The Effect And Mechanism Of CIAPIN1 In Non-small Cell Lung Cancer Metastasis

Objective:(1)To analyze the effect of CIAPIN1 on the prognosis of NSCLC patients;(2)To study the role of CIAPIN1 in the invasion and metastasis of lung cancer cell A549;(3)To explore the mechanism of CIAPIN1’s involvement in in vitro metastasis of A549 cells,so as to provide theoretical basis for NSCLC to find new therapeutic targets.Methods:(1)CIAPIN1 protein expression in NSCLC patients and adjacent tissues was detected by immunohistochemistry and Western blotting.(2)Chi-squared test was used to analyze the correlation between CIAPIN1 expression and the clinicopathological data of NSCLC patients,and kaplan-Meier method and multivariate Cox regression model were used to evaluate the effect of CIAPIN1 on the prognosis of NSCLC patients.(3)The expression of CIAPIN1 in A549 cells was up-regulated by lentivirus infection,and the effect of CIAPIN1 on migration and invasion of A549 cells was detected by cell scratches and Transwell assay.The effects of overexpression of CIAPIN1 on the expression of specific markers of matrix metalloproteinases(MMPs)and epithelial stromal transformation(EMT)in A549 cells were detected by real-time quantitative PCR and Western blotting.(4)The influence of overexpression of CIAPIN1 on the expression of cyclin,cyclin-dependent kinases and apoptotic proteins in A549 cells was detected by Western blotting.(5)Real-time quantitative PCR and Western blotting were used to detect the change of NHE1 expression in A549 cells after overexpression of CIAPIN1,protein chip and Western blotting were used to detect the change of MAPK signaling pathway related protein expression in A549 cells after overexpression of CIAPIN1,and the effect of inhibiting NHE1 activity after overexpression of CIAPIN1 on the expression of phosphorylated ERK1/2 was detected by Western blotting.The regulatory relationship between CIAPIN1 and NHE1 and ERK1/2 was studied.(6)Cell scratches and Transwell assay were used to detect the effect of inhibition of NHE1 activity or/and phosphorylation of ERK1/2 on migration and invasion of A549 cells.(7)Real-time quantitative PCR and Western blotting were used to detect the effect of inhibition of NHE1 activity or/and phosphorylation of ERK1/2 on the expression of specific markers of MMPs and EMT in A549 cells.(8)To observe the effect of CIAPIN1 on the growth of A549 cells in vivo through tumor formation experiment in nude mice.Results:(1)CIAPIN1 expression was decreased in NSCLC tissues;(2)CIAPIN1 expression in cancer tissues is closely related to pathological type,tumor size,lymph node invasion and clinical stage;(3)CIAPIN1 expression in cancer tissues is positively correlated with the overall survival(OS)of NSCLC patients,and is an independent prognostic factor for OS.(4)Overexpression of CIAPIN1 can inhibit migration and invasion of A549 cells,down-regulate the expression of MMP2,MMP9 and MMP14 in A549 cells,inhibit the development of cell EMT and cell cycle,and induce apoptosis.(5)Overexpression of CIAPIN1 can inhibit the expression of NHE1 and phosphorylated ERK1/2 in A549 cells,and inhibition of NHE1 activity can further reduce the phosphorylation level of ERK1/2.(6)After overexpression of CIAPIN1,inhibition of NHE1 activity or phosphorylation of ERK1/2 can inhibit migration and invasion of A549 cells,down-regulate the expression of MMP2,MMP9 and MMP14,and inhibit cell EMT.Meanwhile,inhibition of NHE1 activity and phosphorylation of ERK1/2 can further inhibit migration and invasion of A549 cells,down-regulate the expression of MMP2,MMP9 and MMP14,and inhibit cell EMT.(7)Overexpression of CIAPIN1 can inhibit tumor formation in nude mice.Conclusion: This study demonstrated that low CIAPIN1 expression is an independent risk factor for OS in NSCLC patients,and that overexpression of CIAPIN1 can inhibit the invasion and metastasis of lung cancer cells A549,which is related to the inhibition of NHE1 activity,and the ERK1/2 signaling pathway is involved in this process.This suggests that CIAPIN1 may be a potential therapeutic target for NSCLC.