Study On Expression Changes And Significance Of LMP/TAP Genes In HL-60Cell After Induced Differentiation

Background The mechanism of immune escape is a hot topic in tumor molecular biology research. It has been found that HLA-I molecules are not expressed or expressed at low levels in a variety of tumors, which result that tumor cells can escape the body’s immune surveillance. Low molecular weight protein (LMP) includes two subunits:LMP2and LMP7, it is able to hydrolyze tumor antigens to suitable peptides. Transport of antigenic peptides across the endoplasmic reticulum (ER) membranes is mediated by the transporter associated with antigen procession (TAP) which includes TAP1and TAP2. These peptides bind to MHC class I molecules in ER and they are presented and expressed in cells surface. CD8+T cells recognize antigenic peptides complexes and cause cytotoxic T cell response. Reduction or loss of TAP1、 TAP2、 LMP2or LMP7may well limit the effectiveness of antigen presenting by MHC class I molecules. In the study of LMP/TAP genes associated with tumor, Many scholars conducted a extensive discussion about the correlation between LMP/TAP expression imbalance and tumor’s degree of malignancy、metastasis and survival in solid tumors, such as colon cancer、lung cancer、renal cell carcinoma、esophageal cancer breast cancer and primary melanoma. The series of studies show that TAP or LMP have expression defects in different sub-categories and levels in these cell lines. However, the correlation analysis about LMP/TAP and malignant disease in blood system are reported occasionally.Objective Based on the theoretical background talked above, this project established cell models of ATRA and PMA induced HL-60cell differentiation, Research the expression and significance of LMP/TAP which contains the subunits of TAP1、TAP2、LMP2and LMP7. We meant to learn role of LMP/TAP in the incidence and prognosis of acute leukemia, which can break a path for leukemia treatment.Methods Take logarithmic phase HL-60cell as experimental subject by adding ATRA and PMA for72hours, incubating in37℃,5%CO2saturated humidity, a negative control group was established at the same time.Cell morphology was observed by Wright-Giemsa staining. The expression of CD11b and CD14was measured by flow cytometry. TAP1、TAP2、LMP2and LMP7mRNA were measured by Reverse Transcription-PCR, TAP2proteins were determined by Western blot. Dates are expressed as mean and standard deviations. Comparisons between the treatments and control were performed using the t-est and one-way anova. A P-value<0.05was considered to be statistically significant.Results0.8μM ATRA and50nM PMA treated for72hours can obviously restrain HL-60cell proliferation, and in addition, the cells appeared apparent differentiation tendency under the inverted microscope, which growed like mature granulocyte and mononuclear macrophages respectively; The expression level of cell surface markers CD11b and CD14significantly increased those were detected by flow cytometry(P<0.05); cell differentiation models were detected by Reverse Transcription-PCR:there were significant changes in the express of TAP1、TAP2、 LMP2、and LMP7mRNA(P<0.05); protein expression level of TAP2was obviously decreased which was determined by Western blot(P<0.05).Conclusions LMP/TAP expression levels were increased significantly in cell models of ATRA and PMA induced HL-60cell differentiation, which shows that TAP or LMP may have expression defects in different sub-categories and levels of in acute myeloid leukemia cells; LMP/TAP expression levels were increased significantly in cell models of ATRA and PMA induced HL-60cell differentiation. It proved that leukemia-derived mononuclear cells have certain immune function phenotype.