The Role Of Chemoradiotherapy-induced Nasopharyngeal Carcinoma Cell-derived Exosome LncRNA FAM212B-AS1 In Regulating Macrophage Polarization To Promote Tumor Progression

Objectives:The aim of this study was to investigate the effect of chemoradiotherapy-induced NPC cell-derived exosomes lnc RNA FAM212B-AS1 in regulating of M2 macrophages polarization to promote invasion and proliferation of NPC cells,so as to provide a scientific basis for the validation of the negative problem of the deterioration of biological behavior of NPC cells induced by chemoradiotherapy and to provide scientific basis for further clinical interventions in the future.Methods:(1)NPC cells(CNE1 and CNE2)were selected,and the chemoradiotherapy-induced cell lines for NPC were constructed by concurrent chemoradiotherapy and chemoradiation percussion,0.05 μ g/m L cisplatin and 6Gy6 MV X-rays were administered ten times in a row to obtain chemoradiotherapy-induced cell lines,named CNE1 CRT and CNE2 CRT,respectively.(2)The exosomes derived from CNE1 CRT cells and CNE2 CRT cells were separated by differential hypervelocity centrifugation and named as CNE1CRT-exo and CNE2CRT-exo.Then the exosomes were incubated with THP-1 cell-induced macrophages.The expression of FAM212B-AS1 in macrophages incubated with CNE1CRT-exo and CNE2CRT-exo was detected by q RT-PCR to verify whether chemoradio-induced exosomes of NPC cells promote the expression of FAM212B-AS1 in macrophages.(3)The expression of FAM212B-AS1 was interfered in nasopharyngeal carcinoma cells transfected with si-FAM212B-AS1 and chemoradio-induced exosomes were extracted to obtain CNE1CRT-exo and CNE2CRT-exo knockdown FAM212B-AS1,which were verified by q RT-PCR.(4)Macrophages induced by THP-1 cells were incubated with control exosomes and CNE1CRT-exo and CNE2CRT-exo knockdown FAM212B-AS1,respectively.The expression of M2 marker Arg-1 and M1 marker NOS2 in macrophages was detected by immunofluorescence assay.The expressions of IL-10 and Arg-1 in M2 and TNF-α and IL-6 in M1 were detected by q RT-PCR.The expressions of NOS2 protein and Arg-1 protein were detected by Western blot to verify whether chemoradiation-induced exosomes of NPC cells regulate macrophage polarization through FAM212B-AS1.(5)To confirm whether macrophages mediated by FAM212B-AS1 further regulate the migration,invasion and proliferation of NPC,the culture supernatant of macrophages incubated with FAM212B-AS1-deletion CNE1CRT-exo and CNE2CRT-exo were added to the CNE1 CRT and CNE2 CRT cells.The migration and invasion capacity of CNE1 CRT and CNE2 CRT were determined by transwell assays,and the proliferative capacity of CNE1 CRT and CNE2 CRT was determined by CCK-8 assay.Results:(1)The expression of FAM212B-AS1 in macrophages was significantly up-regulated after CNE1CRT-exo and CNE2CRT-exo incubated with macrophages(CNE1CRT-exo vs.black: P = 0.0019;CNE2CRT-exo vs.black: P < 0.0001),these results suggest that chemoradio-induced NPC cell-derived exosomes can enhance the expression of FAM212B-AS1 in macrophages.(2)The interference efficiencies of three FAM212B-AS1 interfering fragments were detected in CNE1 CRT and CNE2 CRT,and si-1009 was found to have the most significant effect on down-regulating FAM212B-AS1 expression(P = 0.0024;P =0.0011).Therefore,we chose si-1009 for follow-up study.After further extraction of exosomes from NPC cells transfected with si-1009,the expression of FAM212B-AS1 in CNE1CRT-exo and CNE2CRT-exo in the si-1009 knockdown group was significantly decreased,suggesting that we successfully obtained the exosomes with FAM212B-AS1 deficiency.(3)Immunofluorescence results showed that CNE1CRT-exo and CNE2CRT-exo significantly up-regulated the expression of M2 macrophage marker Arg-1 and down-regulated M1 macrophage marker NOS2 in macrophages,while knockdown of FAM212B-AS1 in the exosome significantly reversed these effect(all P < 0.05).The q RT-PCR results showed that CNE1CRT-exo and CNE2CRT-exo significantly up-regulated the expression of M2 macrophage markers IL-10,Arg-1 and down-regulated the expression of M1 macrophage markers TNF-α,IL-6,while knockdown of FAM212B-AS1 in exosomes showed the opposite result(all P < 0.05).Western blot results showed that after CNE1CRT-exo and CNE2CRT-exo incubated with macrophages,the expression of NOS2 protein was significantly down-regulated,and the expression of Arg-1 protein was significantly up-regulated,while knockdown of FAM212B-AS1 in the exosome showed the opposite result(all P < 0.05).These data suggest that chemoradio-induced NPC-derived exosomes induce macrophage M2 polarization via FAM212B-AS1.(4)Transwell assay showed that macrophages incubated with exo-si FAM212B-AS1 significantly reduced the migration and invasion of NPC cells,compared with the macrophages incubated the control exosomes(CNE1CRT:migration: P = 0.0063,invasion: P < 0.0001,CNE2CRT: migration: P = 0.0029,invasion: P = 0.0120).The CCK-8 experiment showed that compared with the macrophages cultured with control exosomes,the macrophages cultured with exo-si FAM212B-AS1 showed the reduced proliferation of NPC cells(CNE1CRT: 24h:P < 0.0001,48h:P < 0.0001,CNE2CRT: 24h: P = 0.0030,48h: P < 0.0001).These data suggest that chemoradio-induced NPC exosomal FAM212B-AS1 mediates macrophage to promote the proliferation,migration and invasion of NPC.Conclusion:Chemoradiotherapy-induced NPC cells exosome lnc RNA FAM212B-AS1 promote invasion and metastasis of NPC cells by regulating M2-type macrophages polarization.