Long Noncoding RNA Pvt1 Regulates The Immunosuppression Of G-MDSCs

Objective: In this study,we tried to explore the role of long noncoding RNA(lncRNA)Pvt1 in regulating the immunosuppressive function of G-MDSCs and analyze the possible working mechanism,which provided the experimental basis for a new target of tumor immunotherapy.Methods:(1)CD11b+ Ly6G+ G-MDSCs derived from spleen of wild-tpye(WT)mouse were sorted by magnetic beads,and CD11b+ Ly6G+ G-MDSCs derived from tumor tissue of Lewis lung adenocarcinoma xenograft model were isolated by magnetic beads combined with flow cytometry(FCM).Then we analyzed the purity of G-MDSCs by FCM.The Arraystar LncRNA microarray was detected by Shanghai Kangcheng Biotechnology Co.,Ltd.The expression of lncRNA Pvt1 was confirmed by q RT-PCR.q RT-PCR was used to measure lncRNA Pvt1 expression in G-MDSCs from bone marrow(BM),spleen and tumor tissue of tumor-bearing(TB)mice.(2)The possible downstream molecule regulated by lncRNA Pvt1 in G-MDSCs was analyzed by Arraystar LncRNA microarray.The level of c-myc m RNA was confirmed by q RT-PCR.q RT-PCR was used to detecet c-myc expression in G-MDSCs from BM,spleen and tumor tissue of TB mice.Specific si RNA was used to inhibit lncRNA Pvt1 expression(si-Pvt1)in G-MDSCs from tumor tissue,after that,the expression of c-myc was measured by q RT-PCR and Western-blot.(3)To confirm whether lncRNA Pvt1 could regulate the immunosuppressive function of G-MDSCs,si-Pvt1 was used to inhibit lncRNA Pvt1 expression in G-MDSCs from tumor tissue.The proliferation of CD4+ T cell co-cultured with G-MDSCs transfected with si-Pvt1 was detected by 3H-Td R incorporation assay.The Arg1 activity was measured by colorimetric assay and ROS production was measured by FCM.To compare the suppressive capacity and lncRNA Pvt1 expression between G-MDSCs isolated from bone marrow directly and G-MDSCs induced by IL-6 and GM-CSF,q RT-PCR was used to detect lncRNA Pvt1 expression.The inhibition of G-MDSCs to the proliferation of CD4+ T cells was measured by CFSE.FCM was used to detect the production of ROS and colorimetric assay was used to measure the activity of Arg1.(4)1×106 G-MDSCs transfected with si-Pvt1 mixed with 0.8×106 Lewis were injected subcutaneously into C57BL/6 mice and the tumor growth was monitored continuously.Furthermore,we detected Th1 and CTL proportion in spleen,draining lymph nodes(d LN),and tumor tissue of TB mice by FCM.(5)In order to investigate whether hypoxic condition in tumor microenvironment could regulate lncRNA Pvt1 expression in G-MDSCs,q RT-PCR was used to detect the level of HIF-1α m RNA in G-MDSCs from tumor tissue and spleen of TB mice.G-MDSCs from spleen were placed in a sealed container with oxygen consumption bag,which provided a hypoxic condition.q RT-PCR and Western-blot were used to detect the expression of HIF-1α.LncRNA Pvt1 expression was measured by q RT-PCR.YC-1,an inbitor of HIF-1α,was added simultaneously to hypoxia treatment,the expression of HIF-1α was detected by q RT-PCR and Western-blot,and the expression of lncRNA Pvt1 was detected by q RT-PCR.Results:(1)LncRNA Pvt1 was up-regulated in G-MDSCs isolated from tumor tissue of TB mice than that from spleen of WT mice(p<0.001).And the expression of lncRNA Pvt1 showed a gradually increasing trend in G-MDSCs from bone marrow,spleen,and tumor tissue of TB mice(p<0.01).(2)C-myc was up-regulated in G-MDSCs from tumor tissue of TB mice than that from spleen of WT mice(p<0.01).In accordance with the change of lncRNA Pvt1,c-myc expression also showed a gradually increasing trend in G-MDSCs from bone marrow,spleen,and tumor tissue of TB mice(p<0.001).After knockdown of lncRNA Pvt1 in G-MDSCs from tumor tissue,the expression of c-myc were decreased(p<0.05).(3)After knockdown of lncRNA Pvt1 in G-MDSCs sorted from tumor tissue,the inhibition of G-MDSCs to CD4+ T cell proliferation was down-regulated(p<0.05),the activity of Arg1 in G-MDSCs was decreased(p<0.05)and the production of ROS in G-MDSCs was also decreased(p<0.01).G-MDSCs induced from bone marrow by IL-6 and GM-CSF had stronger inhibition of CD4+ T cell proliferation(p<0.001),stronger Arg1 activity(p<0.001),and higher level of ROS(p<0.05)than that of G-MDSCs sorted from bone marrow directly.Meanwhile,the expression of lncRNA Pvt1 was up-regulated in G-MDSCs induced from bone marrow by IL-6 and GM-CSF than that in G-MDSCs from bone marrow directly(p<0.001).(4)By using a murine Lewis lung adenocarcinoma xenograft model,tumor growth in mice implanted with G-MDSCs transfected with si-Pvt1(si-Pvt1 group)was found to be drastically slower as compared with that in control group(p<0.05).The proportion of CTL was higher in d LN of si-Pvt1 group than that of control group(p<0.05).The proportion of CTL and Th1 in the spleen and tumor tissue had no significant difference between si-Pvt1 group and control group.(5)The level of HIF-1α m RNA was obviously up-regulated in G-MDSCs from tumor tissue than that from spleen of TB mice(p<0.001).G-MDSCs from spleen exposed to hypoxic condition expressed higher HIF-1α m RNA level(p<0.05)than normoxic condition,and protein level of HIF-1α was also up-regulated.LncRNA Pvt1 expression in G-MDSCs was up-regulated in hypoxic condition(p<0.05).YC-1,which inhibited the expression of HIF-1α,was added to hypoxia treatment of G-MDSCs,lncRNA Pvt1 expression was down-regulated(p<0.001).Conclusions: The expression of lncRNA Pvt1 was up-regulated in G-MDSCs from tumor tissue of tumor-bearing mice.Inhibition of lncRNA Pvt1 expression could weaken the immunosuppressive capacity of G-MDSCs.