A Preliminary Experimental Study On The Regulation Of Mmu-circ-012559 In Myeloid-derived Suppressor Cells

ObjectiveThis study aims to observe the potential role of one murine circular RNA,mmu-circ-012559,on MDSCs immunosuppression,and analyze the possible working mechanism existed in tumor-associated MDSCs activity.Methods(1)Mice were implanted s.c.with Lewis lung carcinoma(LLC)cells in order to establish tumor models.Murine MDSCs were isolated from splenocytes or tumor tissue of tumor-bearing mice and identified by flow cytometry(FCM)to further explore the relative expression levels of mmu-circ-012559 and miR-223.(2)MDSCs isolated from tumor-bearing splenocytes were treated with TCCM or medium control for 24 h.mmu-circ-012559 and miR-223 expression levels were determined by qRT-PCR.3H-TdR incorporation experiment was performed to investigate CD4+ T cell proliferation when co-cultured with TCCM treated MDSCs.Mmeanwhile,Western-Blot was used to detect the total and phosporylation level of STAT3.(3)The TCCM treated MDSCs were transfected with si-NC(negative control)+ miR-inhibitor NC ? si-mmu-circ-012559 + miR-inhibitor NC and si-mmu-circ-012559 + miR-223 inhibitor in order to knockdown mmu-circ-012559 and miR-223 expression.3H-TdR incorporation experiment was performed to investigate CD4+ T cell proliferation when co-culture with these posttransfected MDSCs.In addition,MDSCs were harvested for detection of ROS production,Arginase-1 activity and the STAT3 expression.Results(1)The level of mmu-circ-012559 in tumor tissue MDSCs significantly increased compared with corresponding splenocytes-derived MDSCs(3.59 ± 0.04 vs 0.80 ± 0.08,p<0.01),while the level of miR-223 significantly decreased(0.21 ±0.05 vs 2.57 ± 0.29,p<0.01).These results indicate mmu-circ-012559/miR-223 probably involved in tumor-associated MDSCs.(2)qRT-PCR results discovered enhanced mmu-circ-012559(1.95 ± 0.08 vs1.01 ± 0.13,p<0.01)and reduced miR-223 expression(0.50 ± 0.12 vs 2.13 ± 0.75,p<0.05)respectively.Experiment detecting the immunosuppressive ability on CD4+T cell proliferation showed the counts per minute(CPM)significantly decreased in TCCM treated group(p<0.01).Western-Blot showed upregulated STAT3 and p-STAT3 expression after TCCM treatment(p<0.05).(3)Experiment detecting the immunosuppressive ability on CD4+ T cell proliferation showed the counts per minute(CPM)regained to some extent in the si-mmu-circ-012559 transfected MDSCs group(p<0.05).However,cotransfection of si-mmu-circ-012559 and miR-223 inhibitor decreased the CPM(p<0.05).Meanwhile,level of ROS production(p<0.05),Arginase-1 activity(8.96 ± 1.44 vs12.75 ± 0.36,p<0.05)and STAT3 and p-STAT3 expression in MDSCs were reduced after the transfection of si-mmu-circ-012559.In contrast,cotransfection with miR-223 inhibitor reversed the decrease.These results identified a pro-MDSCs function of mmu-circ-012559,and this effect could be abolished by miR-223.ConclusionTumor-associated MDSCs exhibited enhanced mmu-circ-012559 expression and reduced miR-223 expression.Downregulation of mmu-circ-012559 in MDSCs inhibits both the suppressive capacity and the effector molecules expression of MDSCs,while miR-223 inhibitor could abolished these effects,indicating mmu-circ-012559 might function as a miR-223 sponge,thus promote STAT3 expression in tumor-associated MDSCs.