Effects Of St On HLA Class I Antigen And Tap1 In Ges-1 Cells And The Regulation Mechanisms

Objective:Sterigmatocystin (ST) is the toxic fungal metabolite produced by Aspergillus versicolor and Aspergillus nidulansetc. It was proven that ST is a carcinogenic and mutagenic mycotoxin. The contamination of ST is quite common in human food or in animal feed, and also it is one of the most abundant food-contaminating mycotoxins in the high incidence area of gastric carcinoma in China. In order to explore the possible relationship between the high contamination of ST and the high incidence of gastric cancer, we have done a series of research focusing on ST. We found that ST could induce mutation of p53 and Ki-ras gene in human fetal lung fibroblast cells in vitro; induce the formation of lung adenocarcinoma and dysplasia of glandular stomach in mice, respectivly. These results suggested that ST have certein carcinogenic and mutagenic effects. In addition, ST was also found to have the immun toxiology on mice and human immune cells. For example, ST could inhibit the expression of IL-2 and IFN-γin mouse spleen cells; inhibit the expression of IL-12 in murine peritoneal macrophages; induced apoptosis in Human peripheral blood monocular cells (HPBMc) and inhibit IL-2 secretion; reduce the expression of HLA-I molecules and TAP1 of HPBMc. These results indicated that ST have certain negative effects on immune function.Human leukocyte antigen-I (HLA-I) plays an important role in immuno-surveillance against the tumorigenes and progression of tumours. HLA-I distributes on the surface of all the nucleated cells, it can active the CD8+ T cells to proliferate and to destroy the abnormal target cells through the complex of HLA-I with specific peptides. Therefore, HLA-I molecules has a critical role against the virally infected and malignantly transformed cells. Transporter associated with antigen presentation (TAP, a heterodimer of TAP1 and TAP2) is a key factor in the HLA-I antigen presentation pathway, including the process of assembly, maturation, and transit through the Golgi and angtigen presentation of HLA-I. TAP-deficient cell lines showed reduced assembly and transit through the Golgi of mature HLA-I molecules compared with normal cells. Transfection with TAP1 gene can restore the HLA class I expression and the CTL-mediated immunosurveillance correspondingly in some tumor cells with low level of TAP1. These results presented evidence that loss or decrease in TAP1 protein abundance resulted in loss or decrease in HLA-I protein expression, and these changes could permit cells to avoid CTL-mediated immunosurveillance.Downregulation of TAP1 and HLA-I molecules has been reported in numerous human tumors, such as melanoma, renal cell carcinoma, colorectal cancer, cervical cancer, small cell lung cancer and breast cancer, and low level of TAP1 and HLA-I molecules was associated with the clinicopathologic parameters including grade, stage, lymph node metastasis, CD3+/CD8+ lymphocyte infiltration and prognosis of the patients. Besides the role of TAP1 and HLA-I molecules in tumors, some carcinogenic environmental factors such as tobacco and mycotoxins (Fumonisin B1, DON) also could inhibit the expression of TAP and HLA-I molecules in some types of non-tumor cell lines. The reduction in membrane HLA class I could permit cells to avoid immunosurveillance, and the HLA class I-reduced population of cells would then be able to undergo oncogenic changes and form tumors without being detected by the immune system.Our previous study have detected that ST could reduce the expression of HLA-I molecules and TAP1 protein in human peripheral blood monocular cells as well as the HLA-I abundance in normal human esophageal epithelial cells. Given the importance of HLA-I in the immune surveillance and the severe contamination of ST in the high incidence area of gastric carcinoma in China, so in this study, we select the immortalized gastric epithelium cell line (GES-1) to investigate the effects of ST on the expression of HLA-I molecules and TAP1. Next, we constructed the human TAP1 expression vector and transfected it into cells to explore the effects of TAP gene on HLA-I expression and the regulation mechanisms. We proposed that the results could provide clues on the effects of ST in tumorigenes and progression of tumours.Methods:1 Cell culture and treatment1.1 GES-1 cell culture and treatmentHuman gastric epithelial cell line GES-1 was cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100μg/ml) in an atmosphere of 5% CO2/95% air at 37℃. Cells in logarithmic growth phase were randomly divided into control group, solvent control group and different concentration of ST treated groups. ST was diluted in DMSO and left the final concentration to be 100μg/L, 500μg/L, 1000μg/L and 2000μg/L, respectively. Control and solvent control cells were incubated for the same time with normal saline or DMSO. Cells were harvested by a solution containing 0.25% trypsin at the indicated time below.1.2 HPBMc cultureHuman peripheral blood monocular cells (HPBMc) were isolated from 200 mL heparinic acid antiagglutinated fresh venous blood of healthy donors by a Ficolly-Hypaque density gradient centrifugation. HPBMc were then cultured in RPMI 1640 medium supplemented with 10% new-born calf serum (NBCS), streptomycin (100μg/mL), penicillin (100U/mL) and phytohemagglutinin (PHA, 300μg / mL) at 37℃5% CO2 for 48h. Total RNA of HPBMc was used to construct the human TAP1 expression vector.2 semi-RT PCR2.1 Total RNA extractionTotal RNA was extracted from cells using guanidinium isothiocyanate. The integrity of RNA was identfied on 1% agarose gels, and the concentration was mesured by UV Spectro- photometer.2.2 RT and PCR reaction1ug of RNA was reverse transcribed into cDNA using reverse transcriptase, and the cDNA was used in PCR reaction. The following primers were used: TAP1 Sense: 5'-GCTCAGCCGATACCTTCA-3'; Anti-sense: 5'-CCACTTTCAGCAGCATACC-3'. HLA-A Sense: 5'-CCTACGACGGCA AGGATTACA-3'; Anti-sense: 5'-ACATCACGGCAG CGACCA-3'. HLA-B Sense:5'-CTACGACGGCAAGGATTAC-3'; 5'-GGTGGACTGGGAAGACG -3'. HLA-C Sense: 5'-GCAGTTCGTGCGGTTCG-3'; 5'-GTCTCCTTCC CGTTCTCC-3'.β2m Sense: 5'-TCAT CCATCCGACATTG-3'; Anti-sense 5'-GCAGGCATACTCATCTTTT-3'. GADPH Sense: 5'-GGAAGGTGAAG GTCGGAGT-3'; Anti-sense: 5'-CCTGGAAGATGGTGATGGG-3'. The amplified products were analyzed on 1.5% agarose gels with ethidium bromide and visualized by a UV transilluminator. GAPDH used as the endogenous control in each test, and the ratio of target gene with GAPDH represented the level of target gene expression. In our study, the HLA-A, HLA-B, HLA-C,β2m and TAP1 expression at mRNA level in GES-1 cells were determined using RT-PCR assay.3 Western blotFifty micrograms whole cell protein lysates were electrophoresed through 15% SDS–PAGE gel. Proteins were electrotransferred to PVDF membrane, blocked with 5% nonfat milk in TBST and probed or re-probed with the appropriate primary antibodies and secondary antibodies, and then detected by the ECL system. Band density was quantified using Snygene-Image Systems and normalized toβ-actin.4 Construction of human TAP1 expression vector4.1 Construction of the TAP1 plasmid Human full-length TAP1 gene was obtained from human peripheral blood mononuclear cells by RT-PCR reaction, then TAP1 DNA was inserted into pcDNA3.1/V5-HisB vector to get the TAP1 expression vector (pcDNA3.1/V5-His- TAP1) by recombination technology including digestion with restriction enzymes, ligation and transformation. The vector was proved to be positive by restriction enzyme digestion and PCR assay.4.2 Sequencing and validationThe positive clone was sequenced by Takara company, and the sequencing was blasted with that in Genebank (L21204) to ensure the sequence fidelity.5 Cell transfectionTwelve hours prior to transfection, GES-1 cells were plated into 6-well plates, keep the cell density to be around 60% to 80%. Cells were divided into three groups: control group, pcDNA3.1 vector group(empty vector) and pcDNA3.1/V5-His-TAP1 group (p-TAP1). Cells were transfected according to the instruction of LipofectamineTM 2000. 24hs after transfection, cells were collected to evaluate the efficiency of transfection in GES-1 cells by RT-PCR, Western blot and flow cytometry (FCM).GES-1 cells were transfected with pcDNA3.1 vector (ST+vector) or pcDNA3.1/V5-His-TAP1 (ST+p-TAP1) after 1000μg/L ST pre-treatment. 24 h after transfection, cells from the two groups were then harvested to clarify the effects of TAP on HLA-Iexpression by ST treatment.6 Statistical analysisAll results were expressed as mean±S.D. from each group and the statistical analysis was performed with one-way analysis of variance (ANOVA). All statistical analysis were calculated by SPSS 13.0 statistical software. The P-values less than 0.05 were considered to be significant.Results:Part 1 The effects of ST on the expression of HLA-I and TAP1 in GES-1cells in vitro1 The effects of ST on HLA-I expression1.1 The effects of ST on the expression of HLA-I heavy chain (HLA-A,B,C) at mRNA levelCompared to the solvent control, the level of HLA-A, HLA-B, HLA-C mRNA in ST treated groups was decreased gradually with the concentration ranging from 100μg/L to 2000μg/L. The most significance down-regulation of HLA-A and HLA-B mRNA was seen in 1000μg/L and 2000μg/L ST treatment groups (P<0.05). While the level of HLA-C mRNA was decreased significantly in 2000μg/L ST treatment group (P<0.05). 1.2 The effects of ST on the expression of HLA-I light chain (β2m) at mRNA levelThere was no significant difference in the expression ofβ2m mRNA between the control group and ST treatment groups.1.3 The expression of HLA-I protein detected by Western blot The molecular weight of HLA-I is 43~45KD. The positive band could be detected in cells from all groups including control, sovelt control and the ST treatment groups. The results showed that the expression of HLA-I protein was significantly decreased in 1000μg/L and 2000μg/L ST treatment group compared with control group(p<0.05).2 The effects of ST on TAP1 expression2.1 The effects of ST on the expression of TAP1 at mRNA levelThere was no significant difference in TAP1 mRNA expression between control and 100μg/L, 500μg/L ST treated groups. However, the expression of TAP1 mRNA in 1000μg/L and 2000μg/L ST treatment groups was significantly decreased as compared with that in control and solvent groups (P<0.05).2.2 The effects of ST on the expression of TAP1 proteinThe molecular weight of TAP1 is 74KD. The positive band could be detected in cells from all groups. In comparison with the solvent control, TAP1 protein was reduced in ST-treated GES-1 cells, with the most significance inhibition role seen in 1000μg/L and 2000μg/L ST treatment groups (p<0.05).Part 2 The effects of TAP1 transfection on HLA-I expression in GES-1 cells by ST treatment1 Construction of human TAP1 expression vectorFull-length TAP1 (2247bp) gene was obtained from human peripheral blood mononuclear cells by RT-PCR reaction. Restriction enzyme digestions were performed by incubating TAP1 gene and pcDNA3.1 vector with EcoR I and Xho I enzymes, respectively. DNA ligation was performed by incubating TAP1 DNA fragments with appropriately pcDNA3.1 vector. Then the product of ligations was transformed into DH5αcompetent cells. Pick the colony of bacteria harboring the plasmid and target DNA to be detected by restriction enzyme digestions and PCR reaction. The results showed that the colony was positive one. After that, DNA sequencing was run by the company of Takara, and the sequencing result was blasted with the published sequence of TAP1 from Genebank. Through all the steps above, we finally obtained the human TAP1 expression vector successfully.2 The efficiency of transfection with TAP1 expression vector in GES-1 cellsGES-1 cells were collected 24hs after transfection. RT-PCR analysis showed that TAP1mRNA abundance was increased obviously in p-TAP1 cells compared with that in control and empty vector cells (p<0.05). Western blot assay further confirmed that the over-expression of TAP1 protein after p-TAP1 transfection. So we concluded that GES-1 cells could be used as the target cell for transfection with the plasmid we had.3 The effect of TAP1 transfection on the expression of HLA-I in GES-1cells pretreated with ST for 24hs3.1 The effects on HLA-A, HLA-B, HLA-C andβ2m mRNA expression in GES-1 cells by ST pretreatment combined with TAP1 transfection We firstly detected the change of HLA-A, B, C andβ2m mRNA in GES-1 cells without ST treatment after TAP1 transfection, the results showed that the expression of HLA-A, B, C mRNA were all increased significantly by TAP1 transfection when compared with the control and empty vector group (p<0.05), while there was no any change on theβ2m mRNA expression. We next pretreated the cells with 1000μg/L ST for 24hs followed by TAP1 transfection to see the changes of these HLA-I molecules. Cells in ST combined with TAP1 transfection group showed higher abandance of HLA-A, HLA-B and HLA-C mRNA than that in ST combined with empty vector group (p<0.05), and also there was no effect on theβ2m mRNA expression could be found between the two groups. All the results suggested that TAP1 transfection could relieve the inhibition role of ST on HLA-A, B, C mRNA in GES-1 cells. 3.2 The effect on the expression of HLA-I protein in GES-1 cells by ST pretreatment combined with TAP1 transfectionWe detected the change of HLA-I protein in GES-1 cells without ST treatment after transfection. Western blot assay showed that the expression of HLA-I protein was increased significantly by TAP1 transfection compared with the control and empty vector group (p<0.05). Then cells were left pretreated with ST 1000μg/L for 24hs and followed by transfection with TAP1 or empty vector. The HLA-I protein of cells in ST combined with TAP1 transfection group was upregulated statistically when compared with that in ST combined with empty vector (p<0.05). Consistant with the change at mRNA level, the results confirmed that TAP1 transfection could relieve the inhibition role of ST on HLA- I molecules in GES-1 cells.Conclusion:1 ST could inhibit the expression of HLA-A, B,C both at mRNA and protein level in GES-1 cells depending on the different concentration. The most significantly inhibition role of ST was seen in 1000μg/L and 2000μg/L ST treated groups.2 ST at 1000μg/L and 2000μg/L could significantly decrease the expression of TAP1 at mRNA level and protein level in GES-1 cells in vitro.3 Human TAP1 expression vector (pcDNA3.1/V5-His-TAP1) was successfully constructed. The efficiency of transfection was high enough to study the effects of TAP1 in GES-1 cells.4 The expression of HLA-I molecules could be upregulated by TAP1 transfection in GES-1 cells.5 TAP1 transfection could relieve the inhibition role of ST on HLA-I molecules in GES-1 cells, it suggested that the possible mechanism of the inhibition effect of ST on HLA-I molecules is mediated by the downregulation of TAP1 in GES-1 cells.