Expression And Clinical Significance Of Inhibitory B7 Family Molecules In Acute Myeloid Leukemia

Objective At present,the search for new immune targets,the application of specific protein inhibitors and immunotherapy have become the focus of research on acute myeloid leukemia(AML).Inhibitory B7 family molecules was abnormally expressed in solid tumors and hematological malignancies,and played an important role in the anti-tumor immune response.In the early stage,our research group found that the expression of B7-H3 in hematological malignancies was up-regulated and promoted the proliferation,invasion and metastasis of tumor cells.However,the expression and clinical significance of B7-H3 and its isoforms in AML have not been clearly defined,and methylation of B7-H3 promoter CpG island in AML and its clinical significance have not been reported.The expression characteristics and clinical significance of inhibitory B7 family molecules B7-H1,B7-H4 and B7-H6 in AML have not been fully elucidated.It is not clear which molecules in different types of AML might serve as potential tumor markers and therapeutic targets.In this study,the expression characteristics of B7-H3 and its isoforms in AML were detected by RT-PCR and flow cytometry,and the unmethylation level of B7-H3 in AML was detected by RQ-MSP.In addition,flow cytometry was used to detect the expression characteristics of membrane protein B7-H1,B7-H3,B7-H4 and B7-H6 in AML,providing a basis for further research on the role of B7 family molecules in AML and the selection of targets for targeted therapy.Methods1.The expression of B7-H3 and its isoforms in AML cell line was detected by RT-PCR;2.RT-PCR was used to detect the expression of B7-H3 and its isoforms in blast cells of AML patients and the control group;3.Flow cytometry was used to detect the expression of membrane protein B7-H3 and its isoform 4IgB7-H3 in AML cell lines;4.Flow cytometry was used to detect the expression of blast cell membrane proteinVB7-H3 and its isoform 4IgB7-H3 in de novo AML patients and control group;5.RQ-MSP was used to detect the unmethylation level of B7-H3 in de novo AML patients and the controls;6.The correlation between the unmethylated level of B7-H3 and the expression of B7-H3 isoforms was analyzed;7.Flow cytometry was used to analyze the expression of membrane protein B7-H1,B7-H3,B7-H4 and B7-H6 in AML cell lines;8.Flow cytometry was used to analyze the expression of membrane protein B7-H1,B7-H3,B7-H4 and B7-H6 in different bone marrow blood cells of AML patients and controls;9.Flow cytometry was used to analyze the expression of membrane protein B7-H1,B7-H3,B7-H4 and B7-H6 in bone marrow blast cells of de novo AML patients and controls.Results1.Expression of B7-H3 and its isoforms at mRNA level There are two B7-H3 isoforms in AML cell lines �� 4IgB7-H3(4Ig)and 2IgB7-H3(2Ig).Except for SKM-1,4Ig expression was dominant in AML cell lines,while 2Ig expression was relatively weaker.SKM-1 mainly expressed 2Ig,while the expression of 4Ig was relatively weaker compared with that of 2Ig(P=0.0224).RT-PCR results revealed that both expression rate and expression abundance of 2Ig among the 37 AML patients were significantly higher than those of 14 controls(P=0.009,P=0.0025).Meanwhile,compared with patients with high expression of 2Ig,patients with low expression of 2Ig had a decreasing trend in age and a better prognosis indicated by chromosome karyotype.Kaplan-Meier analysis suggested that the overall survival of AML patients with high expression of 2Ig was significantly lower than that of patients with low expression of 2Ig(P=0.014).Cox regression multivariate analysis indicated that 2Ig was an independent risk prognostic factor in AML(P=0.009).2.Expression of membrane protein B7-H3 and its isoform at the protein level Flow cytometry was used to detect the expression of membrane protein B7-H3 and its isoform 4Ig in 8 AML cell lines.It was found that K562 and SKM-1 mainly expressed2 Ig,while HL-60,THP-1 and HEL dominantly expressed 4Ig.We observed a similar pattern of isoform expression in AML patients,with 4Ig expression,2Ig expression,and both.Compared with the control group,both B7-H3 and its isoform 4Ig were up-regulated in AML blast cells.3.Methylation levels of CpG island in B7-H3 promoter region The result of RQ-MSP demonstrated that there were no significant differences in unmethylation level between AML patients and controls.Bioinformatics analysis of the DiseaseMeth database found no significant difference in methylation levels between the AML group and the control group.In addition,there was no correlation between the unmethylated level of B7-H3 and the isoform expression of B7-H3 in AML.4.The expression characteristic of inhibitory B7 family molecule B7-H1,B7-H3,B7-H4 and B7-H6 in AML Majority of AML cell lines expressed high or detectable level of membrane protein B7-H3 and B7-H6.The expression of B7-H3 was higher in SKM-1 and SHI-1,and B7-H6 had higher expression in K562 and HL-60.B7-H1 was highly expressed only in HEL,while B7-H1 membrane protein was not expressed in other AML cell lines.B7-H4 was undetectable in AML cell lines.The expression rate and MFI ratio of membrane protein B7-H3 and B7-H6 in de novo AML blast cells were significantly higher than that of control(P=0.0369,P=0.0121;P=0.0061,P=0.0121).And peripheral white blood cells of group with high B7-H3 expression were significantly higher when compared with group with low B7-H3 expression.Expression of B7-H3 and B7-H6 was elevated in patients with acute monocytic leukemia.The expression of B7-H1 and B7-H4 in de novo AML patients was low or undetectable,and there was no significant difference compared with control.Conclusions2 Ig was highly expressed in AML and was associated with poor prognosis of patients,suggesting that 2Ig maybe could bring new insight for the selection of targeted therapeutic targets.Membrane protein B7-H3 and its isoform 4Ig were up-regulated inAML.The unmethylation level of B7-H3 in AML was not significantly different from that of the control group,and was not correlated with the expression of B7-H3 and its isoforms.There are differences among expression characteristics of four inhibitory B7 family molecule B7-H1,B7-H3,B7-H4 and B7-H6 in AML cell lines and patients.The abnormally elevated expression of B7-H3 and B7-H6 in de novo AML suggests that the two B7 family molecules may be involved in occurrence and progression of acute myeloid leukemia.