Xuemei Qiu

A functional polymorphism in the DNA methyltransferase-3A promoter modifies the susceptibility in gastric cancer but not in esophageal carcinoma

BackgroundDNA-methyltransferase (DNMT)-3A plays an important role in the development of embryogenesis and the generation of aberrant methylation in carcinogenesis. The aim of this study was to investigate the role of a DNMT3A promoter genetic variant on its transcriptional activity and to evaluate the association between DNMT3A gene polymorphism and the susceptibility to gastric cancer (GC) and oesophagus carcinoma (EC) in the Chinese population.MethodsWe selected one of the single nucleotide polymorphisms (SNPs) -448A>G in the DNMT3A promoter region and evaluated its effect on activity using a luciferase assay. -448A>G polymorphisms of DNMT3A were determined by polymerase chain reaction/restriction fragment length polymorphism and confirmed by sequencing. The distribution of -448A>G polymorphisms was detected in 208 GC patients and 346 healthy controls matched for age and gender. The distribution of -448A>G polymorphisms was also detected in 96 EC patients and matched 241 healthy controls. The association of -448A>G polymorphisms of DNMT3A and the risk of GC and EC was evaluated by stratified analysis according to the patients age and gender.ResultsIn a promoter assay, carriage of the -448 A allele showed a significantly higher promoter activity (> two fold) compared with the -448G allele (P < 0.001). The allele frequency of -448A among GC patients and controls was 32.9% versus 19.9%, respectively. Overall, we found that, compared with GG carriers, the DNMT3A -448AA homozygotes has a > six fold increased risk of GC. Stratification analysis showed that AA homozygotes have a more profound risk in the subgroups of individuals at the age range ≤ 60 years in GC. However, individuals with -448AG and -448AA were not statistically significantly associated with an increased risk of EC compared with those carried the -448GG genotype.ConclusionsThe DNMT3A -448A>G polymorphism is a novel functional SNP and contributes to its genetic susceptibility to GC. -448A>G can be used as a stratification marker to predict an individuals susceptibility to GC, especially in the subgroups of individuals at the age range ≤ 60 years. However, the relative distribution of -448A>G in EC can not be used as a prediction marker in order to evaluate an individuals susceptibility to EC.

HBx-upregulated lncRNA UCA1 promotes cell growth and tumorigenesis by recruiting EZH2 and repressing p27Kip1/CDK2 signaling

It is well accepted that HBx plays the major role in hepatocarcinogenesis associated with hepatitis B virus (HBV) infections. However, little was known about its role in regulating long noncoding RNAs (lncRNAs), a large group of transcripts regulating a variety of biological processes including carcinogenesis in mammalian cells. Here we report that HBx upregulates UCA1 genes and downregulates p27 genes in hepatic LO2 cells. Further studies show that the upregulated UCA1 promotes cell growth by facilitating G1/S transition through CDK2 in both hepatic and hepatoma cells. Knock down of UCA1 in HBx-expressing hepatic and hepatoma cells resulted in markedly increased apoptotic cells by elevating the cleaved caspase-3 and caspase-8. More importantly, UCA1 is found to be physically associated with enhancer of zeste homolog 2 (EZH2), which suppresses p27Kip1 through histone methylation (H3K27me3) on p27Kip1 promoter. We also show that knockdown of UCA1 in hepatoma cells inhibits tumorigenesis in nude mice. In a clinic study, UCA1 is found to be frequently up-regulated in HBx positive group tissues in comparison with the HBx negative group, and exhibits an inverse correlation between UCA1 and p27Kip1 levels. Our findings demonstrate an important mechanism of hepatocarcinogenesis through the signaling of HBx-UCA1/EZH2-p27Kip1 axis, and a potential target of HCC.

Depletion of DNMT3A Suppressed Cell Proliferation and Restored PTEN in Hepatocellular Carcinoma Cell

Promoter hypermethylation mediated by DNA methyltransferases (DNMTs) is the main reason for epigenetic inactivation of tumor suppressor genes (TSGs). Previous studies showed that DNMT1 and DNMT3B play an important role in CpG island methylation in tumorigenesis. Little is known about the role of DNMT3A in this process, especially in hepatocellular carcinoma (HCC). In the present study, increased DNMT3A expression in 3 out of 6 HCC cell lines and 16/25 (64%) HCC tissues implied that DNMT3A is involved in hepatocellular carcinogenesis. Depletion of DNMT3A in HCC cell line SMMC-7721 inhibited cell proliferation and decreased the colony formation (about 65%). Microarray data revealed that 153 genes were upregulated in DNMT3A knockdown cells and that almost 71% (109/153) of them contain CpG islands in their 5′ region. 13 of them including PTEN, a crucial tumor suppressor gene in HCC, are genes involved in cell cycle and cell proliferation. Demethylation of PTEN promoter was observed in DNMT3A-depleted cells implying that DNMT3A silenced PTEN via DNA methylation. These results provide insights into the mechanisms of DNMT3A to regulate TSGs by an epigenetic approach in HCC.

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

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 methylation-specific 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.