The Mechanism And Significance Of Imbalance Of MiR-200a/CD47 Expression In Nasopharyngeal Carcinoma

Introduction and objective:Nasopharyngeal carcinoma is a malignant tumor that originates from nasopharyngeal epithelial cells and is highly prevalent in southern China and Southeast Asia.CD47 as a new immune checkpoint can be used as the cell's "self"logo,releasing the "don't eat me" signal to prevent phagocytosis of macrophages.The high expression of CD47 in tumors suppresses the body's immune response,frees tumors from clearance by the immune system,and thus causes immune escape,while blocking CD47 can activate the immune system in both innate and adaptive immunity.MicroRNAs are a type of endogenous non-encoded single-stranded RNA that regulates gene expression.Studies have shown that MicroRNA can precisely regulate the immune network by regulating key genes in the immune system.miR-200a is an important tumor suppressor factor and is down-regulated in many malignant tumors including nasopharyngeal carcinoma.The bioinformatics predictions revealed that the sequence of the seed region of miR-200a is fully complementary to the 3'UTR of CD47 mRNA.This suggests that CD47 is likely to be the target gene of miR-200a and is negatively regulated by miR-200a.Based on this,we proposed a research hypothesis:CD47 is a key tumor immune checkpoint,miR-200a as a tumor suppressor gene expression imbalance in nasopharyngeal carcinoma,CD47 upregulation of nasopharyngeal carcinoma,the formation of miR-200a/CD47 imbalance,promote nasopharyngeal carcinoma "Immune escape"occurs,and the imbalance of miR-200a/CD47 expression is an important reason for the recurrence of nasopharyngeal carcinoma and a key target of immunotherapy.Materials and Methods:40 cases of nasopharyngeal carcinoma were collected from South Medical University.The expression of miR-200a/CD47 were detected by in situ hybridization and immunohistochemistry respectively.At the same time,we detected the expression level of miR-200a and CD47 in nasopharyngeal carcinoma cell lines and predicted the target gene and dual-luciferase reporter system.Then,the mechanism of miR-200a and CD47 in proliferation,migration,invasion and apoptosis was detected by cell function experment.Finally,by phagocytosis experiments to proveanti-CD47 antibody in nasopharyngeal carcinoma and whether miR-200a can inhibit CD47-mediated phagocytosis.Result:1.Nasopharyngeal carcinoma cell lines miR-200a and CD47 expression levelsThe expression of miR-200a in NPC cell lines detected by qPCR showed that the miR-200a was decreased and the expression of CD47 was higher than NP69.2.The correlation between miR-200a and CD47 in nasopharyngeal carcinomaThe expression of miR-200a in nasopharyngeal carcinoma was significantly lower than that in nasopharyngeal tissue,which was correlated with T and N staging in TNM staging.Spearman correlation analysis showed that the expression of miR-200a was negatively correlated with CD47(r=-0.435,P<0.01)3.CD47 is the target gene of miR-200aBioinformatics predicts that the target gene of miR-200a may be CD47.By constructing a dual luciferase reporter gene assay,it was shown that up-regulation of miR-200a expression in cells transfected with the reporter gene psicheck2-wt(including the CD47 3'UTR)Effectively inhibits the luciferase expression activity,but can not be regulated in psicheck2-mut,indicating that CD47 is the target gene of miR-200a.4.Cell function experimentTransfection of miR-200a mimics and anti-miR-200a cell function experiments showed that,miR-200a can deposit into the apoptosis,inhibition of cell proliferation,migration and invasion.However,the expression of miR-200a could be reversed by transfection with siCD47,indicating that miR-200a can promote the apoptosis of nasopharyngeal carcinoma cells and inhibit the proliferation,migration and invasion of cells,partly through the regulation of CD47 expression.5.In vitro phagocytosis experimentTo further clarify whether anti-CD47 antibodies can specifically act on tumor cells to promote phagocytosis of macrophages,we performed macrophage phagocytosis experiments in vitro.The results showed that compared with the control group,the anti-CD47 antibody significantly promoted the macrophage phagocytosis of CNE1 cells(P<0.05).To further clarify the effect of the corrected imbalance of miR-200a/CD47 on macrophage phagocytosis of nasopharyngeal carcinoma cells,we over-expressed CNE1 in miR-200a co-cultured with macrophages.The results showed that compared with the control group,overexpression of miR-200a significantly increased the phagocytosis of tumor cells by macrophages,and the difference was statistically significant(P<0.05).The indicated miR-200a can inhibit the expression of CD47 and promote phagocytosis of macrophages.Conclusion:The imbalance of miR-200a/CD47 expression in nasopharyngeal carcinoma may affect the proliferation,apoptosis and metastasis of tumor cells,inhibit macrophage phagocytosis,and play an important role in the occurrence and development of nasopharyngeal carcinoma and immune escape.