Clinicopathological significance of RASSF1A reduced expression and hypermethylation in hepatocellular carcinoma

Hu, Lang; Chen, Gang; Yu, Hong‐Ren; Qiu, Xiaoqiang

doi:10.1007/s12072-010-9164-8

Cited by 55 publications

(52 citation statements)

References 34 publications

(56 reference statements)

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“…Ectopic expression of RASSF1A in cancer cell lines, which lack endogenous RASSF1A transcripts, resulted in reduced growth of the cells in vitro and in nude mice (21,(25)(26)(27)(28)(29)(30). In addition, hypermethylation and inactivation of RASSF1A during tumor development was observed in a variety of different tumor types, including HBV-related HCC (6)(7)(8). However, whether HBV or HBx was involved in the downregulation of RASSF1A and the regulation mechanisms during HCC carcinogenesis remain unclear.…”

Section: Discussionmentioning

confidence: 99%

“…Hypermethylation of RASSF1A was frequently found in most major types of human tumors including lung, breast, prostate, pancreas, kidney, liver, cervical, thyroid and several other types of cancer (5). Recent studies have shown that HCC is associated with hypermethylation of RASSF1A (6)(7)(8). However, it is not yet known whether these methylation abnormalities result from hepatitis B virus (HBV), a major risk factor for the development of HCC (9).…”

Section: Introductionmentioning

confidence: 99%

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Upregulation of DNMT1 mediated by HBx suppresses RASSF1A expression independent of DNA methylation

Qiu

Zhang

et al. 2013

Oncology Reports

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Abstract. The hepatitis B virus (HBV) X protein (HBx) plays a key role in the molecular pathogenesis of HBV-related hepatocellular carcinoma (HCC). However, its critical gene targets remain largely unknown. RASSF1A gene (Ras-association domain family 1A, RASSF1A), a tumor-suppressor gene, is frequently found to be hypermethylated and downregulated in HCC. In the present study, we investigated whether HBx is involved in the hypermethylation and downregulation of RASSF1A and we examined the potential regulation mechanisms. RT-PCR analysis was used to determine RASSF1A and HBx expression in 9 liver cell lines and the results showed that RASSF1A expression was relatively low in HBx-positive cells. Notably, RASSF1A was downregulated in HepG2.2.15 cells, as compared to HepG2 cells. Further analysis revealed that HBx transfection suppressed RASSF1A expression and HBx knockdown induced its expression. Enforced HBx suppressed RASSF1A and meanwhile induced DNMT1 and DNMT3B expression. In addition, RASSF1A is negatively regulated by DNMT1. ChIP analysis using an antibody against DNMT1 revealed that HBx enhanced the binding of DNMT1 to the RASSF1A promoter but the inhibition of RASSF1A by HBx is DNA methylation-independent as detected by methylationspecific PCR (MSP). Further studies using MSP and bisulfite genomic sequencing (BGS) revealed that no significant methylation changes were observed for regional methylation levels of RASSF1A in DNMT1 knockdown cells, although methylation levels of specific CpG sites at the predicted binding sites for the Sp1 and USF transcription factors were reduced. Additionally, RASSF1A was downregulated in HBV-associated HCC (HBV-HCC) as detected by RT-PCR and immunohistochemistry suggesting RASSF1A expression may be related to HBx in HCC and the clinical relevance of our observations. Collectively, our data showed that HBx suppressed RASSF1A expression via DNMT1 and offered a new mechanism of RASSF1A inactive in HCC in addition to the widely known DNA methylation, enriching the epigenetic mechanism by which HBx contributes to the pathogenesis of HBV-HCC.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Upregulation of DNMT1 mediated by HBx suppresses RASSF1A expression independent of DNA methylation

Qiu

Zhang

et al. 2013

Oncology Reports

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“…The epigenetic inactivation of tumor-suppressor genes by promoter hypermethylation has been identified as an important mechanism underlying tumorigenesis (55). Hypermethylation has been detected in numerous HCC-associated genes, including cyclin dependent kinase inhibitor 2A (p16INK4a) (56,57), runt related transcription factor 3 (RUNX3) (58,59), suppressor of cytokine signaling 1 (SOCS1) (60,61), secreted frizzled related protein 1 (SFRP1) (62,63), O-6-methylguanine-DNA methyltransferase (MGMT) (64-66), Ras association domain family member 1 (RASSF1A) (67)(68)(69) and glutathione S-transferase pi 1 (GSTP1) (66,70) by investigations using various kinds of HCC cell lines and various stage of clinical HCC samples.…”

Section: Molecular Alterations In Hcc Tumorsmentioning

confidence: 99%

Molecular alterations in the carcinogenesis and progression of hepatocellular carcinoma: Tumor factors and background liver factors

et al. 2016

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Abstract. Although hepatocellular carcinoma (HCC) is associated with poor prognosis worldwide, the molecular mechanisms underlying the carcinogenesis and progression of this disease remain unclear. Several tumor characteristics have previously been demonstrated to be prognostic factors of survival following hepatic resection, or the recurrence of HCC or other types of cancer. Comparisons of normal tissues and HCC tumor tissues have revealed the presence of numerous molecular alterations in HCC, including genetic and epigenetic mechanisms, particularly mutations in certain genes and DNA methylation in the promoter regions of tumor-suppressor genes. A number of studies have previously used array analysis to detect variations in the expression levels of cancer-associated genes and microRNAs, and in DNA methylation. However, an investigation of HCC tumor tissues may not determine the effect of noncancerous liver tissues (background liver) in patients with HCC. As HCC may recur multicentrically following resection, a damaged or chronically diseased HCC background liver may be considered as a pre-cancerous organ. Therefore, the influence of the background liver on HCC requires further study. Detailed studies regarding the background liver may be essential for the improved understanding of the carcinogenesis and progression of this malignancy; however only a few studies have investigated the microenvironment of the HCC background liver. The present review discusses prior molecular studies of hepatocarcinogenesis that focus on HCC and background liver tissues.

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“…Gene silencing by epigenetic mechanisms, including DNA methylation, has been reported to contribute to HCC development (16)(17)(18). These epigenetic mechanisms, alone or in combination with genetic modifications such as mutations, may lead to the inactivation of tumor suppressor genes, including RASSF1a and APC, and therefore promote hepatocarcinogenesis (19)(20)(21)(22)(23).…”

Section: Introductionmentioning

confidence: 99%

DNA methyltransferase 3b silencing affects locus-specific DNA methylation and inhibits proliferation, migration and invasion in human hepatocellular carcinoma SMMC-7721 and BEL-7402 cells

Wang¹,

Wang²,

Fan³

et al. 2015

Oncology Letters

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Abstract. DNA methylation is an important regulator of gene transcription, and its role in carcinogenesis has been a topic of considerable interest in previous years. The present study examined the influence of DNA methyltransferase 3b (DNMT3b) on cell proliferation, migration and invasion, and the methylation status of identified tumor suppressor genes in hepatoma SMMC-7721 and BEL-7402 cells. DNMT3b was silenced by small interfering RNA (siRNA) in human hepatocellular carcinoma cell lines. Transfection efficiency was verified using a fluorescent imaging system, reverse transcription polymerase chain reaction (RT-PCR) and western blotting. A cell proliferation assay was performed to evaluate cell viability. Cell cycle distribution and apoptosis were analyzed by flow cytometry. The migratory and invasive ability of cells was measured using a Transwell assay. Methylation-specific PCR (MSP) was performed to assess methylation in the promoter region of genes. The present data revealed that DNMT3b siRNA successfully inhibited expression of the DNMT3b gene in these two liver cancer cell lines and therefore inhibited the proliferation of the transfected cells, stimulated apoptosis in the cells, led to an accumulation of cells in the G 2 /M phase and decreased cell migration and invasion. It was also found that silencing DNMT3b expression results in hypomethylation of specific sets of gene promoters and increases the expression of distinct set of genes in HCC cell lines. The present study is therefore useful for assessing the specificity of emerging action based on the altered expression of associated regulatory genes, particularly in methylation-silenced genes.

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Clinicopathological significance of RASSF1A reduced expression and hypermethylation in hepatocellular carcinoma

Cited by 55 publications

References 34 publications

Upregulation of DNMT1 mediated by HBx suppresses RASSF1A expression independent of DNA methylation

Upregulation of DNMT1 mediated by HBx suppresses RASSF1A expression independent of DNA methylation

Molecular alterations in the carcinogenesis and progression of hepatocellular carcinoma: Tumor factors and background liver factors

DNA methyltransferase 3b silencing affects locus-specific DNA methylation and inhibits proliferation, migration and invasion in human hepatocellular carcinoma SMMC-7721 and BEL-7402 cells

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