The Research Of Antigen Processing Machinary Components Methylation With Xinjiang Kazakh Esophageal Squamous Cell Carcinoma Development

Aim of study: Esophageal cancer is one of the most common malignancies. Thereare about16²0million people died of the disease each year in china, morbidity andmortality ranking the first in the world. Xinjiang is one of the high-risk areas foresophageal squamous cell carcinoma （ESCC）, in which Kazakh people have the highestincidence of2to3times higher than other minorities. The mortality of Kazakh ESCCoccupied the first place in all minorities and was2.3times higher than the nationalaverage rate. As a common and frequently occurring disease in Xinjiang, ESCC hasseriously affected the quality of life in people who live in the region. Because kazakhpeople live in a distinctive environment and have unique living habits, so the populationis a good object for study. To investigate the reason of ESCC and establish tumormolecular marker system is very important for diagnosis, clinical treatment andprevention of ESCC in Kazakh population. Taking Xinjiang Kazakh ESCC resources tocarry out basic research of ESCC is of great significance. Epidemiological studiesrevealed that the incidence of ESCC was associated with multiple factors, many geneswere involved in the different carcinoma development stage. Investigations found thatthere was a familial aggression phenomenon in high-incidence people and the immunefactors played a important role in the incidence of ESCC. However, the definitemechanism is still not clear. Numerous studies show that low expression or absence ofhuman leukocyte antigen class I （HLA-I） can cause tumor cells escaping the host immunesurveillance, which is closely related with tumor development. Human leukocyte antigenclass I is the basis of the immune response system generation and transmission. It’sprimary function is deriving antigen peptides from tumor cells and presenting it to cytotoxic T lymphocytes, by which realizing self antigens recognition and immune surveillance.This endogenous antigen peptide presenting process must be assisted by antigenprocessing machinery （APM） components to achieve the normal function of the HLA-I.Antigen processing machinery components are a group of antigen presenting proteinsresponsible for assisting the HLA-I molecular assembly, loading and presentingendogenous antigen peptide. Defects in antigen processing machinery componentsexpression may provide tumor cells with a mechanism to escape from recognition anddestruction by HLA class I antigen-restricted tumor antigen-specific cytotoxic T cells.DNA methylation is an important mechanism in silencing the expression of genes. Thefunctional relevance and potential clinical significance of these epigenetic alterationshave been addressed. More recently, epigenetic events associated with tumordevelopment and progression have been found to underlie changes in HLA antigen, APMcomponent, co-stimulatory molecule expression in malignant cells, including cervicalcancer, bladder cancer, head and neck cancer. However, expression and functionalproperties of HLA-I antigens and APM components in malignant cells of ESCC havebeen investigated in a limited extent. To overcome the limitation in the present study, wehave selected Xinjiang Kazakh ESCC species, investigated the methylation level ofhuman leukocyte antigen class I and antigen-processing machinery components （TAP1,TAP2, LMP2, LMP7, ERAP1, Tapasin, and ERp57） as well as functional proteinexpression in25primary esophageal squamous cell carcinomas and in25matchedhealthy esophageal tissues. We want to find out the association of APM componentsmethylation and development of ESCC in Xinjiang Kazakh people through epigeneticangle. Methods:（1）ECa109cells were treated with bisulfate sequeneing PCR （BSP）analysis to evaluate the CpG island methylation status of HLA-B, TAP1, TAP2, LMP2,LMP7, ERAP1, Tapasin and ERp57gene. Using the resources of the online database ofthe human genome project, genetic information was obtained from the genbank database,and specific PCR primers of CpG island fragment were designed through specializedsoftware scanning gene promoter regions. The methylation status of specific target geneswas gained by PCR amplification, cloning and sequencing.（2）50cases of fresh KazakhESCC species and matched healthy esophageal tissues were collected and the DNA wasextracted. HLA-B TAP2LMP7Tapasin and ERp57genes were choosed as candidategenes screened out from the previous step study. MassARRAY technology was employedto analyze the methylation levels of methylated CpG sites in the gene promoter fragmentsbetween ESCC and normal control.（3）The HLA-I, LMP7, Tapasin, and ERp57antigens were detected by immunohistochemical SP method in50cases of esophageal species.The association between the downregulated expression of HLA-I, LMP7, Tapasin,ERp57antigens and clinicopathological features of ESCC was analyzed by theKruskal-waillis test. Correlation of LMP7, Tapasin, ERp57genes promoter methylationlevels with protein expression levels was identified by the Spearman rank correlationcoefficient. The data was processed by SPSS16.0statistical analysis software,“P” valuewith bilateral, signincance at a=0.05. Results:（1） CpG islands in promoter region werehypermethylated in the Eca109cells, methylated CpG sites were found to differentdegrees in HLA-B, TAP2, LMP7, Tapasin and Rp57genes. The proportion of methylatedCpG sites in all CpG sites of each gene were10/14,8/8,2/22,5/12,18/18respectively.No methylated CpG site was found in TAP1, LMP2, ERAP1gene promoterregions.（2）According to MassARRAY methylation level detecting, the methylationlevels of LMP7,Tapasin and ERp57genes in ESCC species were higher than matchedhealthy esophageal tissues （P＜0.05）, while there were no significant difference inHLA-B and TAP2genes （P＞0.05）. Single CpG sites methylation level analysis showedthat: CpG₅, CpG₉, CpG₂0, CpG₂1, CpG₂2in the LMP7gene promoter region;CpG₁, CpG₆in the Tapasin gene promoter region; CpG₁in the ERp57gene promoterregion were statistically different between ESCC and adjacent normal tissues. Analysisbetween LMP7, Tapasin, ERp57gene methylation levels and clinicopathological featuresfound that: LMP7, ERp57genes promoter methylation levels in stage I and stage II grouppatients are higher than stage III and stage IV group; LMP7, ERp57genes methylationlevels in moderately and poorly differientied carcinomas are higher than welldifferientied tissues; ERp57gene methylation level in tumor vascular invasion group ishigher than without vascular invasion group （P＜0.05, respectively）.（3）The expressionof HLA-I, LMP7, Tapasin and ERp57antigens were reduced in tumor lesions comparedwith matched healthy tissues, the difference was statistically significant respectively（P＜0.05）. Downregulated expression of HLA-I is closely associated with tumordifferentiation, depth of tumor invasion; Downregulated expression of LMP7is closelyassociated with tumor differentiation, lymph node metastasis, depth of tumor invasion,vascular invasion, TNM staging; Downregulated expression of Tapasin is closelyassociated with tumor differentiation, depth of tumor invasion; Downregulatedexpression of Erp57is closely associated with lymph node metastasis, depth of tumorinvasion, vascular invasion, TNM staging （P＜0.05, respectively）. Analysis by theSpearman rank correlation coefficient showed that LMP7, Tapasin and ERp57protein expression level were significantly correlated with that of HLA-I （r=0.823, r=0.721andr=0.378, P＜0.05, respectively）. The methylation levels of LMP7and ERp57genepromoters were negatively correlated with protein expression levels （P＜0.05,respectively）. Conclusion:（1）The CpG islands in promoter fragments of HLA-B, TAP2,LMP7, Tapasin and Rp57genes existed different degrees of mehthylation in Eca109cellline. This may be specific epigenetic changes in esophageal squamous cell carcinoma. Itprovides a important mechanism for esophageal cancer development.（2）In XinjiangKazakh ESCC tissue specimens, the methylation levels of LMP7, Tapasin and ERp57genes enhanced abnormally and are closely associated with clinicopathological features.It should be noted as high risk factors of ESCC development in Xinjiang Kazakhpopulation. Aberrant hypermethylation of CpG islands （CpG₅, CpG₉, CpG₂0,CpG₂1, CpG₂2in the LMP7gene promoter region; CpG₁, CpG₆in the Tapasingene promoter region; CpG₁in the ERp57gene promoter region） may be the key sitesof regulating the gene methylation. These CpG sites can affect multiple cellular functionsby silencing transcriptional process.（3）HLA-I, LMP7, Tapasin and ERp57antigens playthe critical role in tumor cell immune surveillance and killing, they can prevent theoccurrence of esophageal cancer and produce a protective effect on the body. Themethylated CpG sites in LMP7and ERp57gene promoter can affect the level of proteinexpression, which play a significant role in hindering HLA-I molecules assembly andpresenting, the latter changes result in the HLA-I down or missing in the surface of tumorcells. The whole process cause tumor cells escape from body’s immune surveillance andlead to carcinoma development. This maybe is the epigenetic mechanism of XinjiangKazakh ESCC.