Associations Of MICA Gene Polymorphism With Susceptibility Of Breast Cancer Cells To NK Cell-mediated Cytotoxicity

Objective: To investigate the associations of MICA gene polymorphism withsusceptibility of breast cancer cells to NK cell-mediated cytotoxicity.Methods:1. MICA full-length cDNA fragments were ampliifed from breast cancercells (including MCF-7，MDA-MB-231，MDA-MB-435S and SK-BR-3) by RT-PCRmethod, and cloned into pMD18-T Vector, then the genotypes were determined bysequencing method.2. The eukaryotic expression vectors of various MICA alleles wereconstructed and transfected into293T cells，and the expressed products weredectected by western blot and lfow cytometry.3. Peripheral blood mononuclear cellswere isolated, then NK cells were ampliifed by combination of IL-2+IL-12+IL-15+IL-18, and highly pure NK cells were isolated by VarioMACS.4. The cytotoxicityof NK cells against the above293T cells transfected with MICA genes were measuredby LDH assay, and the release of PFN and Gzm B were measured by Elispot assaywhen NK cells were induced by the above293T cells transfected with MICA genes.Results:1. DNA sequencing result showed that MICA*008/A5.1and MICA*001/A4were determined in MCF-7cells，MICA*010/A5was determined in MDA-MB-435Scells，MICA*019/A5and MICA*002/A9were determined in both MDA-MB-231andSK-BR-3cells. Protein sequence alignment showed that MICA*019/A5is Arg atposition29of N-terminal, but MICA*010/A5is Pro at the same positon. Structureanalysis showed that Arg/Pro located at P-sheet of MICA structure and was close tothe center.2. MICA*008/A5.1，MICA*001/A4，MICA*019/A5，MICA*002/A9andMICA*010/A5were cloned into pcDNA3. l/myc-His(-) A respectively to constructthe recombinant expression vectors (pMCFA5.1，pMCFA4，p231A5R，p231A9andp435A5P). After the recombinant expression vectors were transfected into293T cells， western blot analyses showed that the level of MICA allele were lowest in pMCFA5.1groups, then in p435A5P groups, and higher in the other three groups. FCM analysesshowed that the level of MICA allele were lowest in p435A5P groups, then inpMCFA5.1groups and MICA*001/A4groups, and higher in p231A5R groups andp231A9groups.3. After17days cultured in combination of IL-2+IL-12+IL-15+—IL-18，the proportion of NK cells (CD3CD56+) was (72.1土2.9)%，the proportion ofNKG2D was (93.7土3.6)%. After isolating, the proportion of NK cells was (95.2土3.0)%，the proportion of NKG2D was (98.1土1.5)%.4. LDH analyses showed thatthe susceptibility of293T cells transfected with MICA genes to NK cell-mediatedcytotoxicity were increased significantly (P<0.05)，p435A5P groups were lowest intransfected groups (P<0.05)，but there were no differerce among the other transfectedgroups (P>0.05). Elispot analyses showed that the ability of inducing NK cells torelease PFN and Gzm B were increased signiifcantly in transfected groups (P<0.05)，the ability were lowest in p435A5P groups (P <0.05)，but there were no differerceamong the other transfected groups (P>0.05).Conclusion: MICA gene polymorphism is associated with susceptibility of breastcancer cells to NK cell-mediated cytotoxicity。NK cells infusion could be individualizedthrough analyzing MICA gene polymorphism to select tumor patients sensitive to NK cellstherapy.