| PurposePart ?:To explore the relationship between the expression of PD-L1 protein and the prognosis of patients with invasive breast cancer.Furthermore,study the relationship between PD-L1 and commonly used clinicopathological features.Part ?:Prognostic and clinicopathological value of programmed death ligand-1(PD-L1)in breast cancer:a meta-analysis(This part has been published in Plos One).Part?:We investigated the effect and mechanism of miR-34a on PD-L1 by in vitro and in vivo experiments.MethodPart ?:The expression of PD-L1 was analyzed by immunohistochemical staining of two tissue microarrays containing 300 patients with invasive breast cancer.Then relationship between the expression of PD-L1 and the prognosis of these breast cancer patients was analyzed.The relationship between PD-L1 and several clinicopathological features was explored by further subgroup analysis.Part ?:We searched the databases from PubMed,EMBASE,Web of Science,and Cochrane libraries for literatures about the role of PD-L1 in the prognosis of patients with invasive breast cancer from inception to February 2016.Meta-analysis was performed using a rigorous inclusion and exclusion criteria to screen for standards-compliant literature.Using the Review Manager software to calculate the risk ratio(RRs)and its 95%confidence intervals(CIs),to analyzed the relationship between PD-L1 and the prognosis of patients with invasive breast cancer.If there was no significant heterogeneity between the studies,a fixed effect model was used to analyze RRs for each study included;whereas if heterogeneity is observed,a random effect model is used.The heterogeneity between the data is evaluated by using the chi-square test and the I2 statistic.The p-value of the chi-square test less than 0.10 or I2>50%is considered to be a significant heterogeneity.Potential publication bias is assessed by funnel chart analysis.Part?:In vitro and in vivo experiments were carried out using human breast cancer cell line MDA-MB-231.In vitro experiments,the expression of miR-34a and PD-L1 were detected by RT-PCR,Western Blot,double luciferase activity assay,CCK-8 and Transwell after transfected by miR-34a mimic,miR-34a mimic NC,miR-34a inhibitor or miR-34a inhibitor NC in MDA-MB-231 cells.In vivo experiments,MDA-MB-231 cells were used to establish Nude Mice Xenografts,and the changes of PD-L1 expression and tumor growth were observed after miR-34a overexpression.ResultPart?:(1).Two tissue microarrays involved 300 patients with invasive breast cancer,286 cases were included to this study and 165(57.69%)of PD-L1 overexpression;(2).There was no significant correlation between PD-L1 and age,tumor location,histological grade,tumor diameter,lymph node metastasis,clinical stage of tumor,CK5/6,P53,EGFR;but there were significant correlation between PD-L1 and ER(P=0.002,X2=10.507),PR(P=0.006,X2=7.784),Her-2(P=0.003,X2=9.167),Ki-67(P=0.005,X2=8.404),molecular subtypes(P=0.000,X2=21.322)(P<0.05);(3).Kaplan-Meier survival analysis showed that expression of PD-L1 was associated with OS in breast cancer patients significantly(p = 0.001).The patients with PD-L1 overexpression have a poor prognosis(The tissue microarray did not provide data of Disease-free survival(DFS)about the patient.Thus we did not analyzed it);(4).There was a statistically significant difference in PD-L1(P = 0.001,HR = 2.299,95%CI:1.389-3.803)by univariate and multivariate COX regression analysis.PD-L1 overexpression may be a specific predictor of poor prognosis in patients with invasive breast cancer;(5)In further subgroup analysis,it was found that ER +,PR +,Her-2+,Ki67>14%and Ki67? 14%patients with PD-L1 overexpression have a poor prognosis.Part ?:(1).A total of 5 articles were included in the meta-analysis;(2).The high expression of PD-L1 was significantly correlated with the total mortality risk(MR)of breast cancer patients(p = 0.007),and the total RR was 1.64(95%CI,1.14-2.34);(3).The high expression of PD-L1 was correlated with the mortality risk of 10 years after surgery(MR10years)significantly,RR was 2.53(95%CI,1.78-3.59),(P<0.0001);(4).The relationship between PD-L1 expression and lymph node metastasis in breast cancer patients was statistically significant(P = 0.02),RR was 1.33(95%CI,1.04-1.70);and the relationship between histology grade was statistically significant(P = 0.003),RR was 1.24(95%CI,1.07-1.43);and the relationship between ER was statistically significant(p = 0.006),RR was 2.45(95%CI,1.31-4.60).Part?:(1)The expression of miR-34a was significantly increased after transfection with miR-34a mimic in human breast cancer cell line MDA-MB-231,and the expression of PD-L1 mRNA was significantly decreased;while,after transfection with miR-34a inhibitor expression of miR-34a was significantly reduced,and the expression of PD-L1 mRNA was significantly decreased(p<0.05);(2)After detected with Western Blot,the expression of PD-L1 protein in human breast cancer cell line MDA-MB-231 transfected with miR-34a mimic was decreased.While,the expression of PD-L1 protein was significantly increased after transfection with miR-34a inhibitor(p<0.05);(3)Double luciferase assay were used to detect the relationship between miR-34a and target gene PD-L1.Results confirmed that miR-34a mimics can down-regulate the luciferase activity of MDA-MB-231 Cell after transfection of wild-type PD-L1(PD-L1 WT)plasmid(p<0.05);(4)Rsults of CCK-8 test showed human breast cancer cell line MDA-MB-231 proliferation ability decreased after transfected miR-34a mimic;and cell proliferation ability increased after transfected miR-34a inhibitor;(5)Result of Transwell test show that the migration and invasion ability of human breast cancer cell line MDA-MB-231 decreased after transfected by miR-34a mimic;(6)In Nude Mice Xenografts,the tumor volume and growth rate of intratumoral injection of miR-34a agomir group were significantly lower than those of miR-34a agomir NC group and Mock group;(7)RT-PCR showed that the expression of miR-34a in miR-34a agomir group was significantly higher than that in miR-34a agomir NC group and Mock group;(8)Western Blot showed that the expression level of PD-L1 in miR-34a agomir group was significantly lower than that in miR-34a agomir NC group and Mock group.(9)Immunohistochemical staining showed that PD-L1 was low in miR-34a agomir group,while miR-34a agomir NC group and Mock group were highly expressed.ConclusionSingle-center tissue microarray and evidence-based Meta-analysis co-indicated that expression of PD-L1 was associated with prognosis in patients with invasive breast cancer,patients with PD-L1 overexpression has a relatively poor prognosis.The expression level of PD-L1 was regulated by miR-34a,miR-34a specifically mediates the transcriptional level of the PD-L1 gene by targeting the 3'UTR terminus of PD-L1 mRNA.Meanwhile,upregulation of miR-34a inhibits the proliferation of breast tumors and the expression of PD-L1 mRNA and protein in nude mice tumor-bearing models. |
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