| Objective: Lung cancer has become a leading cause of male cancer death worldwide.Although advance in the treatment of lung cancer with the integration of targeted therapy, overall outcomes remain poor. Abundant studies have demonstrated that immune escape is the main cause of poor outcomes of antitumor therapy. In our study,to find a new antitumor target, we tried to investigate the regulation of long noncoding RNA (IncRNA) AK036396 to the development and immunosuppression of granulocytic myeloid-derived suppressor cells (G-MDSCs) and its role in the antitumor immune response.Methods:1 The Arraystar IncRNA microarray was used to detect the lncRNA expressing profile in G-MDSCs from tumor tissue of tumor-bearing mice. The expression of IncRNA AK036396 was confirmed by qRT-PCR. The expression of IncRNA AK036396 in different myeloid cells and different developmental stage of G-MDSCs was detected by qRT-PCR.2 The Arraystar lncRNA microarray was used to detect the expressing profile of potential target gene regulated by lncRNA in G-MDSCs from tumor tissue. The expression of target gene was confirmed by qRT-PCR. The expression of target gene in different myeloid cells was detected by qRT-PCR and Western-blot.3 FISH was used to confirm the localization of lncRNA AK036396 in the G-MDSCs from tumor. RIP and RNA pull down were used to confirm the interaction between IncRNA AK036396 and Fcnb. NCX (40μg/mL) was added into culture of G-MDSCs with lncRNA AK036396 knockdown and Western-blot was used to confirm the influence of lncRNA AK036396 on the stability of Fcnb. MG 132 (20μM) was added into culture of G-MDSCs with IncRNA AK036396 knockdown and Western-blot was used to confirm the influence of lncRNA AK036396 on the stability of Fcnb protein.IP was used to confirm the effect of lncRNA AK036396 knockdown on the ubiquitination of Fcnb.4 To confirm the regulation of Fcnb to the development and immunosuppression of G-MDSCs, specific siRNA of Fcnb was used to inhibit the expression of Fcnb in G-MDSCs from tumor tissue firstly. And then CD244 level, which is the marker of G-MDSCs development, was detected by qRT-PCR. To confirm the influence of Fcnb knockdown on the immunosuppression of G-MDSCs, colorimetric assay and FCM were used to confirm the activity of Argl and expression of ROS in G-MDSCs respectively. Additionally, [3H]-thymidine incorporation assay was used to investigate the suppressive function of G-MDSCs post Fcnb knockdown.5 Specific modified siRNA of Fcnb was administrated into tumor tissue of tumor-bearing mice to suppress the expression of Fcnb. And then the tumor progression and weight were measured. FCM was used to detect the proportion of G-MDSCs in tumor tissue after siFcnb administration. To confirm the development of G-MDSCs in tumor post siFcnb administration, the expression of CD244 in G-MDSCs from tumor was detected by qRT-PCR. To investigate the effect of siFcnb administration on the immunosuppression of G-MDSCs, the activity of Argl and expression of ROS in G-MDSCs from tumor tissue were respectively detected by colorimetric assay and FCM. And in the following, FCM was used to confirm the proportion of CD3+CD4+IFN-y+ T cells and CD3+CD8+IFN-y+ T cells in spleen,draining lymph node and tumor tissue.6 To confirm the regulation of lncRNA AK036396 to the development and immunosuppression of G-MDSCs, specific siRNA was used to inhibit the expression of IncRNA AK036396 in G-MDSCs from tumor tissue. And then the maturation of G-MDSCs from tumor tissue was investigated by detecting the expression of FicolinB(Fcnb) with qRT-PCR and Western-blot, detecting the expression of CD244 with qRT,PCR and observing morphological characteristics with Wright Giemsa staining.To make sure whether IncRNA AK036396 can regulate the immunosuppression of G-MDSCs, colorimetric assay and FCM were used to confirm the activity of Argl and expression of ROS in G-MDSCs respectively. And [3H]-thymidine incorporation assay was used to investigate the suppressive function of G-MDSCs.7 Specific modified siRNA of lncRNA AK036396 was administrated into tumor tissue of tumor-bearing mice to suppress the expression of IncRNA AK036396. And then the tumor progression and weight were measured. FCM was used to detect the proportion of G-MDSCs in tumor tissue. To confirm the development of G-MDSCs in tumor post siAK036396 administration, the expression of CD244 and Fcnb in G-MDSCs from tumor was detected by qRT-PCR and Western-blot. To investigate the effect of siAK036396 administration on the immunosuppression of G-MDSCs, the activity of Argl and expression of ROS in G-MDSCs from tumor tissue were respectively detected by colorimetric assay and FCM. And FCM was in following used to confirm the proportion of CD3+CD4+IFN-γ+ T cells and CD3+CD8+IFN-γ+ T cells in spleen,draining lymph node and tumor tissue.8 The expression of human M-ficolin, the homologue of mouse Fcnb, in the PBMCs of lung cancer patients was detected by qRT-PCR and its correlation with the clinicopathological parameters of lung cancer was analyzed.Results:1 LncRNA AK036396 was highly expressed in G-MDSCs from tumor. The expression of lncRNA AK036396 in G-MDSCs from tumor was higher compared to monocyte (P<0.001), granulocyte (P<0.001) and M-MDSCs (P<0.001) from tumor.The expression of lncRNA AK036396 decreased progressively in G-MDSCs in bone marrow (P<0.05), G-MDSCs in tumor (P<0.05) and granulocyte (P<0.05).2 Fcnb was highly expressed in G-MDSCs from tumor. The expression of Fcnb in G-MDSCs from tumor is the highest compared to monocyte (P<0.001), granulocyte(P<0.001) and M-MDSCs from tumor (P<0.001).3 LncRNA AK036396 was found to be localized in the nuclear of G-MDSCs by FISH.Results of RIP and RNA pull down demonstrated the interaction between lncRNA AK036396 and Fcnb. And knockdown of lncRNA AK036396 could decrease the stability of Fcnb via proteasome-ubiquitin pathway.4 Post knockdown of Fcnb in G-MDSCs from tomor tissue, the expression of CD244 decreased significantly (P<0.01). As the suppressive moleculars released by G-MDSCs, the Argl activity (P<0.05) and ROS production (P<0.05) were decreased significantly post Fcnb knockdown. And the suppression of G-MDSCs to the proliferation of CD4+T cells was reversed post Fcnb knockdown (P<0.05).5 After the administration of specific modified siRNA of Fcnb into tumor tissue, the tumor progression was delayed and tumor weight was decreased (P<0.05). Besides that, the proportion of G-MDSCs in tumor tissue was downregulated post siFcnb administration (P<0.05). When we tried to confirm the effect of Fcnb knockdown on the maturation of G-MDSCs in vivo, it was found the expression of CD244 in G-MDSCs from tumor was decreased (P<0.05). And as the main suppressive moleculars released by G-MDSCs, the Argl activity (P<0.05) and ROS production(P<0.01) in G-MDSCs from tumor tissue were decreased significantly. Finally, the proportion of CD3+CD4+IFN-y+ T cells in spleen (P<0.05) and tumor tissue (P<0.05)were enhanced. And CD3+CD8+IFN-γ+ T cells in spleen (P<0.05) and tumor tissue(P<0.05) were enhanced.6 Post knockdown of lncRNA AK036396 in G-MDSCs from tumor tissue, the expression of Fcnb (P<0.05) and CD244 (P<0.01), which are the marker reflecting the development of G-MDSCs, were downregulated. Morphological observation showed that segmented granulocytes were more post knockdown of IncRNA AK036396. At the same time, the immunosuppresion of G-MDSCs decreased after knockdown of lncRNA AK036396. The Argl activity was decreased significantly after the inhibition of lncRNA AK036396 expression (P<0.01) and the suppression of G-MDSCs to the proliferation of CD4+T cells was reversed (P<0.05). However, the ROS production showed no significant difference (ns).7 After the administration of specific modified siRNA of lncRNA AK036396 into tumor tissue, the tumor progression was delayed (P<0.001) and tumor weight was decreased (P<0.01). Besides that, the proportion of G-MDSCs in tumor tissue was downregulated after the administration of siAK036396 (P<0.01). The expression of Fcnb (P<0.001) and CD244 (P<0.01) chosen as the marker of G-MDSCs were decreased in G-MDSCs from tumor. Additionally, the Argl activity (P<0.01) and ROS production (P<0.001) in G-MDSCs from tumor tissue were decreased significantly. At the same time, the proportion of CD3+CD8+IFN-γ+ T cells in spleen (P<0.05),draining lymph node (P<0.05) and tumor tissue (P<0.05) were enhanced.8 Compared to the healthy control, the expression of M-ficolin was increased in the PBMCs of lung cancer patients (P<0.05). M-ficolin level was positively correlated with the proportion of MDSCs (P<0.01) and the expression of Argl (P<0.01). And M-ficolin level was positively correlated with the tumor size (P<0.05), TNM stage(P<0.05) and lymph node metastasis (P<0.001) of lung cancer.Conclusions:The expression of lncRNA AK036396 shows cell- and stage-specific. LncRNA AK036396 can regulate the immunosuppression of G-MDSCs by interacting with Fcnb. |
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