The Mechanism And Clinical Significance Of ECRG4 In Gastric Cancer

Objective: Recently,the incidence and mortality of malignant tumor are increasing,becoming a major global health care burden.Gastric cancer is a common malignant tumor and remains the main leading cause of cancer-related death worldwide,up to now,its incidence and mortality stills high,the prognosis remains poor,despite its advances in diagnosis and treatment.Gastric carcinoma results from the combined forces of both genetic and epigenetic events.DNA methylation is the most widely studied epigenetic event.It has also been shown that aberrant methylation of tumor suppressor genes is a common event in the development and progression of gastric cancer.Esophageal cancer related gene 4(ECRG4)was initially cloned from human normal esophageal epithelium by Peking Union Medical College of China in 1998.ECRG4 is located in human chromosome 2q14.1-14.3,contains 4 exons,and expressed in many human tissues,including esophageal epithelium,colon tissues,breast,prostate and brain.It reported that there are several Cp G islands in the promoter and the first exon and introns of ECRG4,suggesting that its silencing was realted to DNA methylation.ECRG4 is considered to be a potential tumor suppressor gene in esophageal carcinoma and plays a role in suppression of tumor invasion and metastasis.Recent studies have demonstrated that ECRG4 protein was also down-regulated in several tumors,such as breast cancer,prostate carcinoma,colon cancer and glioma,but it is not known to date its expression and functions in gastric cancer.It's meaningful to understand the role of ECRG4 in gastric carcinogenesis.We investigated the expression of ECRG4 in gastric cancer,and the possible role of ECRG4 in the prosess of gastric cancer.We also explored whether an epigenetic mechanism is involved in ECRG4 inactivation.Methods: ECRG4 expression was examined using real-time reverse-transcription polymerase chain reaction,western blot,immunohistochemistry and the methylation status of the ene was evaluated by methylation-specific PCR in cell lines: normal gastric mucosal cell line GES1 and gastric cancer cell lines BGC-823,SGC-7901,AGS,MKN-45.And we did the same research in the human gastric tissues and corresponding nonmalignant gastric tissues.Furthermore,ECRG4 expression was testified whether treatment with the demethylating agent,5-aza-2'-deoxycytidine can restored in cell lines that lacked ECRG4 expression using all the experiments above.Also,CCK-8 assay and Transwell assay were used to test whether ECRG4 silencing can influence the cell viability and invasion capability of BGC823 which is a highly metastatic gastric cancer cell line.Results: Section I.ECRG4 was down-regulated in gastric cancer cells and tissues 1.We showed a heatmap of the 100 most downregulated genes in the GSE63089 dataset of gastric cancer.The expression level of ECRG4 in normal gastric tissues was approximately 2.46 times that of gastric cancer tissues(log fold changes |log FC| = 2.46,p<0.01).2.q RT-PCR showed expression of ECRG4 was down-regulated in 4 gastric cancer cell lines compared with the normal gastric mucosa cell line GES1,its lowest expression was found in MKN45 cells.The results of Western blot analysis were similar to ECRG4 m RNA expression.3.Base on immunohistochemistry analysis of 102 paired gastric cancer tissues and its corresponding nonmalignant gastric tissues,we observed that ECRG4 was positively stained in the cytoplasm and ECRG4 expression was significantly decreased in gastric cancer compared with corresponding nonmalignant gastric tissues.38 out of the 102 cases(37.3%)were negative for ECRG4 in gastric cancer tissues,but only 4(3.9%)in corresponding nonmalignant gastric tissues(P<0.01).High level of ECRG4 expression were 38(37.3%)cases in gastric cancer tissues,whereas 63 cases(61.8%)in corresponding nonmalignant gastric tissues(P<0.01).In addition,Lower protein expression levels of ECRG4 in gastric cancer were related to lymph node metastasis and histological grade(P < 0.05).We also examined the relationship between ECRG4 expression and the clinicopathologic features of the patients,including age,gender,depth of invasion,lymph node status,histological grade,and pathologic staging of the tumor,ut no correlation with any of the clinicopathologic features of the patients was found(P>0.05).Section II.Silencing ECRG4 expression inhibited proliferation and invasion of gastric cancer We use ECRG4-specific si RNA to silence ECRG4 expression in the highly metastatic BGC823 cells in which the level of ECRG4 was relatively high.Results of Transwell analysis and CCK-8 analysis showed that cell invasion and proliferation effect was significantly enhanced after ECRG4 expression was silencing.Section III.The downregulation of ECRG4 expression in gastric cancer is related to DNA methylation 1.Results of MSP showed ECRG4 was hypermethylated in SGC-7901,BGC823,AGS and MKN45,but partially methylated in GES1 cells.2.We then examined the methylation status of the ECRG4 promoter in 74 primary gastric carcinomas as well as nonmalignant gastric tissues from the same individuals by MSP.Hypermethylation of the ECRG4 gene was detected in 49(66.2%)of the 74 primary gastric carcinomas,while hypermethylation in nonmalignant gastric tissue was only found in 14 cases(18.9%).And ECRG4 was partially methylated in 16 cases(21.6%)and non-methylated in 9 cases(12.2%)in primary gastric tumors and 22 cases(29.7%)of partially methylation and 38 cases(51.4%)of non-methylation in noncancerous tissues respectively.3.To confirm that aberrant methylation was responsible for silencing ECRG4 expression,we treated the BGC-823 gastric cancer cell lines with the demethylating agent 5-Aza-d C.Expression of ECRG4 m RNA and protein were restored in BGC-823 cells after treatment with 5-Aza-d C for 72h(P<0.05).Conclusions: 1.Down regulation of ECRG4 expression was downregulated in gastric cancer cell lines and tissues,and its expression was closely related to lymph node metastasis and histological grade.2.The suppression of ECRG4 in BGC823 cells by si RNA transfection inhibited the growth and invasion of gastric cancer cell.3.The down-regulation of ECRG4 in gastric cancer was mainly related to DNA methylation,which suggests that aberrant methylation is one of the mechanisms in ECRG4 silencing.4.The treatments with 5-Aza-d C could restore the expression of ECRG4.