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【Objective】 To investigate the expression level of B7-H6 in peripheral blood and bone marrow of patient with chronic myeloid leukemia (CML), and to analyze its association with clinicopathological characteristics and prognosis. 【Methods】 A total of 120 CML patients from January 2010 to May 2013 were selected as study subjects. The expression of B7-H6 mRNA in CML peripheral blood and bone marrow pre- and 3-, 6-, 12-month-posttreatment was detected by quantitative real time PCR, and its correlation between prognosis and clinicopathological factors was analyzed. 【Results】 The expression level of B7-H6 mRNA in the PB, BMMCs of CML patients was lower than that of the normal population (P0.1%(P<0.0001) or without CCR (P<0.001). The expression level of B7-H6 in BMMCs was negatively correlated with the BCR-ABL1/ABL level (r=–0.260, P<0.05). Signifiant difference in PFS was observed between patients with high expression level of B7-H6 (not reached, HR: 0.06, 95% CI =0.03-0.37) and low expression level (81 months, HR: 15.58, 95% CI =2.68-30.23) in BM (P<0.05). 【Conclusion】 The low expression of the B7-H6 gene in CML is correlated with BCR-ABL1 copy number and responsiveness to treatment, and monitoring of B7-H6 expression may be used to evaluate CML prognosis, progression and treatment efficacy.
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B7-H6 has been discovered as a new member of the B7 family in recent years, it can specifically bind to the NKp30, an activated receptor of natural killer (NK) cells to mediate NK cells' tumor immunity killing effect. B7-H6 expression is upregulated in a wide variety of malignant tumor cells, but expression deficiency in normal tissue is detected. The intrinsic mechanism of B7-H6 regulation has been explored, and the treatment targeting B7-H6 has achieved a good effect in animal experiments, which shows a wide prospect of clinical application. This paper summarizes the latest progress of B7-H6 molecule in malignant tumors.
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Objective@#To investigate the key factor(s) of hepatitis B virus X protein (HBx) promoting B7-H6 gene activation.@*Methods@#The DNA fragments of the B7-H6 promoter were amplified from the human genomic DNA using polymerase chain reaction(PCR). Products of PCR were digested by KpnI and HindⅢ, and inserted into luciferase reporter vector (pGL3-Basic). The correctness of the recombinant plasmid pGL3-B7-H6 was confirmed by sequencing. Human hepatoma cell line HepG2 were co-transfected with pGL3-B7-H6 and the eukaryotic expression vectors (pCMV-HBs、pCMV-HBc and pCMV-HBx), and the relative luciferase activity was detected.The different dose of HBx expression plasmids were transfected into the HepG2 cells, and the expression of B7-H6 protein were determined by Western blot.@*Results@#A period of 2.2 Kb of B7-H6 promoter region were successfully cloned into pGL3-Basic, which was confirmed by DNA sequencing. The relative luciferase activity was significantly higher in the cells transfected with pGL3-B7-H6 than that in the cells transfected with the empty vector pGL3-Basic (5.24±1.25 vs. 1.12±0.31, P=0.005). The relative luciferase activity in HepG2 cells co-transfected with pGL3-B7-H6 and pCMV-HBx was remarkably increased compared with the control group (17.60±3.36 vs. 6.73±1.36, P=0.001). Western blot further demonstrated that HBx protein can significantly enhance B7-H6 protein expression.@*Conclusions@#Hepatitis B Virus X proteins enhanced B7-H6 promoter activity and promoted B7-H6 protein expression.
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Objective:To investigate the effects of Fructus Corni extract on the B7-H6 expression in primary liver cancer cells of rats. Methods:Sixty SD rats were randomly divided into three groups, namely, model, matrine, and Cornus officinalis. The rat model bear-ing the primary liver cancer was induced by diethylnitrosamine, except for the rats in the control group. The rats in both the matrine and Cornus officinalis groups were fed with matrine and Cornus officinalis. The rats in model groups were fed with 0.9％sodium chlo-ride solution. The number of hepatocellular carcinoma nodules was calculated, and the tumor growth inhibition rate was also calculat-ed. The pathological changes of hepatic tissues in rats of each group were observed by hematoxylin and eosin staining. The expression levels of B7-H6 in these three groups were determined by immunohistochemistry and Western blot. Results:The number of liver nod-ules of the matrine and Fructus Corni group rats was lower than that of the model group (P<0.05). The tumor inhibition rate of the Cor-nus group was significantly higher than that of the matrine group (P<0.05). The tumor growth inhibition rate of the Cornus officinalis group was significantly higher than that of the matrine group (P<0.05). Immunohistochemistry showed that the positive expression of B7-H6 in the Cornus officinalis group and the matrine group was significantly higher than that in the model group (P<0.05), and the positive expression of B7-H6 in the Cornus officinalis group was significantly higher than that in the matrine group (P<0.05). Similarly, the protein expression of B7-H6 in the Cornus officinalis and matrine groups was significantly higher than that in the model group (P<0.05) by Western blot, while the protein expression of B7-H6 in the Cornus officinalis group was significantly higher than that in the matrine group (P<0.05). Conclusion:Fructus Corni extract may inhibit the growth of hepatocellular carcinoma through upregulating the B7-H6 expression.
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Objective To prepare mouse anti-human monoclonal antibodies against B7-H6 and to identify their biological characteristics. Methods The B7-H6 gene was cloned by RT-PCR from a human lung adenocarcinoma cell line ( A549 ) and then subcloned into the eukaryote expression vector pCMV3 to construct the recombinant vector pCMV3-B7-H6. The recombinant vector pCMV3-B7-H6 that was verified with enzyme digestion and gene sequencing was transfected into NIH/3T3 cells by electroporation. BALB/c mice were immunized with the successfully transfected cells named 2H8 through intraperitoneal injection. The monoclonal antibodies against human B7-H6 with the advantages of high affinity and specificity were pre-pared by using hybridoma technology. Western blot assay and flow cytometry analysis were used to identify the specificity of prepared monoclonal antibodies. Results The recombinant eukaryotic expression vector encoding B7-H6 was successfully constructed. Two hybridoma clones that stably secreted monoclonal anti-bodies against B7-H6 were screened out by using flow cytometry analysis and the monoclonal antibodies se-creted by them were belonged to IgG2a isotype. Specific reactions between B7-H6 and the secreted mono-clonal antibodies were confirmed by Western blot assay and flow cytometry analysis. Conclusion The mon-oclonal antibodies which recognized B7-H6 specifically were prepared successfully.