鼻咽癌细胞外泌体circSLAIN1通过连接黏附分子A促进肿瘤相关巨噬细胞M2型极化OA

Objective To investigate the role and molecular mechanism of circular RNA(circ)SLAIN1 in M2 polarization of tumor-associated macrophages(TAMs)in nasopharyngeal carcinoma(NPC).Methods QRT-PCR was used to detect the expression level of circSLAIN1 in NPC cell lines(HONE1,HNE1,etc.)and clinical tissue samples from 121 NPC patients.Fluorescence in situ hybridization(FISH)was used to determine its subcellular localization.Kaplan-Meier survival curves were used to evaluate the rela-tionship between circSLAIN1 expression and prognosis.Stable NPC cell lines with circSLAIN1 overexpression and knockdown were constructed.Exosomes were extracted by ultracentrifugation and identified by Western blot(CD63,TSG101)and transmission electron microscopy(TEM).The isolated exosomes were co-cultured with M0,M1,or M2 macrophages derived from the THP-1 cell line.QRT-PCR was used to detect changes in M1[CD86,NOS2,tumor necrosis factor(TNF)-α]and M2(CD163,ARG-1,IL-10)markers.RNA pull-down assay combined with mass spectrometry was used to screen for circSLAIN1-interacting proteins,which were verified by Western blot.Finally,functional rescue experiments were performed using junctional adhesion mol-ecule A(JAMA)-shRNA interference technology to verify the necessity of JAMA in circSLAIN1-mediated regulation of macrophage polarization.Results Compared with the nasopharyngeal epithelial cell line NP69 and inflammatory tissues,circSLAIN1 was highly expressed in NPC cells and tissues(P<0.05),mainly local-ized in the cytoplasm,and patients with high expression had shorter overall survival(OS)and progression-free survival(PFS)(P<0.05).NPC cell models with circSLAIN1 overexpression and knockdown were success-fully constructed,and circSLAIN1 levels in exosomes changed accordingly.Functional experiments showed that exosomes derived from circSLAIN1-overexpressing NPC cells upregulated the expression of M2 markers ARG-1 and IL-10 in M1 macrophages(P<0.05);conversely,exosomes from circSLAIN1-knockdown cells upregulated the expression of M1 markers NOS2 and TNF-α in M2 macrophages(P<0.05).In addition,RNA pull-down combined with mass spectrometry and Western blot confirmed that circSLAIN1 could directly bind to JAMA protein and positively regulate its expression.Functional rescue experiments showed that knockdown of JAMA expression could offset the M2 polarization effect induced by circSLAIN1 overexpression(decreased ARG-1 expression,P<0.05).Conclusion circSLAIN1 is highly expressed in nasopharyngeal car-cinoma and is delivered to macrophages via exosomes.It regulates JAMA expression to promote the polariza-tion of TAMs to the M2 type,thereby possibly participating in the progression of nasopharyngeal carcinoma.