Zhujiang Zhao

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.

Promoter polymorphisms of DNMT3B and the risk of colorectal cancer in Chinese: a case-control study

BackgroundDNA-methyltransferase-3B (DNMT3B), which plays a role in DNA methylation, is usually aberrant expression involved in carcinogenesis. Polymorphisms of the DNMT3B gene may influence DNMT3B activity on DNA methylation in several cancers, thereby modulating the susceptibility to cancer.MethodsDNMT3B -579G>T genotypes and -149C>T were determined by PCR-RFLP and sequencing in 137 colorectal cancer patients and 308 controls matched for age and sex, who did not receive radiotherapy or chemotherapy for newly diagnosed and histopathologically confirmed colorectal cancer. The association between two SNPs of the DNMT3B promoter and the risk of the development of colorectal cancer was analyzed in a population of Chinese.ResultsThe allele frequency of -149C >T among patients and controls was 0.73% versus 0.65%, respectively. The allele frequency of -597G>T for patients and controls was 6.57% versus 11.53%, respectively. Individuals with at least one -149C>T allele were no at a significantly increase risk of colorectal cancer compared with those having a -149TT genotype. However, Individuals with at least one 579G>T allele were decreased risk of colorectal cancer compared with those having a -579TT genotype.ConclusionThe relative distribution of -149C>T DNMT3B SNPs among a Chinese population can not be used as a stratification marker to predict an individuals susceptibility to colorectal cancer. However, the DNMT3B -579G>T polymorphism may contribute to the genetic susceptibility to colorectal cancer.

Reduced miR-29a-3p expression is linked to the cell proliferation and cell migration in gastric cancer

BackgroundMicroRNAs (miRNAs) play an important role in a tumor-suppressive or oncogenic manner in carcinogenesis. Alteration expression patterns of miRNAs in gastric cancer (GC) are associated with cancer initiation and progression. In the present study, we evaluated miR-29a-3p expression pattern and its function in gastric carcinogenesis.MethodsThe expression of miR-29a-3p in GC tissue samples and cell lines was detected by quantitative real-time PCR (qRT-PCR). After transfected with miR-29a-3p mimics or inhibitor, the cell proliferation, cell migration, and invasion ability were assessed by CCK-8 assay, wound healing assay, and Trans-well assay, respectively. The level of CDK2, CDK4, CDK6, and CyclinD1 were determined by qRT-PCR and Western blot.ResultsCompared with the corresponding non-tumor tissues, miR-29a-3p showed a significant down-regulated expression in tumor tissues. In vitro functional assays demonstrated that enforced miR-29a-3p expression inhibited cell proliferation by reducing the expression of CDK2, CDK4, and CDK6. Wound healing and Transwell assays revealed that miR-29a-3p suppressed tumor metastasis in GC.ConclusionsOur preliminary results suggest that altered expression of miR-29a-3p is involved in gastric cancer process. The present study provides the first insight into the specific role of miR-29a-3p in gastric carcinogenesis.

DNA methyltransferase 3A isoform b contributes to repressing E-cadherin through cooperation of DNA methylation and H3K27/H3K9 methylation in EMT-related metastasis of gastric cancer

DNA methyltransferase 3A (DNMT3A) has been recognised as a key element of epigenetic regulation in normal development, and the aberrant regulation of DNMT3A is implicated in multiple types of cancers, especially haematological malignancies. However, its clinical significance and detailed functional role in solid tumours remain unknown, although abnormal expression has gained widespread attention in these cancers. Here, we show that DNMT3A isoform b (DNMT3Ab), a member of the DNMT3A isoform family, is critical for directing epithelial–mesenchymal transition (EMT)-associated metastasis in gastric cancer (GC). DNMT3Ab is positively linked to tumour-node-metastasis (TNM) stage, lymph node metastasis and poor prognosis in GC patients. Overexpression of DNMT3Ab promotes GC cell migration and invasion as well as EMT through repression of E-cadherin. Meanwhile, DNMT3Ab promotes lung metastasis of GC in vivo. Mechanistic studies indicate that DNMT3Ab mediates the epigenetic inaction of the E-cadherin gene via DNA hypermethylation and histone modifications of H3K9me2 and H3K27me3. Depletion of DNMT3Ab effectively restores the expression of E-cadherin and reverses TGF-β-induced EMT by reducing DNA methylation, H3K9me2 and H3K27me3 levels at the E-cadherin promoter. Importantly, DNMT3Ab cooperated with H3K9me2 and H3K27me3 contributes to the transcriptional regulation of E-cadherin in a Snail-dependent manner. Further, gene expression profiling analysis indicates that multiple metastasis-associated genes and oncogenic signalling pathways are regulated in response to DNMT3Ab overexpression. These results identify DNMT3Ab as a crucial regulator of metastasis-related genes in GC. Targeting the DNMT3Ab/Snail/E-cadherin axis may provide a promising therapeutic strategy in the treatment of metastatic GC with high DNMT3Ab expression.

Downregulated PITX1 Modulated by MiR-19a-3p Promotes Cell Malignancy and Predicts a Poor Prognosis of Gastric Cancer by Affecting Transcriptionally Activated PDCD5

Background/Aims: PITX1 has been identified as a potential tumor-suppressor gene in several malignant tumors. The molecular mechanism underlying PITX1, particularly its function as a transcription factor regulating gene expression during tumorigenesis, is still poorly understood. Methods: The expression level and location of PITX1 were determined by quantitative reverse transcription PCR (qRT-PCR) and immunohistochemical staining in gastric cancer (GC). The effect of PITX1 on the GC cell proliferation and tumorigenesis was analyzed in vitro and in vivo. To explore how PITX1 suppresses cell proliferation, we used PITX1-ChIP-sequencing to measure genome-wide binding sites of PITX1 and assessed global function associations based on its putative target genes. ChIP-PCR, electrophoretic mobility shift assay, and promoter reporter assays examined whether PITX1 bound to PDCD5 and regulated its expression. The function of PDCD5 in GC cell apoptosis was further examined in vitro and in vivo. The relationship between the PITX1 protein level and GC patient prognosis was evaluated by the Kaplan-Meier estimator. Meanwhile, the expression level of miR-19a-3p, which is related to PITX1, was also detected by luciferase reporter assay, qRT-PCR, and western blotting. Results: The expression level of PITX1 was decreased in GC tissues and cell lines. Elevated PITX1 expression significantly suppressed the cell proliferation of GC cells and tumorigenesis in vitro and in vivo. PITX1 knockdown blocked its inhibition of GC cell proliferation. PITX1 bound to whole genome-wide sites, with these targets enriched on genes with functions mainly related to cell growth and apoptosis. PITX1 bound to PDCD5, an apoptosis-related gene, during tumorigenesis, and cis-regulated PDCD5 expression. Increased PDCD5 expression in GC cells not only induced GC cell apoptosis, but also suppressed GC cell growth in vitro and in vivo. Moreover, PITX1 expression was regulated by miR-19a-3p. More importantly, a decreased level of PITX1 protein was correlated with poor GC patient prognosis. Conclusion: Decreased expression of PITX1 predicts shorter overall survival in GC patients. As a transcriptional activator, PITX1 regulates apoptosis-related genes, including PDCD5, during gastric carcinogenesis. These data indicate PDCD5 to be a novel and feasible therapeutic target for GC.