| Objective:In this study,We aimed to investigate the role of IL-17A/IL-17 RA in promoting NSCLC progression.By manipulating IL-17 RA expression in NSCLC cells,we detected the role of IL-17A/IL-17 RA in promoting cell proliferation and motility in vitro,and elucidate the possible mechanisms underlying the progression-promoting effect by IL-17A/IL-17 RA in NSCLC.Methods:1.Samples of paraffin-embedded tissue sections and clinicopathological features were obtained to detect the expression of Il-17 A,IL-17 RA,MMP-9.The correlation between Il-17 A,IL-17 RA,MMP-9 and clinicopathologic characteristics was confirmed.Survival curves were drawn using the Kaplan-Meier method.2.Western blot was used to detect the difference in endogenous expression of IL-17 RA protein in different non-small cell lung cancer cell lines.Cell lines with high expression of IL-17 RA and cell lines with low expression of Il-17 RA were screened.The lentiviral packaging vector carrying GFP-interfering IL-17 RA expression was designed and synthesized,and the IL-17 RA overexpression lentiviral vector carrying GFP was designed and synthesized.The cell line H1975 with high expression of IL-17 RA was selected to establish stable interference IL-17 RA cell line by lentivirus infection;cell line A549 with low expression of IL-17 RA was selected to establish a stable over-expressing IL-17 RA cell line by lentivirus infection.3.MTT was used to measure the effect of IL-17A/IL-17 RA on the cell growth in vitro;Wound healing assays were used to investigate the effect of IL-17A/IL-17 RA on the cell motility in vitro;transwell invasion assays were used to investigate the effect of IL-17A/IL-17 RA on the cell invasion ability in vitro.4.The phosphorylation of signal transduction modules of MAPK signaling p38 weredetected after manipulating IL-17 RA expression.p38 MAPK inhibitor SB203580 was used to confirm the effect of p38 MAPK signaling in IL-17A/IL-17RA-induced tumor malignant behavior.5.The expression level of MMP-2/MMP-9 was detected after manipulating IL-17 RA expression and adding p38 MAPK inhibitor SB203580.and adding p38 MAPK inhibitor SB203580 was used to confirm the effect of MMP-2/MMP-9 in IL-17A/IL-17RA-induced tumor malignant behavior.Results:1.Il-17 A,IL-17 RA,MMP-9 expression is up-regulated in NSCLC tissues and associated with prognosisIn 60 cases of clinical specimens,the positive expression rate of IL-17 A was 76.7%,increased IL-17 A expression in NSCLC cells was correlated with decreases in pTNM stage(P < 0.05).In addition,overall survival(OS)rate for IL-17A-positive NSCLC patients were significantly lower compared to patients who were IL-17A-negative.The positive expression rate of MMP-9 was 80.0%,increased MMP-9 expression in NSCLC cells was correlated with decreases in pTNM stage(P<0.05).In addition,overall survival(OS)rate forMMP-9-positive NSCLC patients were significantly lower compared to patients who were MMP-9-negative.In 139 clinical specimens,the positive expression rate of IL-17 RA was 54.7%(76/139),increased IL-17 RA expression in NSCLC cells was correlated with decreases in cell differentiation(P=0.040),tumor size(P=0.034),and advanced stages(P=0.018).In addition,overall survival(OS)rate and disease-free survival(DFS)rate for IL-17RA-positive NSCLC patients were significantly lower compared to patients who were IL-17RA-negative.2.IL-17A/IL-17 RA interaction promotes the motility,migration and invasion of NSCLC cells in vitroTo assess the functional role of IL-17 RA expression in NSCLC cells,we determined IL-17 RA expression in NSCLC cell lines(A549,H1975 and H157).Then,H1975 cells were selected for transfection with shIL-17 RA lentivirus vector to knockdown the expression of IL-17 RA.In addition,A549 cells were transfected with IL-17 RA lentivirus to overexpress IL-17 RA.Knockdown or overexpression of IL-17 RA did not affect cell proliferation in NSCLC cells stimulated with IL-17 A.Cell invasion and migration were markedly elevated after IL-17 A treatment,in a dose-dependent manner.Cell proliferationassay showed that IL-17 A has no significant effect on the proliferation of H1975 and A549 cells.Would healing assay showed that the wound area in IL-17 RA knockdown(IL-17RA-KD)H1975 cells was significantly wider than that in NC group at both 24 h and48h(P<0.05).Furthermore,overexpression of IL-17 RA significantly decreased wound area in IL-17 RA overexpressing(IL-17RA-OE)A549 cells compared with NC group(P<0.05).Matrigel invasion assay revealed that knockdown of IL-17 RA expression significantly reduced the number of cells migrating through the Matrigel(P< 0.05).In contrast,IL-17 RA overexpression significantly increased the number of cells migrating through the Matrigel(P<0.05).3.IL-17A/IL-17 RA interaction results in up-regulated expression of MMP-2 and MMP-9The data showed that Western blot analysis showed that IL–17A stimulation increased the expression of MMP-2 and MMP-9 in H1975 and A549 cells,and protein levels of MMP-2 and MMP-9 in IL-17 RA knockdown(KD)cells were reduced compared with those in NC group,while those in IL-17 RA overexpression(OE)cells were higher than those in NC group.The data showed that the levels of phosphorylated p38 in IL-17 RA knockdown H1975 cells were decreased compared with those in NC group,while the levels of phosphorylated p38 in IL-17RA-overexpressing A549 cells were increased compared with those in NC group.4.The p38 MAPK activity is crucial for IL-17A/IL-17 RA interaction to promote motility,migration and invasion of NSCLC cellsThe motility and migration and invasion capabilities of H1975 cells,which endogenously express IL-17 RA,were significantly reduced compared to those of untreated H1975 cells.Similarly,Wound healing and Matrigel assays showed that SB203580 inhibited IL-17RA-OE A549 cells invasion and migration induced by IL-17 A.In addition,Western blotting analysis revealed that pre-treatment with SB203580 abrogated IL-17A-induced up-regulation of MMP-2 and MMP-9 in H1975 and IL-17RA-OE A549 cells.Conclusion:1.IL-17 A,IL-17 RA and MMP-9 expression are up-regulated in NSCLC tissues and are unfavorable prognostic factors for NSCLC patients.2.IL-17A/IL-17 RA interaction promotes the motility,migration and invasion of NSCLC cells in vitro3.IL-17A/IL-17 RA interaction results in up-regulated expression of MMP-2 and MMP-9;The p38 MAPK activity is crucial for IL-17A/IL-17 RA interaction to promote motility,migration and invasion of NSCLC cells. |
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