Methylation Of RASSF10Gene In Esophageal Squamous Cell Carcinoma

ObjectiveIncidence of esophageal squamous cell carcinoma (ESCC) is increasing. It is a serious threat to health of human being, but in recent years the knowledge of the epigenetic mechanisms related to this disease remains limited. In this study, we identified RASS10as a candidate tumor suppressor gene (TSG) in esophageal carcinoma, and investigated its function in ESCC cell lines. We hope to explore the theoretical basis of RASSF10in the early diagnosis, treatment and prognosis of ESCC.MethodsUsing immunohistochemical compares the different expression of RASSF10protein between esophageal Squamous carcinoma tissue specimens and adjacent tissues. The RASSF10methylation statu was detected by Methylation-specific PCR(MSP) in tissue specimens. The RASSF10expression was detected by semi-quantitative RT-PCR before and after5-aza-dc treatment in different esophageal carcinoma cell lines KYSE70, KYSE140, KYSE150, KYSE180, KYSE450and TE8. MSP was used in these esophageal Squamous carcinoma cell lines to detect the RASSF10methylation statu. Applicating Lentiviral eukaryotic expression system to recovery RASSFIO gene expression in esophageal cancer cell lines, and detect cell cycle, apoptosis, colony-forming ability and cell proliferation ability.Results1We selected32cases of paraffin sections of ESCC and paired adjacent specimens, which were examined by immunohistochemistry. The expression of RASSF10in adjacent tissues and cancer tissues were significantly different (P=0.000).2The result of MSP detection of specimens of patients with esophageal squamous carcinoma showed RASSFIO genes were down-regulated in44.3%case (39/88), and only one case in five of adjacent tissues was methylated20%(1/5). Further statistical analysis showed that RASSFIO methylation was significantly associated with lymph node metastasis (P=0.001).3The RT-PCR result before and after the induction by the demethylation drug5-aza-dc showed that KYSE150cell lines loss expression of RASSFIO, but the expression of RASSFIO was recovered after the induction. KYSE140, KYSE450, TE8cell lines found no significant expression changes before and after the induction.4The methylation status of RASSFIO gene in esophageal squamous carcinoma cell line was detected by MSP, the result showed that KYSE150was full methylation, KYSE180, KYSE70awas partial methylation, KYSE140, KYSE450, TE8was unmethylation.5The Bisulfite genomic sequencing of RASSFIO was detected in Esophageal squamous carcinoma cell lines KYSE150KYSE140KYSE180, the results shown KYSE150is completely unmethylated KYSE180partially methylated the KYSE140unmethylated.6The cell function detection was tested in KYSE150. The clone number of the group that RASSFIO infected (94.67%±6.11%) was significantly lower than the empty lentiviral group (225.00%±15.00%)(P=0.006).7The growth curve of MTT test showed after recovery of RASSFIO expression the proliferation of KYSE150cells was inhibited.8After recovery the expression of RASSFIO, the proportion of cells in the G1phase (36.68%±1.03%) was significantly lower than the empty lentiviral group (67.17%±1.23%)(P=0.001). The proportion of G2-M phase cells of the RASSFIO recovered group (35.03%±2.018%) was significantly higher than the empty lentiviral group (15.65%±0.728%)(P=0.003).9The apoptosis rate of RASSFIO expression restored group (16.55%±0.92%), the empty lentiviral group (22.00%±1.13%), P=0.166. ConclusionsRASSF10is a tumor suppressor gene and the expression of it was regulated by promoter methylation. The gene can effectively inhibit the proliferation of esophageal squamous cell carcinoma cells lines.