Investigation Of The Mechanism Of Abnormal HLA Molecules Expression In Hepatocellular Carcinoma

Human Leukocyte Antigen (HLA) is essential in anti-tumor immune response. HLA molecules bind antigenic peptides generated by antigen processing machinery and present these peptides to T cell receptor on the cell surface. The recognition of these peptides by CTLs triggers a serie of events that can result in tumor cell lysis. In the present study, the expression of genes involved in HLA class I antigen processing and presenting pathway were investigated in Hepatocellular carcinoma (HCC) cell lines and tissues. The purpose of this study is to explore the molecular mechanisms underling the abnormal HLA class I expression in HCC and the relationship between the HLA class I expression and its clinical significance.Using a panel of antibodies, the expression of HLA class I molecules was studied in HCC tissues by immunohistochemical technique. Our research also extended to the analysis the molecular mechanisms of aberrant HLA class I expression in different HCC cell lines. Our results include the following several parts:1) We investigated HLA molecule expression in HCC tissues based on the long-term collaboration with Qidong Liver Cancer Research Institue in Jiangsu province. The result indicated that the expression of most molecules that involved in HLA class I precessing and presenting pathway were upregulated in 165 HCC tissues except LMP2, at the same time HLA class I A locus exhibit the highest percentage of upregulation (54%). In addition, various HLA molecules were co-upregulated in most samples, which demonstrated that some upstream molecules might activate HLA class I presenting pathway in HCC tissues. This was confirmed by the results of investigation of molecules involved in HLA class I presenting pathway in fresh HCC tissues.2) The expression of surface HLA class I complex was investigated by flow cytometry in eight hepatocellular carcinoma cell lines (QGY7701, BEL7402, BEL7405, QGY7703, SMMC7721, BEL7404, HepG2, Q05), one normal liver cell line L-02 and one cell line QSG7701, which was derived from peripheral tissue of liver carcinoma. Either upregulated or downregulated HLA class I expression were found in different cell lines. We also found upregulated HLA-A heavy chain in high HLA class I expression cell lines QGY7701 and BEL7405 and lacked the expression of LMP2 and LMP7 in low HLA class I expression cell line BEL7404 by real-time PCR. Next step would be studying of the molecular mechanisms under these abnormal HLA class I expression.3) Upregulated HLA-A heavy chain might be the main cause of HLA class I upregulation in HCC cell lines QGY7701 and BEL7405. To identify regulatory elements that involved in HLA-A gene transcription, a series of truncated HLA-A promoter-reporter constructs were transfected into HCC cell lines with high and low levels of endogenous HLA-A respectively. These experiments showed that the regulation of HLA-A expression in HCC cell lines was mostly mediated by interferon stimulated response element (ISRE) located at 150bp upstream of the transcription initiation site. Electrophoretic mobility shift analysis (EMSA) further demonstrated that binding of interferon regulatory factor 1(IRF-1) to the HLA-A ISRE was increased in high HLA-A expression cell line. By using RT-PCR and western Blot assay, we also showed by that the expression of IRF-1 was upregulated in high HLA-A expression cell line at both the protein and mRNA levels. Furthermore, we investigate the expression of HLA-A and IRF-1 in 10 pairs of HCC and corresponding normal liver tissues. The enhanced expression of IRF-1 was fully concordant with HLA-A upregulation. In conclusion, our results indicate that IRF-1 acted as an important regulator for constitutional upregulation of HLA-A antigen in HCC. However, the correlation between HBV infection and the activated signal pathway of HLA-A upregulation in HCC need further study.4) This study also investigated the involvement of those components in the downregulation of HLA class I expression in human hepatocellular carcinoma cell line BEL7404. BEL7404 cells down regulated the expression of HLA class I antigen and lacked the expression of LMP2 and LMP7. Interferon (IFN)-γtreatment could increase the expression of LMP2 but not LMP7. LMP2-transfected BEL7404 cells or LMP2 and LMP7-cotransfected cells restored surface HLA class I expression while LMP7-transfected cells did not.