The Role And Mechanism Of Visfatin In NSCLC Migration And Invasion

Background:Currently,the incidence and mortality of lung cancer ranks first in China's malignant tumors,of which about 80%are non-small cell lung cancer.Most NSCLC patients have local invasion and/or distant metastasis at the time of presentation.Invasion and metastasis is the most essential biological feature of malignant tumors,and is the main cause of death in patients with cancer.In recent years,molecular targeted therapy has greatly improved the prognosis of patients with NSCLC who are positive for EGFR drug-sensitive mutations,but the overall 5-year survival rate of patients with NSCLC is still below 15%.Invasion and metastasis are still the main factors affecting the prognosis and treatment of patients with NSCLC.Therefore,it is important to explore the molecular markers related to the regulation of invasion and metastasis of NSCLC and to elucidate its mechanism.Visfatin,also known as pre-B cell colony-stimulating factor and nicotinamide phosphoribosyltransferase,plays a role in regulating cell signaling,insulin resistance,apoptosis,oxidative stress,and inflammation;Moreover,during tumor progression,Visfatin also has the ability to regulate a variety of key signaling pathways,such as PI3K/AKT,ERK1/2,STAT3,NF-?B,Toll-like receptor signaling pathways etc.Visfatin plays a significant role in inhibiting apoptosis,promoting proliferation,malignant transformation,tumor angiogenesis,invasion and migration,changing tumor microenvironment and escape immune surveillance.Visfatin overexpression is associated with tumorigenesis.Overexpression of visfatin is found in the serum or tissues of various solid tumors such as prostate cancer,gastric cancer,breast cancer,colorectal cancer,pancreatic cancer,and liver cancer.Studies have shown that visfatin can promote the malignant progression of endometrial cancer through PI3K/AKT and MAPK/ERK pathway;visfatin-Notch1 pathway promotes the growth of breast tumor by activating NF-?B;up-regulated visfatin plays an important role in the occurrence and metastasis of prostate cancer;visfatin promotes osteosarcoma cell migration and invasion through NF-?B/Snail-1/EMT pathway;elevated visfatin levels in serum of small cell lung cancer patients promote brain microvascular endothelial migration,and visfatin is a prospective target for small cell lung cancer brain metastasis.Visfatin is up-regulated in a variety of malignancies.The involvement of visfatin in the malignant biological behavior of tumors is closely related to the occurrence,development and poor prognosis of tumors,and can provide new targets and new ideas for the research and treatment of tumors.The visfatin inhibitor FK866 inhibits gastric cancer cell migration and anchorage-independent growth.FK866 also increases the chemosensitivity of gastric cancer cells to fluorouracil by inhibiting cell proliferation and inducing apoptosis.STF-118804,a highly specific new generation of visfatin inhibitors,significantly improved the survival rate of high-risk acute lymphoblastic leukemia in situ xenograft models.Recently,it has been shown that STF-118804 can significantly inhibit the survival and growth of pancreatic cancer in vivo and in vitro models.In short,visfatin plays an important role in the occurrence,development,prognosis evaluation and treatment of tumors,and may provide new targets and new ideas for the research and treatment of tumors.However,there are little research of visfatin in NSCLC,and the mechanism of its involvement in the development of NSCLC is not clear.This study sought to investigate the expression of visfatin in patients with NSCLC and its relationship with clinical features and prognosis,and further explore the effect of visfatin expression on invasion and metastasis of NSCLC cell lines and possible mechanisms to find potential invasion and metastasis for NSCLC patients.First.The relationship between expression of visfatin in NSCLC tissues and clinicopathological features and prognosis of patients with NSCLCObjective:1.Immunohistochemistry was used to detect the expression of visfatin in NSCLC tissues and adjacent tissues.2.To investigate the relationship between visfatin expression and clinical pathological features(gender,age,smoking index,tumor size,pathological type,degree of differentiation,lymph node metastasis,distant metastasis and EGFR mutation status)in NSCLC patients,and provide ideas of screening a new molecular marker for NSCLC.3.To analyze the relationship between the expression of visfatin and clinical pathological features and the prognosis of patients with NSCLC,aiming to provide a basis for the prognosis evaluation of NSCLC.Materials and methods:A total of 136 patients with primary NSCLC were selected for analysis.Among them,66 were ?-?A surgical resectable specimens,and 70 were paraffin-embedded specimens of stage ?B-? percutaneous lung puncture or bronchoscopy without surgical indications.The control group was the specimen of adjacent tissues that were surgically removed.Among them,75 cases of adenocarcinoma and 61 cases of squamous cell carcinoma;patients with pathologically confirmed lung adenocar-cinoma have been tested for EGFR gene mutation status.The expression of visfatin in NSCLC tissues was detected by immunohistochemistry.The expression of Visfatin in NSCLC tissue and its relationship with clinicopathological features of NSCLC patients were analyzed.Kaplan-Meier method,Log-rank test and Cox regression analysis were used to investigate the relationship between visfatin expression,clinical features and Overall Survival(OS)of patients with NSCLC.All data were statistically analyzed using SPSS 19.0 software.Results:1.Expression of Visfatin in NSCLC tissues and adjacent tissuesImmunohistochemistry showed that visfatin mainly expressed in the cytoplasm of lung cancer cells of NSCLC tissues.The positive expression rate of visfatin in NSCLC tissues was 64.7%,and that of surgically resected adjacent tissues was only 19.7%.The positive expression rate of visfatin in NSCLC cancer tissues was significantly higher than that in adjacent tissues,and the difference was statistically significant(P<0.05).2.The relationship between visfatin expression and clinical pathological features of patients with NSCLCThe positive expression rates of Visfatin in lung adenocarcinoma and squamous cell carcinoma were 72.0%and 55.7%,respectively;the positive expression rates of N1?3 and N0 were 74.7%and 46.9%,respectively;the positive expression rates in distant metastasis and no distant metastasis were 77.1%and 58.0%,respectively,and those differences were statistically significant(P<0.05).There was no significant difference with gender,age,smoking index,tumor size and differentiation(P>0.05).Among 75 cases of lung adenocarcinoma,35 cases were wild type of EGFR and 40 cases were EGFR sensitive mutants.The positive expression rates of visfatin in wild type and mutant of lung adenocarcinoma were 60.0%and 82.5%,respectively,and the difference was statistically significant(P<0.05).3.The relationship between visfatin expression,clinical pathological features and prognosis of patients with NSCLCThe median OS of visfatin positive group was 20.3 months(95%CI:13.9-26.7 months),while the median OS was not reached in the negative expression group.Univariate analysis was performed by Kaplan-Meier method,and the difference was analyzed by Log-rank test.The results showed that the OS of the visfatin positive expression group were shorter than the negative expression group.The difference was statistically significant(p<0.05)(?2=5.03,P=0.03).The univariate analysis of visfatin expression and clinical features in patients with NSCLC using COX regression showed that there was no statistical difference with gender,age,smoking index,tumor size,degree of differentiation,pathological type and EGFR mutation status(P>0.05);and a statistically significant OS difference between with or without lymph node metastasis,with or without distant metastasis,and visfatin-positive or negative expression in NSCLC patients(P<0.05);and further multivariate analysis found that visfatin expression,lymph node metastasis and distant metastasis were independent prognostic factors for OS in patients with NSCLC.Conclusions:1.The positive expression of visfatin in NSCLC tissues is significantly higher than that in adjacent tissues.2.Positive expression of visfatin is related to pathological type,lymph node metastasis,distant metastasis,EGFR mutation in NSCLC patients.There is no significant correlation between positive expression of visfatin in NSCLC patients and gender,age,smoking index,tumor size,the degree of differentiation.3.Visfatin expression,lymph node metastasis,and distant metastasis are independent prognostic factors for OS in patients with NSCLC.It is suggested that patients with positive expression of visfatin in NSCLC have shorter overall survival and worse prognosis.Visfatin may be a useful biomarker for NSCLC clinical prognosis assessment.Second.Effect of Visfatin on the migration and invasion of NSCLC cellsObjective:To investigate the effect of visfatin on the proliferation,migration and invasion of NSCLC cells in vitro.Materials and methods:100 ng/mL visfatin and its specific inhibitor were added exogenously into NSCLC cell line A549 and H358 cell culture medium.The effect of visfatin on the proliferation of NSCLC cells was detected by CCK8 assay.The effects of Visfatin on migration and invasion of NSCLC cells were examined by wound healing assay and Transwell assay.Visfatin-specific interfering RNA was transfected to observe the effect of blocking Visfatin on migration and invasion of NSCLC cells.Results:Exogenous addition of 100 ng/mL visfatin did not affect the proliferation of A549 and H358 cells,but significantly increased the number of cells entering the scratch area.Transwell experiments also showed that exogenous addition of visfatin promoted the migration and invasion of NSCLC cells.Visfatin-specific interfering RNA was transfected with liposome,which inhibited the migration and invasion of NSCLC cells.Conclusions:1.Exogenously added visfatin has no significant effect on the proliferation of NSCLC cell lines A549 and H358.Overexpression of visfatin does not affect the proliferation of A549 and H358 cells in vitro.2.1n the exogenously added visfatin A549 and H358 cells,the wound healing assays show that visfatin can promote the migration of A549 and H358 cells.Transwell experiments further confirm that overexpression of visfatin can promote the migration and invasion of A549 and H358 cells in vitro.3.Silencing of Visfatin via siRNA transfection reveals that migration of A549 and H358 cells can be significantly inhibited in vitro.Third.Visfatin promotes invasion and migration of NSCLC cells through IKK?/NF-?B/MMPs signaling pathwayObjective:We have previously identified the role of visfatin in promoting invasion and migration of A549 and H358 cells.This section aims to explore the possible mechanism by which visfatin promotes invasion and migration of NSCLC cells.Materials and methods:qRT-PCR and WB were used to detect the expression of MMP family in NSCLC cells after treatment with visfatin,and the effect was verified by MMP family-specific siRNA transfection.Using ERK1/2,p38-MAPK,PI3K/Akt,NF-?B signaling pathway specific inhibitors,explore the possible signaling pathways of visfatin to promote migration and invasion of NSCLC cells.Results:1.Visfatin treatment significantly increased the mRNA and protein levels of MMP-2/MMP-9 in NSCLC cells without affecting the expression of MMP-1,3,7,12.2.Transfection of si-MMP-2 or si-MMP-9 or both before 24h visfatin treatment significantly knocked down the expression of MMP-2 or MMP-9.The results showed that si-MMP-2 and si-MMP-9 could attenuate visfatin-induced NSCLC cell migration and invasion and have a synergistic effect.3.Inhibitors of NF-?B signaling pathway can inhibit migration and invasion of A549 and H358 cells induced by visfatin,while inhibitors of ERK1/2,p38-MAPK and PI3K/Akt signaling pathway can not.NF-?B inhibitors also attenuated visfatin-induced overexpression of MMP-2 and MMP-9 in A549 cells.4.Visfatin increased I?B?,p65 phosphorylation and NF-?B transcriptional activity in NSCLC cells.In addition,visfatin also increased IKK-?phosphorylation,and IKK-?inhibitor ACHP blocked visfatin-induced p65 activation and MMP-2 and MMP-9 overexpression.Conclusions:Visfatin can promote the migration and invasion of NSCLC cells by activating IKK-?,enhancing the phosphorylation of I?B? and p65,activating the NF-?B signaling pathway and up-regulating the expression of MMP-2/MMP-9.The visfatin/NF-?B/MMPs signaling pathway may provide a new direction for NSCLC therapy,and visfatin may become a new therapeutic target for NSCLC.