| Objective: Lung cancer is currently one of the malignant tumors with the highest morbidity and mortality in the world,and it is a great threat to the health and life of the population.According to statistics,about 80% of lung cancers are non-small cell lung cancer(NSCLC).For early stage non-small cell lung cancer,surgery is still the best treatment at this stage.However,for advanced lung cancer,the current treatment methods are still limited.Local recurrence or systemic metastasis may occur even after surgical treatment.This is also the main reason for the high mortality rate of lung cancer.The treatment of lung cancer has developed rapidly in recent years,and new treatment methods including targeted therapy and immunotherapy have emerged.These new treatments have indeed improved the 5-year survival rate of lung cancer patients,but they are far from enough.Therefore,it is imperative to discover new therapeutic targets and explore new therapeutic methods.It is still extremely important and valuable to explore the occurrence and development of NSCLC.Analysis of the Gene Expression Profiling Interactive Analysis(GEPIA)database revealed that Kelch-like 17(KLHL17)protein is overexpressed in non-small cell lung cancer(NSCLC)including adenocarcinoma and squamous cell carcinoma.However,there are few studies on KLHL17 at present,especially its biological function and mechanism of action in non-small cell lung cancer are still unclear.Therefore,this study aims to explore the biological function and mechanism of KLHL17 in non-small cell lung cancer,and to find new therapeutic targets for non-small cell lung cancer.Methods: Firstly,we used GEPIA database to mining data and analyze the differences of gene expression between NSCLC tumor tissues and normal tissues.At the same time,we used oncomine database to analyze each data set of NSCLC gene expression array.The m RNA expression of KLHL17 in clinical NSCLC specimens was analyzed and compared with control group.It was observed that the expression level of KLHL17 in lung adenocarcinoma or lung squamous cell carcinoma was significantly higher than that in normal lung tissue.Therefore,we hypothesized that KLHL17 may play a role in the occurrence and development of lung cancer.In the follow-up study,we compared the expression of KLHL17 in 173 clinical tissue samples by immunohistochemical method,and randomly selected 39 pairs of fresh tissue samples(cancer tissues and adjacent tissues)from the 173 tissue samples for Western blot experiment,and then analyze the expression level of KLHL17.We used Kaplan-Meier plotter to analyze the survival time of the KLHL17 high expression group and low expression group,and compared the differences between the two groups.In the second part of this study,we first detected the expression of KLHL17 in the five NSCLC cell lines(A549,SK,H460,H1299 and H292)and normal bronchial epithelial cell line(HBE)by Western blot,and observed the cell localization of KLHL17 in the above five NSCLC cell lines and normal bronchial epithelial cell line by immunofluorescence and cytochemical experiments.After that,we overexpressed the expression of KLHL17 by transfecting KLHL17-Flag plasmid and inhibited the expression of KLHL17 by si KLHL17 in the NSCLC cell line.We used MTS assay and colony formation assay to detect the proliferation of NSCLC cells,and used Transwell cell migration test to detect the migration ability of NSCLC cells.In the third part of this study,we first used the GEO database for GSEA analysis to find the signal pathways and genes related to NSCLC enrichment.It was found that KLHL17 and MAPK signal pathways were closely related to NSCLC.Subsequently,we conducted Western blot assay to explore the changes of the expression of proteins related to Ras/MAPK signal pathway(RAS,p-ERK)and its downstream proteins related to cell function(Cyclin D1,CDK4,MMP2,Rho A)in NSCLC cells after up-regulating or down-regulating the expression of KLHL17 by KLHL17-Flag plasmid or si KLHL17.In particular,the NSCLC cells were treated with Salarasib,a kind of specific inhibitor of Ras,after transfected by KLHL17-Flag,and Western blot was conducted to detect the changes of proteins related to Ras/MAPK signal pathway and its downstream proteins related to cell function.Similarly,we used MTS experiment,colony formation experiment and Transwell migration experiment to observe the proliferation and migration ability of NSCLC cells,which was treated with Salarasib after transfected by KLHL17-Flag.Results: 1.In the process of information mining in the GEPIA database,we observed that the expression level of KLHL17 in NSCLC was significantly higher than that in normal lung tissue.From the Oncomine database,we found that the expression level of KLHL17 in NSCLC is higher than other tumors.Kaplan-Meier analysis showed that the survival time of lung cancer patients with high expression of KLHL17 was shorter than that of low expression group.2.The expression of KLHL17 in 173 clinical tissue specimens was compared by immunohistochemistry.The results showed that the expression of KLHL17 was related to the degree of NSCLC differentiation(P<0.05),and was significantly related to tumor size,lymph node metastasis,and TNM staging(P<0.005).3.The results of cellular immunofluorescence and cellular immunochemistry showed that KLHL17 is highly expressed in NSCLC cell lines and low in HBE cells,and KLHL17 is mainly located in the nucleus in HBE and NSCLC cell lines.4.The results of MTS analysis showed that the proliferation ability of NSCLC(A549,H1299 and SK cells)was significantly enhanced after KLHL17 overexpression by transfection of KLHL17-Flag.In contrast,knockdown of KLHL17 by si KLHL17 in the NSCLC cell lines suppressed the proliferation of tumor cells.5.Colony formation experiments showed that the migration ability of NSCLC(A549,H1299 and SK cells)was significantly enhanced after KLHL17 overexpression by transfection of KLHL17-Flag.In contrast,knockdown of KLHL17 by si KLHL17 in the NSCLC cell lines suppressed the migration of tumor cells.6.Transwell experiments showed that the high expression of KLHL17 promoted the migration ability of NSCLC(A549,H1299 and SK cells),while the inhibition of KLHL17 by si KLHL17 reduced the migration ability of NSCLC(A549,H1299 and SK cells).7.Through the GSEA analysis by using GEO database,we found that KLHL17 and MAPK signal pathways are closely related to NSCLC.Overexpression of KLHL17 in NSCLC(A549,H1299 and SK cells)promoted the expression of Ras,p-ERK and downstream proteins related to cell function(Cyclin D1,CDK4,MMP2,Rho A).In contrast,knockdown KLHL17 by si KLHL17 in NSCLC(A549,H1299 and SK cells)inhibited the expression of Ras,p-ERK and downstream proteins related to cell function(Cyclin D1,CDK4,MMP2,Rho A).After treating KLHL17-Flag transfected NSCLC(A549,H1299 and SK cells)with the Ras specific inhibitor Salarasib,it was found that the expression of Ras/MAPK signaling pathway related proteins(Ras,p-ERK)and their downstream functional proteins(Cyclin D1,CDK4,MMP2,Rho A)decreased.It was observed that the proliferation and migration ability of NSCLC(A549,H1299 and SK cells)were inhibited through MTS experiment,colony formation experiment and Transwell migration experiment.Conclusion: 1.KLHL17 expression is significantly higher in the tumor tissues from patients with NSCLC,compared with normal lung tissues;upregulated KLHL17 expression is positively correlated with tumor size,lymph node metastasis and TNM stage,and affected the overall survival of patients with NSCLC.2.KLHL17 expression is higher in various cell lines of NSCLC(A549,H1299,H460,SK,and H292 cells)as compared to normal human bronchial epithelial cells(HBE).3.Overexpression of KLHL17 in the cell lines of NSCLC(A549,H1299 and SK cells)promotes the proliferation and migration of tumor cells,which is associated with elevated activation of Ras/MAPK signaling and increased expression of cyclin D1,CDK4,MMP2 and Rho A.In contrast,knockdown of KLHL17 in the cell lines of NSCLC suppresses the proliferation and migration of tumor cells,in association with reduced activation of Ras/MAPK signaling and decreased expression of cyclin D1,CDK4,MMP2 and Rho A.4.Treatment of tumor cells with Ras inhibitor Salirasib in the cell lines of NSCLC(A549,H1299 and SK cells)transfected by KLHL17-Flag,prevents KLHL17-induced Ras/MAPK activity as well as tumor proliferation and migration. |
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