Effect Of MICA Protein On Anti-breast Cancer Mediated By NK Cells

Objective To value the expressions of MICA(MHC class I chain-related gene A) on breast tumor cellular membrane and that of soluble MICA (sMICA) in serum of patients with breast tumor;explore the effect of membrane MICA(mMICA)and soluble MICA on NKG2D;investigate the roles MICA on anti-breast cancer.Methods 1.PBMCs were purified by negative depletion using immumomagnetic beads(NK cell isolation kit. Melteny, Germany) by Vario MACS system according to the manufacturer's recommended conditions.2.Flow cytometry(FCM)were performed to identify the expression of membrane MICA on breast tumor tissues and cell lines,and the expression of NKG2D on NK cells.The effect of mMICA,sMICA and IL-15 on the expression of NKG2D were observed by FCM. RT-PCR was used to identify membrane MICA on breast tumor tissues.Immunohistochemistry was used to detect the expression and distribution of soluble MICA and membrane MICA on breast tumor tissues. ELISA was performed to examine the soluble MICA in peripheral blood.3.Study the reaction between MICA and NKG2D by purified-antibody blockade.4.Observe the cytotoxicity of NK cells to breast cancer by MTT test.Results 1.mMICA was not detected on normal breast tissues,but (38.5±7.5)% on breast benign tumor and (53.2±5.6)% on breast malignant tumor.2.sMICA was not detected in the serum of healthy person,but(76.8±22.3)pg/ml in breast benign tumor patients and(205.36±71.27)pg/ml in breast malignant tumor patients.There were positive correlation between sMICA levels with breast cancer stage.3.Cytotoxicity of NK cells was decreased because of NKG2D or MICA antibody blockade,from(76.5±11.8)% to(31.6±2.2)% with NKG2D antibody,(29.8±1.9)% with MICA antibody and(25.9±1.8)% with NKG2D and MICA antibody blockade.The expression of NKG2D was decreased from (92.5±7.5)%to(62.5±7.6)% after NK cells cultured with sMICA,and decrease NK cells cytotoxicity from(76.2±10.5)% to(49.9±10.14)%.4.The expression of NKG2D on NK cells was increased from (55.12±2.4)% at 0h to(81.18±4.7)%at 23-24h after NK cells co-incubated with MCF-7.IL-15 up regulated the expression of NKG2D on NK from(54.9±6.8)% to(83.7±5.6)%,and increase NK cells cytotoxicity activity form(46.5±6.3)% to(63.5±4.8)%.Conclusion 1. MICA was expressed on lots of breast tumor cellular membrane but not on normal breast tissues, and might be regarded as tumor associated antigen (TAA).2. MICA expressed malignant cells were recognized by NK cells via the activating immunoreceptor NKG2D.This implies that MICA-NKG2D play a critical role in tumor immune surveillance.3.Soluble MICA level was positive correlation with breast cancer stage.sMICA reduced the expression of NKG2D,impaired NK-mediated immune surveillance and leads to immuneescape of breast tumor.4.IL-15 can up regulate the expression of NKG2D and increase NK cells cytotoxicity activity.