The Effect Of CD47 On Stemness Of Nasopharyngeal Carcinoma Cell And Its Mechanism

Introduction and objective:Nasopharyngeal carcinoma(NPC)is a malignant tumor with unique biological characteristics.Recurrence and metastasis are the main causes of treatment failure and death of NPC.Cancer stem cells(CSCs)are a small subset of cancer cells with self-renewal and multi-lineage differentiation potential.They are considered to be the"seed cell" for tumor recurrence and metastasis.CD47,is a newly found immune checkpoint that mediating immune escape.CD47 is up-regulated in a variety of malignant tumors,especially in CSCs.Anti-CD47 monoclonal antibody is also considered to be a potential new anti-tumor drug.Our previous studies have shown that CD47 is highly expressed in NPC cells and tissues,which enhances the proliferation and migration of NPC and is related to poor prognosis.In the present study,we found that CD47 is the important factor to maintain and enhance the sternness of NPC,and may eventually promote the recurrence and metastasis of NPC.Materials and Methods:NPC stem-like cells were enriched from NPC cell lines CNE1,CNE2 and 5-8F by serum-free suspension sphere formation and CD 133 was selected as the surface marker of NPC stem-like cells.At the same time,the expression level of CD47 in NPC stem-like cells was detected.Then the cell population of CD47+ and CD47-were sorted by flow cytometry.The relationship between the expression of CD47 and self-renewal and proliferation was detected by cell function study.The xenograft tumor formation in Balb/C nude mice was used to evaluate the difference of tumorigenicity.The classical colony assay and comet assay were used to verify the radio sensitivity of the two subclone of cells.RT-qPCR and Western blot were used to detect the expression of stem related genes in the two cell subsets.Result:1.NPC CNE2 cell showed increased level of CD47 along with enhanced capacity of sphere formation.After 7 days of serum-free suspension culture of NPC cells,it was found that the poorly differentiated NPC cell CNE2 had the strongest sphere formation ability,and the morphological structure of the cell spheres was more round and dense;the second was highly metastatic NPC cell 5-8F,while the well-differentiated NPC cell CNEI did not form a sphere.The results of RT-qPCR showed that the expression of CD47 in suspended tumor cell spheres(CNE2S)was higher than that in CNE2(P<0.05).2.Positive correlation of CD47 and CD133 was observed in CNE2 cell.CD 133 was selected as the surface marker of NPC stem-like cells.Flow cytometry result show that CD133 expression in NPC CNE2 cell ranged from 1%-2%The expression of CD47 in CNE2/CD133+ cell group was higher than that in CNE2/CD133-cell group(90%vs 64.5%,P<0.05).3.CNE2/CD47+ cell has stronger self-renewal abilityThe CNE2/CD47+ and CNE2/CD47-cell populations were sorted by flow cytometry.The results of serum-free suspension sphere formation assay showed that the efficiency of CNE2/CD47+ cell population was higher than that of CNE2/CD47-cell population in forming suspended cell spheres(P<0.05).The results showed that CNE2/CD47+ cell population had stronger self-renewal ability.4.CNE2/CD47+ cell has increased capacity of proliferation and cloning.EDU proliferation assay showed that the proliferation ability of CNE2/CD47+cell population was higher than that of CNE2/CD47-cell population(0.6301±0.02151 vs 0.3639±0.01925,t=9.219,P<0.0001).Plate clone formation assay showed that the clone formation ability of CNE2/CD47+ cell population was higher than that of CNE2/CD47-cell population(t=11.27,P=0.0004).The results showed that the high expression of CD47 made the tumor cells have stronger ability of clone and proliferation.5.CD47 promotes tumorigenesis of NPC cells in vivoCNE2/CD47+ and CNE2/CD47-cell populations were inoculated subcutaneously in Balb/c nude mice,the tumor growth was monitored regularly and the tumor volume was recorded.The results showd that CNE2/CD47+ cell population showed stronger tumorigenic ability in vivo than CNE2/CD47-cell population,and the tumor grew faster and larger(1504.5±714.42mm3 vs 471.8±502.7mm3,t=2.319,P=0.034).These results suggest that CNE2/CD47+ cell population has stronger tumorigenic ability and may contain more NPC stem-like cells.6.CD47 enhances radiation resistance of NPC cellsCNE2/CD47+ and CNE2/CD47-cells were irradiated with different radiation doses.The radiosensitivity of the two groups of cells was evaluated by classical clone assay and comet assay.The results showed that the radiosensitivity of CNE2/CD47-cells was higher than that of CNE2/CD47+ cells by classical clone formation analysis and click the multi-target model curve(dose-survival curve)of CNE2/CD47+ and CNE2/CD47-cells(P<0.05).Comet assay showed that DNA damage was aggravated with the increase of radiation dose.At the same radiation dose,the DNA damage of CNE2/CD47-cells was higher than that of CNE2/CD47+ cells(P<0.05),which indicated that the radio sensitivity of CNE2/CD47-cells was higher than that of CNE2/CD47+ cells,that is to say,the high expression of CD47 enhanced radioresistance.7.Up-regulation sternness-related genes in CNE2/CD47 + cell populationThe expression of stem cell related transcription factors such as OCT-4,SOX-2,NANOG,c-MYC,KLF-4 and surface marker CD133/CD44/CD24/ABCG2 in CNE2/CD47+ and CNE2/CD47-cell populations were detected by RT-qPCR and Western blot.These results showed that CNE2/CD47+ cell population expressed higher stemness-related genes,indicating that overexpression of CD47 may maintain the stemness of NPC by up-regulating sternness-related genes.Conclusion:NPC stem-like cells have higher CD47 level.Overexpression of CD47 boosts NPC cell sternness.Overexpression of CD47 may be a key factor in the formation and maintenance of nasopharyngeal carcinoma stem-like cells,and targeting CD47 may become a new way to treat recurrent refractory nasopharyngeal carcinoma.