Gang Kuang

Associations between polymorphisms of HOTAIR and risk of gastric cardia adenocarcinoma in a population of north China

As an important long noncoding RNA, Hox transcript antisense intergenic RNA (HOTAIR) is involved in the development and progression of various carcinomas. However, the role and genetic alterations of HOTAIR in gastric cardia adenocarcinoma (GCA) occurrence and progression have not been elucidated. We performed a case-control study in a population of north China to evaluate the possible association between haplotype-tagging SNPs (htSNPs) of the whole HOTAIR sequence and the risk of GCA as well as functional effect of the susceptibility single nucleotide polymorphism (SNP) rs12826786 on gene expression. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to examine the genotype of htSNPs in 515 GCA patients and 654 control subjects, and the quantitative real-time reverse transcription PCR (RT-PCR) method was used to examine the expression of HOTAIR in 102 GCA patients. A family history of upper gastrointestinal cancer (UGIC) significantly increased the risk of developing GCA. Among three htSNPs of the HOTAIR gene (rs12826786 C>T, rs4759314 A>G, and rs10783618 C>T), only the T allele of rs12826786 was found to increase the risk of developing GCA and was associated with smoking habit and tumor-node-metastasis (TNM) stage. In addition, higher expression levels of HOTAIR were found in tumor tissues and rs12826786 SNP has a genotype-specific effect on HOTAIR expression. A high HOTAIR expression level was associated with poor GCA patients’ survival. These results indicate that functional genotype alteration of rs12826786 SNP may influence the expression of HOTAIR, and HOTAIR may be a useful marker to predict the biological behavior of tumors and potentially a therapeutic target in GCA treatment.

Polymorphisms of the DNA repair gene XPA and XPC and its correlation with gastric cardiac adenocarcinoma in a high incidence population in North China.

Goals Investigated the association of DNA repair gene xeroderma pigmentosum group A(XPA) and C(XPC) polymorphisms with the risk of gastric cardiac adenocarcinoma (GCA) in a high incidence region in north China. Background Polymorphisms of a number of DNA repair genes may contribute to variations in DNA repair capacity and genetic susceptibility to different cancers. Study Two single nucleotide polymorphisms of XPA and 3 single nucleotide polymorphisms of XPC were genotyped in 253 GCA patients and 612 healthy controls. Results Family history of upper gastrointestinal cancer may increase the risk of developing GCA. Compared with A/A genotype, A/G+G/G genotype of XPA A23G significantly decreased the risk of developing GCA especially in nonsmoker group. The genotype and allelotype distributions of XPC intron 9 PAT+/− and exon 15 Lys939Gln in GCA patients were not significantly different from that in healthy controls (P>0.05). T allelotype frequencies of XPC exon 8 Val499Ala in GCA patients was significantly lower than that in healthy controls (P<0.05). The C/T genotype frequency of XPC exon 8 in GCA patients (35.6%) was significantly different from that in healthy controls (46.1%) (P=0.01). Compared with individuals with C/C genotype, individuals with T allele (C/T or T/T genotype) had significantly lower risk in developing GCA. We also found that polymorphisms of this 3 XPC locus were in linkage disequilibrium. Conclusions XPA A23G and XPC exon 8 Val499Ala polymorphisms may be useful markers for identifying individuals at risk of developing GCA in the high incidence region of north China.

Aberrant CpG Island Hypermethylation of RASSF1A in Gastric Cardia Adenocarcinoma

Ras-association domain family 1A (RASSF1A) gene, a candidate tumor suppressor gene, is inactivated in several human tumors, usually by hypermethylation of its promoter region. RASSF1A induces cell cycle arrest through inhibition of cyclin D1 accumulation. In this work, the promoter methylation status of the RASSF1A in 92 gastric cardia adenocarcinoma (GCA) and corresponding normal tissues were investigated using Methylation-specific PCR (MSP) approach, immunohistochemistry method and RT-PCR were used respectively to examine the protein expression and mRNA expression of RASSF1A in tumors and corresponding normal tissues. Cyclin D1 expression was examined by immunohistochemistry. RASSF1A was methylated in 54/92 (58.7%) tumor specimens, which was significantly higher than that in corresponding normal tissues (p <.001). Methylation frequencies of stage III and IV tumor tissues were significantly higher than that in stage I and II tumor tissues (p <.05). By immunostaining, 43/92 (46.7%) tumor tissues demonstrated heterogeneous, positive immunostaining of tumor tissues was significantly reduced with comparison to matched normal tissues (p <.001). mRNA expressions of RASSF1A in GCA tumor tissues were reduced significantly with comparison to the corresponding normal tissues (OD value: 0.2376 ± 0.2315 vs 0.6874 ± 0.2668, p <.001). RASSF1A mRNA expression in methylation group of GCA was significantly different from that in unmethylation group (p <.001). Cyclin D1 hyper-expression was found in 72/92 (78.3%) cases and correlated with RASSF1A methylation (p <.05). Our data suggested that epigenetic silencing of RASSF1A gene expression by promoter hypermethylation may play an important role in GCA.

Aberrant methylation and decreased expression of the TGF-β/Smad target gene FBXO32 in esophageal squamous cell carcinoma.

F‐box protein 32 (FBXO32) (also known as atrogin‐1), a member of the F‐box protein family, has recently been identified as a transforming growth factor beta (TGF‐β)/Smad target gene involved in regulating cell survival, and it may be transcriptionally silenced by epigenetic mechanisms in some kinds of carcinomas, yet its role in esophageal squamous cell carcinoma (ESCC) has not been defined.

Concordant Promoter Methylation of Transforming Growth Factor-Beta Receptor Types I and II Occurs Early in Esophageal Squamous Cell Carcinoma

Introduction:Transforming growth factor-&bgr; (TGF-&bgr;) is a pleiotropic growth factor with multiple functions through its type I (TGFBR1) and type II (TGFBR2) receptors. A reduction or loss of expression of TGFBRs enables cancer cells to escape the growth inhibitory effect of TGF-&bgr; and to gain a growth advantage. Objective:The promoter methylation status and expression of TGFBR1, TGFBR2 and Smad4 gene were investigated in esophageal squamous cell carcinoma (ESCC). Design:Methylation-specific polymerase chain reaction approach was used to detect the methylation status. Immunohistochemistry and reverse transcription-polymerase chain reaction method were used to examine the protein and messenger RNA expression, respectively. Results:Both the ESCC and the high-grade dysplastic tissues showed hypermethylation of TGFBR1 and TGFBR2, and the hyper-methylation of TGFBR1 and TGFBR2 in ESCC tissues was significantly associated with decreased messenger RNA and protein expression (P < 0.05). When stratified for tumor lymph node metastasis stages, TGFBR1 and TGFBR2 gene methylation was more frequent in stage III and stage IV tumor tissues than that in stage I and stage II tumor tissues (P < 0.05). Simultaneous methylation of the 2 receptors was progressively increased along with the increasing of tumor lymph node metastasis stage and decreasing of histological differentiation. Smad4 hypermethylation was only detected in 7 ESCC tumor tissues and associated with the loss of Smad4 expression. The decreased protein expression of TGFBR1, TGFBR2 and Smad4 was correlated with increased expression of TGF-&bgr;1 in ESCC. Conclusions:These data suggest that promoter methylation of TGFBR1 and TGFBR2 may exist in the early stage of ESCC and play important roles in TGFBR1 and TGFBR2 gene silencing.

Aberrant Methylation of Thrombospondin-1 and Its Association with Reduced Expression in Gastric Cardia Adenocarcinoma

Aim. Investigate the promoter methylation of the Thrombospondin-1 (TSP1) gene in gastric cardia adenocarcinoma (GCA). Methods. MSP approach, immunohistochemistry method, and RT-PCR were used respectively to examine the promoter methylation of TSP1, its protein and mRNA expression in tumors and corresponding normal tissues. The expression and concentration of TGF-β1 were examined respectively by immunohistochemistry and ELISA method. The status of T cell immunity was examined by Flow cytometry analysis. Results. TSP1 was methylated in 34/96 (35.4%) tumor specimens, which was significantly higher than that in corresponding normal tissues (P < .001). Protein and mRNA expression of TSP1 in GCA tumor tissues were reduced significantly and were associated with TSP1 methylation. The protein expression of TGF-β1 was significantly higher in tumor tissues (P < .001) and was associated with TNM stage and histological differentiation. The concentration of active and total TGF-β1 did not show significant difference between the GCA patients with hypermethylation of TSP1 and without methylation of TSP1 (P > .05). The function of T cell immunity was significantly different between the GCA patients with hypermethylation of TSP1 and without methylation of TSP1. Conclusions. Epigenetic silencing of TSP1 gene by promoter hypermethylation may play an important role in GCA.

Decreased expression of RASSF1A and up-regulation of RASSF1C is associated with esophageal squamous cell carcinoma

The Ras-Association Domain Family 1 (RASSF1) gene, which is located on the small arm of chromosome 3, contains two CpG islands and generates seven transcripts (RASSF1A-RASSF1G) by differential promoter usage and alternative splicing. As the main transcript, RASSF1A, B and C may play different roles in tumorigenesis. The present study was to detect the role of RASSF1A, B and C in esophageal squamous cell carcinoma (ESCC) and clarify the critical CpG sites of RASSF1A, in order to clarify more information on the role of RASSF1 with regard to the pathogenesis of ESCC. Frequent silencing of RASSF1A but not RASSF1B and RASSF1C were found in esophageal cancer cell lines and the silencing of RASSF1A may be reversed by 5-Aza-dC treatment. The aberrant promoter and exon 1 especially exon 1 methylation of RASSF1A induces silencing of its expression in TE13 cell line. Decreased mRNA and protein expression of RASSF1A was observed in ESCC tumor tissues and was associated with RASSF1A promoter and exon 1 methylation status. Unlike RASSF1A, methylation and expression variation of RASSF1B was not found in ESCC tissues. However, RASSF1C is highly expressed in ESCC tissues. RASSF1A methylation and protein expression were independently associated with ESCC patients’ survival. These data indicated that the inactivation of RASSF1A through promoter and exon 1 methylation may play an important role in ESCC carcinogenesis and reactivation of RASSF1A gene may has therapeutic potential and may be used as a prognostic marker for ESCC patients.

Decreased expression of WWOX in the development of esophageal squamous cell carcinoma

The WW domain‐containing oxidoreductase (WWOX) gene, located on chromosome 16q23.3–24.1 in the region recognized as the common fragile site FRA16D is considered to be a tumor suppressor gene involved in various carcinomas. The present study was to investigate the alterations of WWOX expression and its correlation with polymorphism, the level of WWOX loss of heterozygosity (LOH), and methylation status in esophageal squamous cell carcinoma (ESCC). Immunohistochemistry and RT‐PCR methods were used, respectively, to examine the protein and mRNA expression of WWOX in ESCC tissues. PCR‐RFLP, PCR‐SSLP, and MSP approach were used, respectively, to detect polymorphisms of rs3764340, rs2548861, and rs1079635 site, the level of LOH, and WWOX methylation status. Family history of upper gastrointestinal cancer (UGIC) significantly increased the risk of developing ESCC. Protein and mRNA expression of WWOX was reduced in ESCC tumor tissues and was associated with LOH and hypermethylation of the gene. The G allele of rs3764340 significantly elevated the risk of developing ESCC and was associated with TNM stage. LOH at the WWOX loci was observed in 41.4% tumors. The hypermethylation of promoter and exon1 of WWOX was found to be occurred in dysplastic tissues and the methylation frequency of WWOX in ESCC tumor tissues was significantly higher than that in corresponding normal tissues and was associated with UGIC family history. In all, these results indicate that the WWOX gene may play an important role in the development of ESCC especially in individuals with UGIC family history.

Decreased expression and aberrant methylation of Gadd45G is associated with tumor progression and poor prognosis in esophageal squamous cell carcinoma

The growth arrest DNA damage-inducible gene (Gadd45) family, which is composed of Gadd45A, Gadd45B, and Gadd45G, is involved in DNA damage response and cell growth arrest. The present study was to detect the role of Gadd45 gene family in esophageal cancer and the relationship of Gadd45G methylation to a series of pathological parameters in a large esophageal squamous cell carcinoma (ESCC) sample, in order to elucidate more information on the role of Gadd45 gene family with regard to the pathogenesis of ESCC. Frequent silencing of Gadd45G but not Gadd45A and Gadd45B were found in esophageal cancer cell lines and the silencing of Gadd45G may be reversed by 5-Aza-dC or TSA treatment in Eca109 cell line. The aberrant proximal promoter methylation of Gadd45G induces silencing of Gadd45G expression in Eca109 cell line. Gadd45A mRNA and protein expression in ESCC tumor tissues was significantly different compared to corresponding normal tissues. Decreased mRNA and protein expression of Gadd45G was observed in ESCC tumor tissues and was associated with Gadd45G proximal promoter methylation. Gadd45A or Gadd45B expression was not correlated with ESCC patients survival, while Gadd45G methylation status and protein expression were independently associated with ESCC patients’ survival. These data indicated that Gadd45G may be a functional tumor suppressor and its inactivation through proximal promoter methylation may play an important role in ESCC carcinogenesis and reactivation of Gadd45G gene may has therapeutic potential and may be used as a prognostic marker for ESCC patients.

Promoter hypermethylation‐mediated downregulation of miR‐770 and its host gene MEG3, a long non‐coding RNA, in the development of gastric cardia adenocarcinoma

Maternally expressed gene 3 (MEG3) is an imprinted gene located at 14q32 which encodes an lncRNA and is downregulated in an expanding list of cancer cell lines and primary human cancers. The miR‐770 is transcribed from the intronic sequence of MEG3 and MEG3 may be the host gene for miR‐770. However, the biological role of MEG3 and miR‐770 in gastric cardia adenocarcinoma (GCA) development and prognosis is poorly defined. The present study was to investigate the function and methylation status of MEG3 in GCA, and further to detect the functional association of miR‐770 and its host gene MEG3 in GCA carcinogenesis and prognosis. MEG3 and miR‐770 was significantly downregulated in GCA patients and cell lines, and their expression was associated with TNM stage and lymph node metastasis. Overexpression of MEG3 and miR‐770 inhibited gastric cancer cell proliferation and invasion in vitro. Furthermore, the expression level of MEG3 and miR‐770 was significantly increased in cancer cells after treated with 5‐Aza‐dC. The aberrant hypermethylation of proximal promoter and enhancer region of MEG3 was detected in GCA tissues. In addition, the proximal promoter and enhancer region hypermethylation and dysregulation of MEG3 and miR‐770 were associated with poorer GCA patients’ survival. These findings suggest that miR‐770 and its host gene MEG3 may play tumor suppressor role and hypermethylation of proximal promoter and enhancer region may be one of the critical mechanisms in inactivation of MEG3 and miR‐770 in GCA development. MEG3 and miR‐770 may be used as potential biomarkers in predicting GCA patients’ prognosis.