| The classical human leukocyte antigen (HLA) class I genes, including HLA-A,-B, and-C loci, are the most polymorphic system. The products of HLA class I are present on almost all the nucleate cells, which play a central role in both innate and adaptive immunity by providing ligands for Cytotoxic T-Lymphocytes (CTL) as well as Natural Killer (NK) cells.CTL and NK cells are essential in tumor surveillance. TCRs on CTLs recognize antigenic peptide combined with classical HLA I molecules on the target cells, which is known as "MHC restriction". Its common that cancer cells escape from CTLs by down-regulating HLA class I expression. Meanwhile, the expression of classical HLA I molecules inhibits NK cells activation mainly through recognition of killer cell immunoglobulin-like receptors (KIRs) on NK cells. Hence cells which fail to express self HLA class I molecules are vulnerable to the attack of NK cells.Most of tumor cells escape from CTLs by down-regulating HLA class I expression but it is opposite in liver cancer. Different from the peripheral blood and other organs, both NK cells and CTL account for about 30% of the leucocytes in human liver. So tumor cells in the liver have to develop unique mechanism to escape the surveillance from both NK cell and CTLs. Both HLA and KIR gnes are highly polymorphic, and different HLA class I antigens encoded by different HLA I alleles recognize different KIR molecules, which leads to different NK cells functions. We hypothesize that liver tumor cell may selectively modulate expressions of HLA class I alleles to escape from killing of both NK cells and CTLs.In this study, we designed HLA allele-specific primers after genotyping the HLA and KIR genes, and compared classical HLA class I mRNA levels encoded by different alleles on hepatoma carcinoma tissues with that on autologous adjacent liver tissue in 72 HCC tissue samples. The results are as follows:1. HLA-A、-B、-C loci of patients with primarty hepatocellular carcinoma (40 patients for HLA-A,31 patients for HLA-B, and 72 patients for HLA-C) were genotyped by PCR-Sequencing-Based Typing(PCR-SBT) method.12 loci of KIR genes in all the HCC patients were detected by PCR-Sequence Specific Primer(PCR-SSP) method.2. For HLA-A and-C loci, allele-specific primers were designed and tested (20 pairs of primers for HLA-C and 4 pairs for HLA-A loci).3. mRNA levels of 80 HLA-A alleles,62 HLA-B alleles, and 144 HLA-C alleles were quantified and compared between HCC tissue with that on autologous adjacent liver tissue by real-time quantitative PCR. The results show that comparing to their adjacent liver tissues, 36.11% of HLA-A alleles are up-regulated in liver cancer tissue,47.92% of them are constant, while 15.97% are down-regulated; For HLA-B alleles,26.43% are up-regulated,43.57% are constant and 30% are down-regulated; For HLA-C alleles,27.78% are up-regulated,41.67% are constant,while 30.55% are down-regulated.4.45.46% of HLA-Bw4 alleles(inhibitory ligand for KIR3DL1) are upregulated on HCC tissues, which is higher than HLA-Bw6 alleles (non-ligand for KIR3DL1) do(20.95%) in KIR3DL1+ individuals(P=0.006).The HLA-C*03 molecules which are up-regulated are more frequent in patients with KIR-BX genotype than that in patients with KIR-AA genotype (P=0.011).In conclusion, we have designed HLA allele-specific primers and quantified the mRNA expression level of classical HLA class I alleles in 72 liver cancer tissue and their adjacent non-cancer tissue samples. Our results indicate that selectively modulate expressions of HLA class I alleles are present on liver tumor cells, which might underlie the escape of tumor cells from the surveillance of both NK cell and CTLs in liver.The quantification method on allele-specific HLA I mRNA we have built in this project will be useful in the research on virus immunity, tumor immunity, autoimmune and other fields which are related to HLA expression. |
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