Study On UCA1 Involved In The Mechanism Of NK Cell-mediated Immune Tolerance In Breast Cancer

OBJECTIVE:1.To investigate the biological role and related molecular mechanism of LncRNA-UCA1 in NK cell-mediated immune tolerance in breast cancer.2.To explore the correlation between the expression of lncRNA-UCA1in breast cancer tumor tissues and prognosis of breast cancer.3.To investigate the the level of 25(OH)D in the peripheral blood of patients and analyze its correlation among clinicopathologic characteristic,prognostic indicators and the number of peripheral blood NK cells in BC patients.METHODS:1.Construct the NK resistant BC cell lines such as MDA-MB-231/R-NK and MCF-7/R-NK by multiple attacking of NK-92 cells.2.The primary peripheral blood monocyte cells(PBMC)were isolated from leukocyte concentrates(buffy coats)of 3 healthy volunteers(routine blood transfusion donors)through Ficoll density gradient centrifugation.The primary primary NK cells were isolaeted from 3 healthy donor PBMC by MACS and verified by FCM.To evaluate the sensitivity of MDA-MB-231/R-NK and MCF-7/R-NK and their parent cells to NK-cell including NK-92 and primary NK cells by CytoTox 96?Non-Radioactive Cytotoxicity kit.3.The MDA-MB-231/R-NK and MDA-MB-231 cells were analyzed by microarray and verified by QRT-PCR.We over-expressed the UCA1 in both MDA-MB-231 and MCF-7 verified by the QRT-PCR and detected their sensitivity to NK-92cells compared with their parent cells.4.Detect the difference of transcriptome between the MDA-MB-231/UCA1 and its parent cells.The only one significantly changed member-ULBP2 of the NK killing effect signaling pathway was verified by QRT-PCR.Compare the expression of ULBP2between MDA-MB-231/UCA1,MCF-7/UCA1 and their parent cells by QRT-PCR respectively.Compare the level of ULBP2 and sULBP2 level between the MDA-MB-231/UCA1,MCF-7/UCA1 and their parent cells respectively.5.Explore the mechanism of UCA1 promoting ULBP2 shedding from cell membrane through bioinformatie prediction miRNA which could interact with UCAl and prediction the target gene to miRNA and verified by dual-luciferase reporter gene assay and RIP assay.6.The AB type serum from 3 BC patients with bone metastasis containing high sULBP2 was collected and then added the serum into healthy NK-92 cells.The killing capacity of NK-92 cells incubated with the riched sULBP2 serum on MDA-MB-231and MCF-7 cells was detected and compared to untreated NK-92 cells.The levels of perforin and granzyme were also detected.7.In situ hybridization was used to detect the expression of UCA1 in primary local tissues from 417 BC patients(92 of which had paired bone metastasis),and to explore the correlation between the expression of UCA1 in primary local tissues from BC patients and overall survival(OS)of patients with BC.Compare the expression of LncRNA-UCA1 in primary and bone metastasis BC tissue in 92 BC patients who had paired bone metastasis.8.The level of 25(OH)D in the peripheral blood of 109 BC patients were detected by chemiluminescence immunoassay method and the expression of VDR in breast cancer tissues were detected by immunohistochemistry.Analyze the relationship between clinicopathologic characteristic and the level of 25(OH)D in the peripheral blood.Detect the expression of VDR and analyze its correlation among clinicopathologic characteristics at the same time.The relationship between the level of 25-hydroxyvitamin D and the level of NK cells in peripheral blood of breast cancer patients was further studied.RESULTS:1.Build the NK killing effect resistant model by multiple attacking of NK92cell:MDA-MB-231/R-NK and MCF-7/R-NK.The killing effect of NK cells(including NK-92 cells and primary NK cells)on MDA-MB-231/R-NK and MCF-7/R-NK was significantly lower than that of their parent cells(P<0.05)when E/T is 8:1,16:1,32:1 and64:1.The ELISA results showed that the Perforin and Granzyme B level in the MDA-MB-231/R-NK and MCF-7/R-NK group were significantly lower than that in their parent cells.(p<0.05)The cytotoxic test suggested parent cells were more sensitive to NK target cell killing than MDA-MB-231/R-NK and MCF-7/R-NK cell.2.MDA-MB-231/R-NK and its parental cells were analyzed by LncRNAs microarray and found that LncRNA-UCA1 expression was significantly up-regulated and verified on MDA-MB-231/R-NK?MCF-7/R-NK and their parent cells by QRT-PCR respectively.Build UCA1 over-expressed cell lines.The killing effect of NK-92cells on the two UCA1 over-expressed cell lines was significantly lower than that of their parent cells(p<0.05)which indicates UCA1 is associated with immune tolerance.3.Detect the difference of transcriptome between the MDA-MB-231/UCA1 and its parent cell.UCA1 up-regulated the NKG2D ligands(ULBP2)expression.The level of ULBP2 expression was up-regulated in both MDA-MB-231/UCA1 and MCF-7/UCA1 cell verified by QRT-PCR.Shedding soluble NKG2D ligands(sULBP2)would give rise to immune escape.The level of soluble NKG2D ligand(sULBP2)in the culture supernatant of UCA1 over-expressed cell lines was significantly higher than that of their parent cells by ELISA(P<0.05).4.UCA1 up-regulated the ADAM17 through miR-26b-5p for shedding of ULBP2verified by by dual-luciferase reporter gene assay and RIP assay.5.The LHD Cytotoxicity Assay suggested that compared to untreated NK-92cells,the killing capacity of NK-92 cells incubated with the riched sULBP2 serum on MDA-MB-231 and MCF-7 cells reduced significantly.The ELISA results show the Perforin and Granzyme B level in the untreated NK group were higher than sULBP2treated NK-92 cells group dramatically(P<0.05)which indicates that sULBP2 is involved in immune escape.6.The 5-year overall survival rate of patients with high UCA1 expression was lower significantly than that of patients with low UCA1 expression.The 5-year survival rates of breast cancer patients were 57.1%and 75.8%,respectively(P<0.05).Among the 92 paired bone metastasis BC tissues and primary BC tissues,the expression of UCA1 in bone metastasis BC tissue was significantly higher than that of paired primary BC tissue(16.48±3.73 VS 13.86±3.21,P<0.05).7.Compared with healthy volunteers and breast benign tumor patients,peripheral blood 25(OH)D level decreased significantly in BC patients(P<0.05).The 25(OH)D level of the peripheral blood in patients with breast cancer was significantly associated with T cell immune function(CD4~+/CD8~+),clinical stage,the metastasis of lymph node,bone metastasis,the level of PR,but not with tumor size,the level of ER.There was a statistically significant correlation between the expression of VDR in breast cancer tissues and the level of hormones receptor(ER and PR)(P<0.05).There was no significant correlation between the level of peripheral blood 25(OH)D and the number of peripheral blood NK cells in BC patients(P>0.05).CONCLUSION:1.UCA1 is involved in NK cell-mediated immune tolerance in breast cancer.UCA1 can up-regulate the expression of ULBP2 and promote the secretion of ULBP2,which is soluble sULBP2.sULBP2 leads to the occlusion of NKG2D and induces immune escape.UCA1 up-regulated the ADAM17 through miR-26b-5p for shedding of ULBP2.2.High expression of UCA1 is closely related to poor prognosis of breast cancer.3.Vitamin D might play an important role in the occurrence and development of breast cancer.While the level of serum 25(OH)D had no relation to the number of peripheral blood NK cells in BC patients.