell fractions, CD146+CD31−CD45− cells showed significantly increased colony-forming capacity in vitro, consistent with TICs. This study demonstrates that HT-FC screening can be successfully applied to primary human HCC and reveals CD146 to be a novel TIC marker in this disease.

The transcription factor FOXN3 inhibits cell proliferation by downregulating E2F5 expression in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide, and the mechanisms underlying the development of HCC remain to be elucidated. Forkhead box N3 (FOXN3) is an important member of the FOX family of transcription factors that plays an essential role in several cancers but has not been investigated in HCC. In this study, we demonstrate that FOXN3 is downregulated in human primary HCC tissues compared with their matched adjacent liver tissues. Functional tests of FOXN3 demonstrated that FOXN3 inhibits the proliferation of HCC cells in vitro and in vivo. Additionally, FOXN3 repressed the mRNA and protein expression of E2F5, a reported potential oncogene, by inhibiting the promoter activity of E2F5. Collectively, our findings indicate that FOXN3 functions as a tumor suppressor in HCC by downregulating the expression of E2F5.

Overexpression of WWP1 promotes tumorigenesis and predicts unfavorable prognosis in patients with hepatocellular carcinoma.

WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) has been speculated to play important roles in the development of several kinds of cancers. However, the role of WWP1 in hepatocellular carcinoma(HCC) is not clear. In the present study, we investigated the expression and prognostic role of WWP1 in primary hepatocellular carcinoma (HCC) using cell lines and 149 archived HCC samples. Correlation between the functions of WWP1 in HCC was also explored. We used human HCC cell lines (BEL-7402, SMMC-7721, Hep-G2, Hep-3B, SK-hep1 and Huh7) and a normal hepatocyte cell line (LO2) along with HCC samples from patients who had undergone resection for HCC previously at our hospital. A battery of methods (real-time quantitative polymerase chain reaction; western blotting; immunohistochemical analyses; cell proliferation and colony formation assays; cell migration and cell invasion assays) were employed to assess various aspects of WWP1. We found that WWP1 expression was upregulated aberrantly at mRNA and protein levels in human primary HCC tissues. Amplified expression of WWP1 was highly correlated with poor outcome. Silencing of WWP1 expression by siRNA inhibited the proliferation, colony formation, migration and invasion of HCC cells in vitro, and resulted in significant apoptosis and cycle arrest in HCC cells. Our findings suggest that WWP1 might have an oncogenic role in human primary HCC, and that it could be used as a prognostic marker as well as a potential molecular target for the treatment of HCC.

SIP1 is a downstream effector of GADD45G in senescence induction and growth inhibition of liver tumor cells.

Cellular senescence evasion caused by the inactivation of tumor suppressive programs is implicated in tumor initiation and therapeutic resistance. Our previous study has shown that the downregulation of growth arrest and DNA damage 45G (GADD45G) contributes to senescence bypass in hepatocellular carcinoma (HCC). Here, we report that the Smad-interacting protein-1 (SIP1) is transcriptionally activated and functions critically in the GADD45G-induced tumor cell senescence. Knockdown of SIP1 significantly abrogates the suppressive effects of GADD45G on the growth of xenografted liver tumor in vivo. The essential role of SIP1 in GADD45G activities is further validated in the model of the proteasome inhibitor MG132-induced cell senescence. We further show that JNK but not p38 MAPK activation is involved in the GADD45G-mediated SIP1 upregulation, and that JNK inhibition counteracts the GADD45G-induced cellular senescence. More importantly, we show that GADD45G and SIP1 expression are coincidently downregulated in primary human HCC tissues. Together, our results establish that the dowregulation of GADD45G-SIP1 axis may contribute to cellular senescence evasion and HCC development.

Expression of NEDD9 in hepatocellular carcinoma and its clinical significance.

Neural precursor cell expressed, developmentally downregulated 9 (NEDD9) plays an integral role in natural and pathological cell biology. Overexpression of NEDD9 protein has been correlated with poor prognosis in various types of cancer. However, few available data address the precise function of the NEDD9 gene in hepatocellular carcinoma (HCC). In the present study, we investigated NEDD9 expression in 40 primary human HCC tissues compared with matched adjacent non-tumor hepatic tissues using RT-qPCR and western blot analysis. Immunohistochemistry was performed to analyze the correlations between NEDD9 expression and clinicopathological factors. Statistical analyses were applied to derive prognostic values of NEDD9 in HCC. The results showed that the NEDD9 mRNA and protein expression levels in HCC tissues were significantly higher than those in matched adjacent non-tumor hepatic tissues. High NEDD9 expression was correlated with larger tumor size, advanced tumor grade, metastasis, intrahepatic venous invasion and high UICC TNM stages in HCC patients. Patients with high NEDD9 expression levels exhibited poorer recurrence-free and overall survival than those with a low NEDD9 expression. Additionally, NEDD9 expression status was an independent prognostic factor for survival. This correlation remained significant in patients with early-stage HCC or with normal serum AFP levels. The results of this study suggest that NEDD9 may be a valuable prognostic biomarker for HCC, including early-stage and AFP-normal patients.

Expression and prognostic role of ubiquitination factor E4B in primary hepatocellular carcinoma.

Ubiquitination factor E4B (UBE4B) has been speculated to have contradictory functions upon tumorigenesis as an oncogene or tumor suppressor in different types of cancers. We investigated the expression and prognostic role of UBE4B in primary hepatocellular carcinoma (HCC) using cell lines and 149 archived HCC samples. Correlation between the functions of UBE4B in HCC was also explored. We used human HCC cell lines (HepG2, Hep3B, SK-Hep1, Huh7, SMMC-7721, BEL-7402) and a normal hepatocyte cell line (LO2) along with HCC samples from patients who had undergone resection for HCC previously at our hospital. A battery of methods (real-time quantitative polymerase chain reaction; Western blotting; immunohistochjemical analyses; cell proliferation and colony formation assays; cell migration and cell invasion assays) were employed to assess various aspects of UBE4B.We found that UBE4B expression was upregulated aberrantly at mRNA and protein levels in human primary HCC tissues. Amplified expression of UBE4B was highly correlated with poor outcome. Silencing of UBE4B expression by siRNA inhibited the proliferation, colony formation, migration and invasion of HCC cells in vitro, and resulted in significant apoptosis that was associated with downregulation of expression of Bcl-2 and upregulation of expression of total p53, p-p53, Bax and Cleaved-Caspase3 in HCC cells. Our findings suggested that UBE4B might have an oncogenic role in human primary HCC, and that it could be used as a prognostic marker (as well as a potential molecular target) for the treatment of HCC.

Pioglitazone, a PPARγ agonist, inhibits growth and invasion of human hepatocellular carcinoma via blockade of the rage signaling.

Pioglitazone (PGZ), a synthetic PPARγ ligand, is known to have anti-tumor activity. However, it is unclear how it acts against hepatocellular carcinoma (HCC). We hypothesized that the pathological receptor for advanced glycation end products (RAGE) is involved in the PGZ anti-tumor process. To test this notion, human primary HCC tissues and corresponding adjacent non-cancerous tissues (ANCT) from 75 consecutive cases were analyzed. The expression and clinical significance of RAGE was assessed by immunohistochemical assay through tissue microarray. After HCC cells were pretreated with different concentrations of PGZ, cell proliferation, apoptosis, cell invasion, and cell cycle distribution were evaluated by multiple assays. The results showed that, the positive expression of RAGE was significantly higher in HCC tissues than in ANCT (66.7% vs. 36.0%, P < 0.001), and was closely associated with pathological staging (P = 0.014) and lymph-vascular space invasion (P = 0.003). Moreover, PGZ inhibited proliferative activity and invasive potential, and induced apoptosis and cell cycle arrest in HCC cells resulting in increased expression of PPARγ and decreased expression of RAGE, NF-κB, HMGB1, p38MAPK, Ki-67, MMP-2, and CyclinD1. Furthermore, knockdown of RAGE or NF-κB by siRNA effectively suppressed cell proliferation and invasion, and mediated the inhibitory effects of PGZ in HCC cells. Taken together, our findings suggest that, RAGE is overexpressed in human HCC tissues, and is closely associated with the pathological staging and tumor invasion of HCC. In addition, PGZ as a PPARγ agonist may inhibit growth and invasion of HCC cells via blockade of the RAGE signaling.

Mir-17-5P Promotes Migration of Human Hepatocellular Carcinoma Cells Through the P38 Mitogen-Activated Protein Kinase-Heat Shock Protein 27 Pathway

miR-17-5p is overexpressed in hepatocellular carcinoma (HCC), but the specific regulatory mechanisms of miR-17-5p in HCC remain unknown. We investigated the molecular basis of miR-17-5p as an oncogene in human HCC cell lines. Our in vivo and in vitro data indicate that miR-17-5p up-regulates the migration and proliferation of HCC cells. Interestingly, proteomic and western blotting assays revealed that miR-17-5p significantly activates the p38 mitogen-activated protein kinase MAPK pathway and increases the phosphorylation of heat shock protein 27 (HSP27). Our results also suggest that E2F1-dependent down-regulation of Wip1 regulates miR-17-5p-p38-HSP27 signaling. Furthermore, suppression of HSP27 expression by small interfering RNA or the p38 MAPK pathway-specific inhibitor SB203580 decreases the migration of HCC cells overexpressing miR-17-5p but does not reduce their proliferation. Finally, we show that miR-17-5p expression correlates well with HSP27 status in primary human HCC tissues with metastasis. Our findings suggest that the p38 MAPK pathway plays a crucial role in miR-17-5p-induced phosphorylation of HSP27 and, as a consequence, phosphorylated HSP27 enhances the migration of HCC cells. Our data highlight an important role of miR-17-5p in the proliferation and migration of HCC cells and support the potential application of miR-17-5p in HCC therapy.

CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity

Recently increasing reported data have suggested that only a small subset of cancer cells possess capability to initiate malignancies including leukemia and solid tumors, which was based on investigation in these cells displaying a distinct surface marker pattern within the primary cancers. CD133 is a putative hematopoietic and neuronal stem-cell marker, which was also considered as a tumorigenic marker in brain and prostate cancer. We hypothesized that CD133 was a marker closely correlated with tumorigenicity, since it was reported that CD133 expressed in human fetal liver and repairing liver tissues, which tightly associated with hepatocarcinogenesis. Our findings showed that a small population of CD133 positive cells indeed exists in human hepatocellular carcinoma (HCC) cell lines and primary HCC tissues. From SMMC-7721 cell line, CD133+ cells isolated by MACS manifested high tumorigenecity and clonogenicity as compared with CD133- HCC cells. The implication that CD133 might be one of the markers for HCC cancer stem-like cells needed further investigation.

Hypermethylation of NAD(P)H: quinone oxidoreductase 1 (NQO1) gene in human hepatocellular carcinoma

NAD(P)H: quinone oxydoreductase 1 (NQO1) and glutathione S-transferase P1 (GSTP1) belong to phase II xenobiotic-metabolizing enzymes. GSTP1 inactivation via CpG island hypermethylation in hepatocellular carcinoma (HCC) was previously reported, but the involvement of NQO1 in HCC is not well known. In this study, we assessed the transcription and status of methylation of NQO1 gene in human hepatoma cells and primary human HCC tissues. NQO1 transcription and DNA hypermethylation in hepatoma cells with or without 5-aza-deoxycytidine (5-Aza-CdR) treatment were investigated by reverse-transcription PCR (RT-PCR), sodium bisulfite sequencing and methylation-specific PCR (MSP). The methylation status of NQO1 and GSTP1, and NQO1 mRNA in 44 HCC cases was also analyzed by MSP and real-time PCR, respectively. NQO1 transcription was down-regulated and the CpG island DNA was hypermethylated in Hep3B and HuH6 cells. After treatment with 5-Aza-CdR, NQO1 transcription was restored and CpG island DNA was demethylated in these cells. MSP analysis revealed that NQO1 hypermethylation occurred in 50.0% of HCC. All of the tumors that exhibited lesser amounts of NQO1 mRNA than corresponding non-tumorous tissues showed NQO1 hypermethylation. NQO1 transcription might be inappropriately suppressed by promoter hypermethylation in a subset of HCC, as well as GSTP1 gene.

Underexpression of mRNA in human hepatocellular carcinoma focusing on eight loci

Genetic alterations associated with human hepatocellular carcinoma (HCC) have been reported previously, but are not sufficient to specify differences of HCCs from precancerous diseases of the liver, such as hepatitis, hepatic fibrosis, and cirrhosis. In the present study, we performed differential gene display analysis (DGDA) to clarify the specific genetic alterations associated with gene expression changes in the course of development of HCC from chronic viral hepatitis. Four pairs of surgically resected HCCs and hepatitis tissues were investigated. We found 1,028 expression sequence tags (ESTs) that were decreased or increased in HCC tissues compared with hepatitis tissues in the same patient. Nucleotide sequencing showed that they included 55 EST clones in the GenBank database, which were considered candidates for specific messenger RNA (mRNA) expression alterations in HCCs. After excluding 9 ESTs that code mitochondrial DNA, we performed quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) for the 46 remaining EST clones. We found 8 mRNAs underexpressed in primary HCC tissues in 20 patients in higher percentages than found in previous studies, including 18 cases (90%) for aldolase B (ALDOB), 15 cases (75%) for carbamyl phosphate synthetase 1 (CPS1), albumin (ALB), plasminogen (PLG), and EST 51549, 13 cases (65%) for cytochrome P450 subfamily 2E1 (CYP2E1), 12 cases (60%) for human retinol-binding protein 4 (RBP4), and 11 cases (55%) for human organic anion transporter C (OATP-C) gene. In conclusion, underexpression of key gene products may be important in the development and/or progression of HCC. (H EPATOLOGY 2002;36:433-438.)

Deletion mapping of chromosome 16q24 in hepatocellular carcinoma in Taiwan and mutational analysis of the 17-beta-HSD gene localized to the region.

Human chromosome band 16q24 commonly undergoes loss of heterozygosity (LOH) in human hepatocellular carcinoma (HCC). To further localize the region of deletion on 16q24 and to evaluate the genetic role of 17-beta-HSD, which is near 16q24, in HCC, we examined the pattern of loss of heterozygosity in 88 HCC patients. DNAs from 88 pairs of HCCs and corresponding non-tumor parts were prepared. Loss of heterozygosity on chromosomes 16q24 was investigated by 11 sets of microsatellite markers. Mutation analysis of type II 17-beta-HSD was performed by automatic sequencing. LOH on 16q24 for at least 1 locus was found in 43 of the 88 tumor DNAs (49%). Three non-overlapping regions of frequent LOH were defined in these 43 tumors with partial deletions. The first region was between D16S516 loci and D16S507, encompassed by a 1-cM region, defined by the D16S504. The second region was defined by the 17HSDB2 locus between D16S505 and D16S422, encompassed approximately by a 1-cM region. The third region was between D16S520 and D16S413, defined by D16S3048, encompassed approximately by a 4-cM region. Homozygous deletions of any exons in 17HSDB2 gene were identified in 7 of 27 cases (26%). Automated sequencing analysis of 17HSDB2 failed to demonstrate mutations in any of these specimens. Our data suggest that the 17HSDB2 locus is a frequent target of deletion in HCC but the inactivation of 17HSDB2 may not involve sequence mutations. Furthermore, the presence of the other 2 frequent LOH regions suggest that the putative tumor suppressor genes at these locations might be involved in the development of HCC.