| Background:Myeloid-derived suppressor cells(MDSCs)are a heterogeneous group of cells derived from bone marrow progenitor cells and immature myeloid cells,mainly including polymorphonuclear myeloid-derived suppressor cells(PMN-MDSCs)and monocytic myeloid-derived suppressor cells(M-MDSCs).PMN-MDSCs account for 80%of the total MDSCs.Studies have shown that the proportion of MDSCs in the peripheral blood of healthy people is low.In contrast,the percentage of MDSCs in the peripheral blood of patients with lung cancer,or breast cancer,or nasopharyngeal cancer,or rectal cancer,or bladder cancer is increased significantly.The high amount of MDSCs is closely related to the shortened survival time in cancer patients.Previous studies have reported that MDSCs play an essential role in the immunosuppressive state of the tumor microenvironment by secreting large amounts of secretory immunosuppressive factors such as interleukin 10(IL-10),arginase 1(ARG1),and reactive oxygen species(ROS),and inducible nitric oxide synthase(NOS2).MDSCs inhibit the proliferation and cytotoxicity of CD8~+T cells,promoting the proliferation of tumor-associated macrophages(TAMs)and Regulatory T cells(Tregs),and eventually help tumor cells to escape immune surveillance,leading to tumor progression.Recent studies have reported that MDSCs can regulate the biological behavior of tumor cells.MDSCs can promote the proliferation of multiple myeloma cells by up-regulating the expression of IL-6 and activating the MAPK signaling pathway.Besides,MDSCs can promote the proliferating,migrating,and invading of oral squamous carcinoma cells.MDSCs can also promote metastasis of prostate cancer.The number of MDSCs was positively correlated with the number of lung metastases in mice with prostate cancer.These results suggest that MDSCs not only exert a powerful immunosuppressive function but also have a regulatory effect on the proliferating,migrating,and invading of tumor cells.In the previous study,we observed that the number of MDSCs increased over time in peripheral blood and transplanted tumors of Lewis lung cancer(LL2)-bearing mice,the expression levels of proliferation-related protein Ki67 in transplanted tumors were up-regulated,the expression of metastasis-related proteins Vimentin,MMP2,and MMP9 in transplanted tumors were up-regulated,while the expression of E-cadherin was down-regulated.These results suggest that MDSCs may be closely related to the proliferation and metastasis of LL2 cells.However,whether PMN-MDSCs or M-MDSCs are involved in regulating the proliferating,migrating,and invading of LL2 cells in vitro,and the related molecular mechanisms are still unknown.This series of questions have yet to be clarified.IL-17A is one member of the interleukin 17 families.More and more studies have shown that IL-17A is closely related to the occurrence and development of tumors.IL-17A participates in the regulation of malignant biological behavior of tumor cells by binding to its receptor IL-17RA,activating downstream genes related to proliferation and metastasis such as TRAF1,TRAF6,MMP9,MMP3,and C-Jun,etc.Recently,studies have shown that spleen-derived MDSCs express IL-17A in breast cancer tumor-bearing mice.Our previous studies have also confirmed that LL2 cells expressed IL-7RA.PMN-MDSCs derived from the spleen of LL2-bearing mouse expressed and secreted IL-17A.Further studies have found that IL-17A can enhance the proliferating,migrating,and invading ability of LL2 cells in vitro.IL-17A up-regulates the expression levels of proliferation and metastasis-related proteins in LL2 cells,including TRAF1,MMP3,MMP9,etc.The above research results suggest that PMN-MDSCs may regulate the biological behavior of LL2 cells by secreting IL-17A.Based on these results,we speculate that PMN-MDSCs promote the proliferating,migrating,and invading of LL2 cells through up-regulating proliferation and metastasis-related target genes by activating the IL-17A/IL-17RA signaling pathway in a paracrine manner.Blocking the expression of IL-17RA in LL2 cells may weaken the promoting effect of PMN-MDSCs on proliferating,migrating,and invading in LL2 cells.Objectives:1.To explore the effect of PMN-MDSCs on the proliferating,migrating and invading of LL2 cells;2.To verify the role of IL-17A/IL-17RA signaling pathway in the promoting effect of PMN-MDSCs on proliferating,migrating,and invading in LL2 cells.Methods:(1)Flow cytometry was applied to detect the quantity change of CD11b~+Gr-1~+MDSCs in peripheral blood of LL2-bearing mice at day 1,7,14,and21 after transplanted tumor volume reached 0.1 cm~3.(2)Immunofluorescence staining was applied to detect the fluorescence intensity changes of Gr-1 in LL2-bearing mice at day 1,7,14,and 21 after transplanted tumor volume reached0.1 cm~3.(3)Western Blot was used to detect the levels of Ki67,E-cadherin,Vimentin,MMP2,and MMP9 protein in transplanted tumors of LL2-bearing mice at day 1,7,14 and 21 after transplanted tumor volume reached 0.1 cm~3.(4)Flow cytometry was applied to detect the purity of PMN-MDSCs in myeloid cells derived from the spleen of LL2-bearing mice in different cytokine concentration groups.(5)CCK-8 assay was used to detect the proliferation capability of LL2 cells after co-cultured with PMN-MDSCs for 48 hours at a cell concentration of 1:1,1:3,and1:5(LL2:PMN-MDSCs).(6)Transwell assay was applied to detect the migration capability of LL2 cells after co-cultured with PMN-MDSCs for 48 hours at a cell concentration of 1:1,1:3,and 1:5(LL2:PMN-MDSCs).(7)Transwell assay was applied to detect the invasion capability of LL2 cells after co-cultured with PMN-MDSCs for 48 hours at a cell concentration of 1:1,1:3,and1:5(LL2:PMN-MDSCs).(8)Western Blot was applied to detect the expression levels of Ki67,E-cadherin,Vimentin,MMP2,and MMP9 protein in LL2 cells after co-cultured with PMN-MDSCs for 48 hours at a cell concentration of 1:1,1:3,and1:5(LL2:PMN-MDSCs).(9)Transcriptome sequencing was used to detect the changes in gene expression in LL2 cells after co-cultured with PMN-MDSCs at a cell concentration of 1:3(LL2:PMN-MDSCs)after 48 hours.(10)KEGG analysis was used to find the possible signal pathways of the differentially expressed genes which detected by transcriptome sequencing.(11)q RT-PCR was applied to detect the m RNA levels of genes,including TRAF1,TRAF3,TRAF6,PI3K,AKT MMP3,MMP9,and C-Jun.(12)Western Blot was used to detect the protein levels of TRAF1,TRAF3,TRAF6,PI3K,AKT,MMP3,MMP9,and C-Jun.(13)Flow cytometry was used to detect the expression levels of IL-17A and IL-17RA in PMN-MDSCs and LL2 cells,respectively.(14)Immunofluorescence staining was used to detect the expression levels of IL-17A and IL-17RA in PMN-MDSCs and LL2 cells,respectively.(15)sh RNA-IL-17RA lentivirus was transfected into LL2cells and establish cell line LL2/IL-17RA(-).(16)q RT-PCR was used to detect the m RNA expression levels of IL-17RA in LL2/IL-17RA(-)cells.(17)Western Blot was used to detect the protein levels of IL-17RA in LL2/IL-17RA(-)cells.(18)ELISA assay was used to detect the levels of IL-17A secreted by PMN-MDSCs after PMN-MDSCs cultured in vitro for 24,48,and 72 hours.(19)CCK-8 assay was used to detect the proliferation capability of LL2 and LL2/IL-17RA(-)cells in different groups.(20)Transwell assay was used to detect the migration capacity of LL2 and LL2/IL-17RA(-)cells in different groups.(21)Transwell assay was used to detect the invasion capacity of LL2 and LL2/IL-17RA(-)cells in different groups.(22)Western Blot was used to detect the TRAF1,TRAF6,MMP3,MMP9,and C-Jun protein levels of LL2 and LL2/IL-17RA(-)cells in different groups.Results:1.The results of flow cytometry showed that CD11b~+Gr-1~+MDSCs ratios in peripheral blood of LL2-bearing mice on day 1,7,14 and 21 were significantly different by F test(P<0.01);Pairwise comparison:there was significant difference among CD11b~+Gr-1~+MDSCs ratios in the peripheral blood of LL2-bearing mice on day 1 and on day 7,14,21(P<0.05,P<0.05,P<0.01);there was a significant difference between CD11b~+Gr-1~+MDSCs ratios in peripheral blood of LL2-bearing mice on day 7 and day 21(P<0.01);there was a significant difference between CD11b~+Gr-1~+MDSCs ratios in peripheral blood of LL2-bearing mice on day 14 and day 21(P<0.05);2.Immunofluorescence staining results showed that Gr-1 fluorescence intensity in the transplanted tumor of LL2-bearing mice on day 1,7,14,and 21 were significantly different by F test(P<0.001).Pairwise comparison:there was significant difference among Gr-1 fluorescence intensity in the transplanted tumor of LL2-bearing mice on day 1 and day 7,14,21(P<0.05,P<0.01,P<0.001);there was a significant difference among Gr-1 fluorescence intensity in the transplanted tumor of LL2-bearing mice on day 7 and on day 14,21(P<0.01,P<0.001);there was a significant difference between Gr-1 fluorescence intensity in the transplanted tumor of LL2-bearing mice on day 14 and day 21(P<0.01);3.Western Blot results showed that,Ki-67,MMP9,E-cadherin,MMP2 and Vimentin protein levels in transplanted tumor of LL2-bearing mice on day 1,7,14and 21 were significantly different by F test(P<0.01,P<0.01,P<0.001,P<0.001,P<0.01);Pairwise comparison:there was significant difference among the protein levels of E-cadherin,MMP2 in transplanted tumor of LL2-bearing mice on day 1and day 7(P<0.05,P<0.01);there was significant difference between the protein levels of Ki-67,MMP9,E-cadherin,MMP2 and Vimentin in transplanted tumor of LL2-bearing mice on day 1 and day 14(P<0.05,P<0.05,P<0.05,P<0.001,P<0.05);there was significant difference between the protein levels of Ki-67,MMP9,E-cadherin,MMP2 and Vimentin in transplanted tumor of LL2-bearing mice on day1 and day 21(P<0.05,P<0.05,P<0.01,P<0.001,P<0.05);there was significant difference between the protein levels of Ki-67,MMP9,MMP2 and Vimentin in transplanted tumor of LL2-bearing mice on day 7 and day 14(P<0.05,P<0.05,P<0.01,P<0.05);there was significant difference between the protein levels of Ki-67,MMP9,E-cadherin,MMP2 and Vimentin in transplanted tumor of LL2-bearing mice on day 7 and day 21(P<0.05,P<0.05,P<0.01,P<0.001,P<0.05);there was significant difference between the protein levels of Ki-67,MMP9,E-cadherin,MMP2 and Vimentin in transplanted tumor of LL2-bearing mice on day14 and day 21(P<0.05,P<0.05,P<0.01,P<0.05,P<0.05);4.The results of flow cytometry showed that the cell purity of PMN-MDSCs,which derived from spleen of LL2-bearing mice,cultured with 20 ng/ml GM-CSF plus 5 ng/ml IL-6 in DMEM medium was significantly higher than that in the 10ng/ml GM-CSF plus 2.5 ng/ml IL-6 group(P<0.05);5.The CCK-8 results showed that,the OD value of LL2 cells in the blank control group,the LL2+GM-CSF plus IL-6 group,the co-culture group(LL2:PMN-MDSCs 1:1),the co-culture group(LL2:PMN-MDSCs 1:3)and the co-culture group(LL2:PMN-MDSCs 1:5)were significantly different by F test(P<0.05);Pairwise comparison:there was significant difference among the OD value of LL2cells in the blank control group and the co-culture group(LL2:PMN-MDSCs 1:1),the co-culture group(LL2:PMN-MDSCs 1:3),the co-culture group(LL2:PMN-MDSCs 1:5)(P<0.05,P<0.01,P<0.01);there was significant difference among the OD value of LL2 cells in the LL2+GM-CSF plus IL-6 group and the co-culture group(LL2:PMN-MDSCs 1:1),the co-culture group(LL2:PMN-MDSCs1:3),the co-culture group(LL2:PMN-MDSCs 1:5)(P<0.05,P<0.01,P<0.01);there was significant difference among the OD value of LL2 cells in the co-culture group(LL2:PMN-MDSCs 1:1)and the co-culture group(LL2:PMN-MDSCs 1:3),the co-culture group(LL2:PMN-MDSCs 1:5)(P<0.05,P<0.01);6.Transwell migration assay results showed that the number of migrated LL2cells in the blank control group,the LL2+GM-CSF plus IL-6 group,the co-culture group(LL2:PMN-MDSCs 1:1),the co-culture group(LL2:PMN-MDSCs 1:3)and the co-culture group(LL2:PMN-MDSCs 1:5)were significantly different by F test(P<0.001);Pairwise comparison:there was a significant difference among the number of migrated LL2 cells in the blank control group and the co-culture group(LL2:PMN-MDSCs 1:1),the co-culture group(LL2:PMN-MDSCs 1:3),the co-culture group(LL2:PMN-MDSCs 1:5)(P<0.05,P<0.001,P<0.001);there was a significant difference among the number of migrated LL2 cells in the co-culture group(LL2:PMN-MDSCs 1:3)and the LL2+GM-CSF plus IL-6 group,the co-culture group(LL2:PMN-MDSCs 1:1)(P<0.001,P<0.01);7.Transwell invasion assay results showed that the number of invaded LL2 cells in the blank control group,the LL2+GM-CSF plus IL-6 group,the co-culture group(LL2:PMN-MDSCs 1:1),the co-culture group(LL2:PMN-MDSCs 1:3)and the co-culture group(LL2:PMN-MDSCs 1:5)were significantly different by F test(P<0.001);Pairwise comparison:there was a significant difference among the number of invaded LL2 cells in the blank control group and the co-culture group(LL2:PMN-MDSCs 1:1),the co-culture group(LL2:PMN-MDSCs 1:3),the co-culture group(LL2:PMN-MDSCs 1:5)(P<0.01,P<0.001,P<0.001);there was a significant difference among the number of invaded LL2 cells in the co-culture group(LL2:PMN-MDSCs 1:3)and the LL2+GM-CSF plus IL-6 group,the co-culture group(LL2:PMN-MDSCs 1:1)(P<0.001,P<0.01);8.Western Blot results showed that,Ki67,MMP9,E-cadherin,MMP2 and Vimentin protein levels in the blank control group,the co-culture group(LL2:PMN-MDSCs 1:1),the co-culture group(LL2:PMN-MDSCs 1:3)and the co-culture group(LL2:PMN-MDSCs 1:5)were significantly different by F test(P<0.001,P<0.001,P<0.001,P<0.05,P<0.001);Pairwise comparison:there was significant difference between Ki67,MMP9,E-cadherin,MMP2 and Vimentin protein levels in the blank control group and the co-culture group(LL2:PMN-MDSCs 1:3)(P<0.001,P<0.01,P<0.001,P<0.05,P<0.001);there was significant difference between Ki67,MMP9,E-cadherin,MMP2 and Vimentin protein levels in the blank control group and the co-culture group(LL2:PMN-MDSCs 1:5)(P<0.001,P<0.001,P<0.001,P<0.05,P<0.001);there was significant difference between Ki67,MMP9,E-cadherin,MMP2 and Vimentin protein levels in the co-culture group(LL2:PMN-MDSCs 1:1)and the co-culture group(LL2:PMN-MDSCs 1:3)(P<0.001,P<0.01,P<0.01,P<0.05,P<0.05);there was significant difference between Ki67,MMP9,E-cadherin,MMP2 and Vimentin protein levels in the co-culture group(LL2:PMN-MDSCs 1:1)and the co-culture group(LL2:PMN-MDSCs 1:5)(P<0.001,P<0.001,P<0.01,P<0.05,P<0.05);9.Transcriptome sequencing results showed that 330 genes were significantly up-regulated and 345 genes were significantly down-regulated in LL2 cells after co-cultured with PMN-MDSCs for 48 hours at a cell concentration ratio of 1:3(LL2:PMN-MDSCs);10.The results of KEGG pathway enrichment analysis showed that the differentially expressed genes in transcriptome sequencing were closely related with the IL-17A signaling pathway;11.The q RT-PCR results showed that after co-cultured with PMN-MDSCs for48 hours at 1:3 cell concentration ratio(LL2:PMN-MDSCs),the m RNA levels of TRAF1,TRAF6,PI3K,Akt,MMP3,MMP9,and C-Jun of LL2 cells were significantly higher than that in the blank control group(P<0.01,P<0.05,P<0.01,P<0.001,P<0.001,P<0.001,P<0.01),these genes were related to tumor proliferation and metastasis;12.Western Blot results showed that,after co-cultured with PMN-MDSCs at1:3 cell concentration ratio(LL2:PMN-MDSCs)for 48 hours,the protein levels of the co-culture group LL2 cells in TRAF1,TRAF3,TRAF6,PI3K,Akt,MMP3,MMP9,and C-Jun were significantly higher than the blank control group(P<0.05,P<0.001,P<0.001,P<0.001,P<0.001,P<0.01,P<0.05,P<0.01),these genes were related to tumor proliferation and metastasis;13.Flow cytometry results showed that:IL-17A~+PMN-MDSCs accounted for18.3%±6.23%of total PMN-MDSCs;IL-17RA~+LL2 accounted for 63.91%±8.29%of total LL2 cells;14.The results of immunofluorescence staining showed that the fluorescence intensity of IL-17A in PMN-MDSCs was 2.36±0.41;the fluorescence intensity of IL-17RA in LL2 cells was 5.61±0.73;15.ELISA results showed that,after PMN-MDSCs were cultured in vitro for 24hours,48 hours and 72 hours,the concentration of IL-17A secreted by PMN-MDSCs was significantly different by F test(P<0.01);Pairwise comparison:there was a significant difference among the concentration of IL-17A secreted by PMN-MDSCs in the 24 hours group and the 48 hours group,the 72 hours group(P<0.01,P<0.05);there was a significant difference between the concentration of IL-17A secreted by PMN-MDSCs in the 48 hours group and the 72 hours group(P<0.01);16.The q RT-PCR results showed that the m RNA expression level of IL-17RA in LL2/IL-17RA(-)cells was significantly lower than that in the blank control group LL2 cells(P<0.001);17.Western Blot results showed that the protein level of IL-17RA in LL2/IL-17RA(-)cells was significantly lower than that in the blank control group LL2 cells(P<0.001);18.The CCK-8 results showed that,the OD value of LL2 cells in the blank control group,the LL2+IL-17A group,the LL2+PMN-MDSC group,the LL2/IL-17RA(-)group,the LL2/IL-17RA(-)+IL-17A group and the LL2/IL-17RA(-)+PMN-MDSC group were significantly different by F test(P<0.001);Pairwise comparison:there was significant difference among the OD value of LL2 cells in the blank control group and the LL2+IL-17A group,the LL2+PMN-MDSC group,the LL2/IL-17RA(-)+PMN-MDSC group(P<0.01,P<0.01,P<0.05);there was significant difference among the OD value of LL2 cells in the LL2+IL-17A group and the LL2+PMN-MDSC group,the LL2/IL-17RA(-)group,the LL2/IL-17RA(-)+IL-17A group,the LL2/IL-17RA(-)+PMN-MDSC group(P<0.05,P<0.01,P<0.01,P<0.05);there was significant difference among the OD value of LL2 cells in the LL2+PMN-MDSC group and the LL2/IL-17RA(-)group,the LL2/IL-17RA(-)+IL-17A group,the LL2/IL-17RA(-)+PMN-MDSC group(P<0.01,P<0.01,P<0.01);there was a significant difference between the OD value of LL2 cells in the LL2/IL-17RA(-)group and the LL2/IL-17RA(-)+PMN-MDSC group(P<0.05);there was a significant difference between the OD value of LL2 cells in the LL2/IL-17RA(-)+IL-17A group and the LL2/IL-17RA(-)+PMN-MDSC group(P<0.05);19.Transwell Migration assay results showed that,the number of migrated LL2cells in the blank control group,the LL2+IL-17A group,the LL2+PMN-MDSC group,the LL2/IL-17RA(-)group,the LL2/IL-17RA(-)+IL-17A group and the LL2/IL-17RA(-)+PMN-MDSC group were significantly different by F test(P<0.001);Pairwise comparison:there was significant difference among the number of migrated LL2 cells in the blank control group and the LL2+IL-17A group,the LL2+PMN-MDSC group,the LL2/IL-17RA(-)+PMN-MDSC group(P<0.01,P<0.001,P<0.05);there was significant difference among the number of migrated LL2 cells in the LL2+IL-17A group and the LL2+PMN-MDSC group,the LL2/IL-17RA(-)group,the LL2/IL-17RA(-)+IL-17A group,the LL2/IL-17RA(-)+PMN-MDSC group(P<0.01,P<0.001,P<0.001,P<0.05);there was significant difference among the number of migrated LL2 cells in the LL2+PMN-MDSC group and the LL2/IL-17RA(-)group,the LL2/IL-17RA(-)+IL-17A group,the LL2/IL-17RA(-)+PMN-MDSC group(P<0.001,P<0.001,P<0.001);there was significant difference between the number of migrated LL2 cells in the LL2/IL-17RA(-)group and the LL2/IL-17RA(-)+PMN-MDSC group(P<0.05);there was significant difference between the number of migrated LL2 cells in the LL2/IL-17RA(-)+IL-17A group and the LL2/IL-17RA(-)+PMN-MDSC group(P<0.05);20.Transwell invasion assay results showed that,the number of invaded LL2cells in the blank control group,the LL2+IL-17A group,the LL2+PMN-MDSC group,the LL2/IL-17RA(-)group,the LL2/IL-17RA(-)+IL-17A group and the LL2/IL-17RA(-)+PMN-MDSC group were significantly different by F test(P<0.001);Pairwise comparison:there was significant difference among the number of invaded LL2 cells in the blank control group and the LL2+IL-17A group,the LL2+PMN-MDSC group,the LL2/IL-17RA(-)+PMN-MDSC group(P<0.001,P<0.001,P<0.05);there was significant difference among the number of invaded LL2 cells in the LL2+IL-17A group and the LL2+PMN-MDSC group,the LL2/IL-17RA(-)group,the LL2/IL-17RA(-)+IL-17A group,the LL2/IL-17RA(-)+PMN-MDSC group(P<0.01,P<0.001,P<0.001,P<0.05);there was significant difference among the number of invaded LL2 cells in the LL2+PMN-MDSC group and the LL2/IL-17RA(-)group,the LL2/IL-17RA(-)+IL-17A group,the LL2/IL-17RA(-)+PMN-MDSC group(P<0.001,P<0.001,P<0.001);there was significant difference between the number of invaded LL2 cells in the LL2/IL-17RA(-)group and the LL2/IL-17RA(-)+PMN-MDSC group(P<0.05);there was significant difference between the number of invaded LL2 cells in the LL2/IL-17RA(-)+IL-17A group and the LL2/IL-17RA(-)+PMN-MDSC group(P<0.05);21.Western Blot results showed that,the protein levels of MMP9,TRAF6,MMP3,TRAF1 and C-Jun of LL2 cells in the blank control group,the LL2+IL-17A group,the LL2+PMN-MDSC group,the LL2/IL-17RA(-)group,the LL2/IL-17RA(-)+IL-17A group and the LL2/IL-17RA(-)+PMN-MDSC group were significantly different by F test(P<0.01,P<0.001,P<0.01,P<0.001,P<0.001);Pairwise comparison:there was significant difference between the protein levels of TRAF6,MMP3,TRAF1 and C-Jun of LL2 cells in the blank control group and the LL2+IL-17A group(P<0.001,P<0.05,P<0.001,P<0.01);there was significant difference between the protein levels of MMP9,TRAF6,MMP3,TRAF1 and C-Jun of LL2 cells in the blank control group and the LL2+PMN-MDSC group(P<0.001,P<0.001,P<0.01,P<0.001,P<0.001);there was significant difference between the protein levels of MMP9,TRAF6,MMP3,TRAF1 and C-Jun of LL2 cells in the blank control group and the LL2/IL-17RA(-)+PMN-MDSC group(P<0.01,P<0.001,P<0.05,P<0.001,P<0.01);there was significant difference between the protein levels of MMP9,TRAF6,MMP3,TRAF1 and C-Jun of LL2 cells in the LL2+IL-17A group and the LL2+PMN-MDSC group(P<0.001,P<0.05,P<0.05,P<0.05,P<0.05);there was significant difference between the protein levels of TRAF6,MMP3,TRAF1 and C-Jun of LL2 cells in the LL2+IL-17A group and the LL2/IL-17RA(-)group(P<0.001,P<0.05,P<0.001,P<0.01);there was significant difference between the protein levels of TRAF6,MMP3,TRAF1 and C-Jun of LL2cells in the LL2+IL-17A group and the LL2/IL-17RA(-)+IL-17A group(P<0.001,P<0.05,P<0.001,P<0.01);there was significant difference between the protein levels of MMP9,TRAF6,MMP3,TRAF1 and C-Jun of LL2 cells in the LL2+PMN-MDSC group and the LL2/IL-17RA(-)group,the LL2/IL-17RA(-)+IL-17A group(P=0.000～0.001);there was significant difference between the protein levels of MMP9,TRAF6,MMP3,TRAF1 and C-Jun of LL2 cells in the LL2+PMN-MDSC group and the LL2/IL-17RA(-)+PMN-MDSC group(P=0.017～0.036);there was significant difference between the protein levels of MMP9,TRAF6,MMP3,TRAF1 and C-Jun of LL2 cells in the LL2/IL-17RA(-)group and the LL2/IL-17RA(-)+PMN-MDSC group(P<0.01,P<0.001,P<0.05,P<0.001,P<0.01);there was significant difference between the protein levels of MMP9,TRAF6,MMP3,TRAF1 and C-Jun of LL2 cells in the LL2/IL-17RA(-)IL-17A group and the LL2/IL-17RA(-)+PMN-MDSC group(P<0.01,P<0.001,P<0.05,P<0.001,P<0.01).Conclusions:(1)PMN-MDSCs promoted the proliferating,migrating and invading of LL2cells in vitro,and significantly increased the expression levels of proliferation and metastasis-related proteins in LL2,such as Ki67,Vimentin,MMP2,MMP9,etc.;(2)Transcriptome sequencing and KEGG signaling pathway enrichment analysis showed that,after co-cultured with PMN-MDSCs,the most differentially expressed genes related to proliferation and metastasis enriched in IL-17A signaling pathway in LL2;(3)Blocking the IL-17RA expression could weaken the promotion effect of PMN-MDSCs and IL-17A on the proliferating,migrating and invading of LL2 cells in vitro,also could weaken the up-regulation effect of PMN-MDSCs and IL-17A on the expression of proliferation and metastasis-related proteins including TRAF1,TRAF6,MMP3,and C-Jun in LL2 cells. |
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