Hypermethylation And Aberrant Expression Of P73and HOXA11Genes In Gastric Cancer

Background and objectiveGastric carcinoma (GC) is one of the most frequent malignancies and remains the second leadingcause of cancer-related mortality in the world. Gastric carcinogenesis is thought to be a multistep processthat involves multiple genes and epigenetic alterations. The combination of the two sides may provide anew viewpoint to better understand the pathogenesis of gastric carcinoma. As a well-defined epigeneticmechanism, DNA methylation plays a major role in cancer development through transcriptional silencingof critical growth regulators such as tumor suppressor genes. A number of genes have been found to beaberrantly methylated in gastric cancer.As a tumor suppressor gene, p53mutation may play an important role in tumorigenesis anddevelopment. p73, a novel p53family member, shows substantial structural and functional homology withp53. p73gene encodes two different proteins that are expressed under the control of two independentpromoters and that have opposite activities: the transcriptionally active full-length TA-p73showspro-apoptotic effects, while the shorter ΔNp73, which lacks the N-terminal transactivating domain, has anevident anti-apoptotic function. Researches show that methylation of p73gene can be found in glioma,multiple myeloma and colon cancer, but the related studies in gastric cancer was rare.As a specialized kind of transcription factors, HOX genes play crucial roles in modulating embryonicmorphogenesis and cell differentiation of the mammal, and are closely correlated to tumorigenesis. Thegene promoter region of p53has binding sites of HOX gene sequences, HOX gene can regulate theexpression of p53, and the protein of HOX can enhance the function of p53. Among HOXA genes,HOXA11hypermethylation and low expression have recently been reported in reproductive system cancers.By upregulating the expression of p73gene, HOXA11may inhibit tumorigenesis.In this study, we have analyzed the methylation status of p73and HOXA11gene promoter andevaluated their correlation with gene expression level, as well as with different clinicopathologicalparameters in GC. Materials and methodFrom May2012to March2013,32patients were histologically diagnosed with gastricadenocarcinoma, and were prospectively recruited into this study from Huaihe Hospital of HenanUniversity (Henan, China). All patients had given informed consent for specimen collection. The mean ageof the patients (20males and12females) was63.4years (range,36to85years, Standard Deviation12.1),and none of the patients has received preoperative chemo-or radiotherapy. The cancerous tissue,peri-cancer tissue (located5cm from the primary tumor) and normal gastric mucosa (superficial gastritispatients) were bisected. One part was stored at-80℃immediately and the other part was fixed in4-10%buffered formaldehyde, embedded in paraffin for further use.The methylation status and expression level of p73and HOXA11gene were determined by Bisulfitesequencing PCR (BSP). Total RNA was extracted with TRIzol reagent (Takara, China), and first-strandcDNA was generated using a first strand cDNA synthesis kit. Quantitative polymerase chain reaction(RT-PCR) was carried out to examine the mRNA expression of p73and HOXA11gene.Immunohistochemistry (IHC) and western blot were used respectively to detect the protein expression ofp73and HOXA11gene. In addition, we analyzed and correlated the findings with clinicopathologicalfeatures. Data were analyzed by the computer program SPSS17.0, using analysis of t test. Two sided P＜0.05was considered to be significant.Results1. The methylation frequencies of p73and HOXA11gene in gastric cancer tissue, peri-cancer tissuespecimen were higher than those in normal gastric mucosa (P＜0.05), but no difference was seen betweenthe peri-cancer specimen and normal gastric mucosa (P＜0.05). The methylation level of p73and HOXA11gene were higher in TNM III and IV patients of GC than that in TNM I and Ⅱp atients (P＜0.05).2. The expression of TAp73mRNA was significantly lower in human gastric cancer tissue than innormal gastric mucosa (P＜0.05). And the expression of ΔNp73mRNA was significantly higher in humangastric cancer tissue than in normal gastric mucosa (P＜0.05). The expression levels of HOXA11mRNAwere significantly lower in human gastric cancer tissue than in normal gastric mucosa (P＜0.05).3. Protein expression was then determined by IHC. The expression of TAp73protein was lower inhuman gastric cancer tissue than in normal gastric mucosa, and the expression of ΔNp73protein was higher in human gastric cancer tissue than in normal gastric mucosa (P＜0.05). HOXA11protein expression wasthen determined by IHC. The expression levels of HOXA11protein was lower in human gastric cancertissue than in normal gastric mucosa (P＜0.05).4. p73protein expression was then determined by western blot. The expression of TAp73protein wassignificantly lower in human gastric cancer tissue than in normal gastric mucosa (P＜0.05). But theexpression of ΔNp73protein was not significantly different between the two groups (P＞0.05).Conclusion1. Hypermethylation of p73and HOXA11may associate with the occurrence and development ofgastric cancer.2. The mRNA and protein expressions of TAp73and HOXA11in GC tissues are lower than those innormal gastric mucosa.3. Aberrant promoter methylation of TAp73and HOXA11is the main reason causing loss ordown-regulation of gene expression, which may be involved in the carcinogenesis of GC.