Expression And Significance Of Immune Inhibitory Receptor LAIR-1in Patients With Uterine Cancer And Ovarian Cancer

Objective:To investigate the expression of LAIR-1in patients with uterine cancer and ovarian cancer, and to explore the role of LAIR-1in tumor immune responses and the relationship between LAIR-1expression and clinicopathological features in epithelial ovarian cancers.Through RNA interference to silence LAIR-1on ovarian cancer cells, we examined the impact of LAIR-1on the proliferation and apoptosis capacity of ovarian cancer cells, to lay a foundation for the deep research on the diagnosis and treatment of tumors.Methods:(1)Peripheral blood mononuclear cells (PBMCs) from patients with cervical cancer,endometrial carcinoma, hysteromyoma and precancerous lesion,were isolated by ficoll-hypaque density gradient centrifugation.Then flow cytometry(FCM) was used to test the expression of LAIR-1on T cells.(2) Expressions of LAIR-1were detected in ovarian cancer tissues, lymph node metastasis tissues and adjacent normal tissue by immunohistochemistry (IHC) and tissue microarray technology.(3) The expression and position of LAIR-1on ovarian cancer cell lines were identified by reverse transcription polymerase chain reaction(RT-PCR), Real Time PCR,Western Blot,laser scanning confocal microscopy (LSCM) and FCM.(4) After human LAIR-l(CD305)-shRNA Lentivirus vector was constructed and packed, they were transfected into ovarian cancer cell line HO8910and COC1.The LAIR-1silencing efficiency were conformed at mRNA and protein level respectively using quantitative real time PCR (qRT-PCR) and Western Blot.Then the CD305-shRNA with the best silencing efficiency was employed to infect HO8910and COC1cell line and the stable CD305-shRNA cell line was obtained.(5) After silencing the expression of LAIR-1, the proliferation and apoptosis of HO8910or COC1cells were detected by CCK-8and FCM with Annexin V-PE staining, respectively. Results:(1)In the peripheral blood of tumor patients,the expression of LAIR-1on CD3+CD4+T cells from patients with cervical cancer and endometrial carcinoma was higher than that of patients with hysteromyoma (P<0.05) and precancerous lesion (P<0.01).Moreover,the results of mean fluorescence intensity (MFI) showed that the expression of LAIR-1on CD3+CD8+T cells of patients with cervical cancer was higher than that of patients with hysteromyoma (P<0.05) and precancerous lesion (P<0.001),on CD3+CD4+T cells was higher than that of precancerous lesion patients (P<0.01);the expression of LAIR-1on CD3+CD8+T cells of patients with endometrial carcinoma was higher than that of patients with precancerous lesion (P<0.05),on CD3+CD4+T cells was higher than that of patients with hysteromyoma (P <0.05) and precancerous lesion (P<0.01).(2) By immunohistochemistry, LAIR-1was predominantly expressed on the cytoplasm and nuclei of cancer cells in ovarian cancers tissues and lymph node metastasis tissues, whereas, LAIR-1protein was not detected in normal ovary tissue or cancer adjacent normal ovary tissue.Future more,LAIR-1expression was significantly correlated with stage(P<0.01),lymph node metastasis(P<0.05) and depth of invasion (P<0.05).(3) LAIR-1was expressed on the cytoplasm of HO8910and the membrane of COC1at the level of mRNA and protein,moreover,LAIR-1was highly expressed on COC1cells.Analysis of RNA-Seq of COC1cells results showed COC1LAIR-1gene was as same as human LAIR-la gene in GenBank.(4) Human CD305-shRNA Lentivirus vector was constructed and packed successfully, and they could be effectively transfected into ovarian cancer cell line HO8910and COCl,then the stable CD305-shRNA HO8910cell line and CD305-shRNA COC1cell line were obtained. Quantitative real time PCR and Western Blot showed CD305-shRNA could highly efficiently knock down the expression of LAIR-1on HO8910.(5) After silencing the expression of LAIR-1,the proliferation rate of CD305-shRNA HO8910was higher than that in NC-shRNA HO8910(P<0.0001), but the apoptosis of which were no statistical significant (P>0.05); However, the proliferation and apoptosis of either CD305-shRNA COC1or NC-shRNA COC1were no statistical significant (P>0.05) Conclusion:(1)The expression of LAIR-1is up-regulated in peripheral blood from patients with uterine cancer, which prompt the expression of LAER-1may contribute to tumor immune escape.(2) The expression of LAIR-1on cancer cells in ovarian cancers tissues was correlated with clinical and pathological characteristics.(3) The expression of LAIR-1on ovarian cancer cell lines COC1and HO8910was positive,and COC1cells highly express LAIR-1.(4) The stable CD305-shRNA HO8910cell line was obtained,which could highly efficiently knock down the expression of LAIR-1.(5) The expression of LAIR-1in HO8910could significantly inhibit the cell proliferation, but there was no obvious affection on the apoptosis of HO8910.