| Objective:Xinjiang is one of the high incidence area of esophageal cancer, the prevalence and mortality of Kazakh people has been very high and beyond the average level of other nations. It has been confirmed riboflavin deficiency is an important risk factor for esophageal cancer, riboflavin supplement can significantly reduce the risk, but the effect of dietary supplements of riboflavin intervention showed obvious individual differences, showed that an interaction of environmental and genetic factors in occurrence of esophageal cancer. In our previous esophageal cancer genome-wide association study found that riboflavin transporter gene (C20orf54) is one of susceptibility genes, but only indirectly recognized the genetic changes may be the Key target on interaction of environmental and genetic factors of esophageal cancer. For the purpose of further research the regulation mechanism of C20orf54gene on riboflavin metabolism in esophageal cancer tumor tissues.In this study, we will collect Paraffin-embedded and/or fresh tissue specimens of Kazakh patients with esophageal cancer and precancerous disease, analyze the C20orf54gene mutation and expression by Mass ARRAY, PCR-RFLP and immunohistochemical methods to find the collelations between esophageal cancer and C20orf54gene expression regulation.In order to further understanding the molecular mechanism of interaction of environmental and genetic factors in occurrence and ethnic differences of esophageal cancer. The result of this study can also provide experimental basis for specific new target on high risk group screening and individualized prevention of esophageal cancer. Methods:1) Paraffin-embedded and/or fresh tissue specimens of Kazak esophageal cancer patients were collected for the detection of protein and semi-quantitative mRNA expression level of C20orf54genes by immunohistochemistry (IHC) and RT-PCR;2) Blood samples from the Kazak patients and normal controls were collected by "Human based case-control study", Plasma riboflavin levels were detected by High-performance liquid chromatography (HPLC) in Kazak patients with esophageal squamous cell carcinoma (ESCC) and healthy controls;3) By DNA Sequenom MassARRAY approach for quantitative detection of methylated DNA, we analyzed the cervical tissue DNA for CpG content of candidate genes provided by the analysis in "Part I" of this study using gene specific primer pairs to compare methylation level of target fragments and CpG sites among different tissues. Results:1) Immunohistochemical analysis showed that with the development of esophageal squasous cell carcionma (ESCC), the protein expression level of C20orf54gene was altered from normal expression to partial loss or total loss of expression, with statistically significance except for calnexin (P<0.05). RT-PCR results suggested that the change in mRNA expression level of C20orf54gene was also significant between esophageal cancer biopsy and normal esophagus epitum, and correlated with the protein expression described above. From the analysis of the association of clinical prognostic parameters for esophageal cancer with candidate gene expression, we found a reverse correlation of the downregulation of C20orf54protein expression with tumor differentiation state (P<0.05) but the lymphnode metastasis wasn’t had a statistically significance (P>0.05);2) The average blood concentration of riboflavin was (2.6468±1.3474ng/ml) in ESCC patients lower than control group (4.2960±3.2293ng/ml, P=0.015). A tendency of decreasing blood riboflavin level was found to be associated with ESCC development. However, no significant difference was observed among other parameters. We also analyzed the relationship between the blood riboflavin level and expression of the C20orf54gene in ESCC. A positive association was found between changes in the blood riboflavin levels and changes in C20orf54protein expression in Kazak ESCC patients;3) According to the data generated by Sequenom Mass ARRAY DNA detection, total methylation level of target fragments of C20orf54genes tissues was higher in esophageal cancer tissues than normal tissues with statistical significance (P<0.05). The single-site methylation level analysis showed that the single-site methylation was significant for7CpG sites among all CpG sites of C20orf54genes in esophaeal cancer tissues compared to the normal. Comparative analysis of target fragment methylation and the data of altered protein expression described in "part I" of this study, we found a reverse correlation of C20orf54gene methylation level with the protein expression. Conclusion:1) The transcription downregulation and loss of protein expression of C20orf54was closely associated with development of the esophageal squasous cell carcionma in Kazak patients, and may become important candidates for the establishment of tumor molecular marker profile;2) The expression of C20orf54and plasma riboflavin levels in Kazak esophageal squamous cell carcinoma, and identified down-regulated expression of C20orf54mRNA and protein as being closely related to the progression of Kazak esophageal squamous cell carcinoma and also found a positive relationship between plasma riboflavin levels and C20orf54protein expression as well as between plasma ri-boflavin levels and development of Kazak ESCC;3) The target CpG fragments of C20orf54gene was CpG site methylated at various degrees in esophageal cancer cells, may most probably the cause of the transcription downregulation or protein loss expression regulated at epigenetic level. |
PDF Full Text Request