The Study Of The Target Inhibition Of MiR-338-3p In Exosomes With CHL1 Gene In Non-small Cell Lung Cancer By Regulating MAPK Signaling Pathway

Objective: In the worldwide,lung cancer is one of the common malignant carcinomas.In China,the incidence and mortality of patients with lung cancer are very high and the prognosis of patients is poor,which is a serious public health problem to the residents.According to the pathological classification,lung cancer can be divided into small cell lung cancer and non-small cell lung cancer,of which non-small cell lung cancer accounts for 85%.The pathogenesis of NSCLC(Non-small Cell Lung Cancer)is complex,and in-depth research on the progress of non-small cell lung cancer put forward the novel targets.Exosomes,wrapped by lipid bilayer membrane structure,carry cellular important active substances,such as mi RNAs,to extracellular microenvironment in the manner of endocytosis-fusion-endocytosis and then participate in the biological activities of cells as cellular information carriers.MAPK is a class of intracellular serine/threonine protein kinases which could be activated by intracellular and/or extracellular signals and plays a vital role in the biological processes such as proliferation,migration and apoptosis of tumor cells.Single nucleotide polymorphism refers to DNA polymorphism caused by a single nucleotide mutation.Multiple studies have showed that single nucleotide polymorphisms are closely associated with the risk of cancers.Therefore,our study verified the effects of exosomal mi R-338-3p/CHL1 gene/MAPK pathway on the biological process of NSCLC cells through bioinformatic analysis and functional experiments in vitro and explored the association between CHL1 gene polymorphism and NSCLC susceptibility.Methods:In the first part of this study,a total of 14 serum samples was selected and differently expressed exosomal mi RNAs was obtained by sequencing analysis.q RTPCR assay was conducted to explore the expression levels of exosomal mi R-338-3p.DIANA TOOLS online database was used to predict the pathway of mi R-338-3p.Exosomes were obtained by differential high-speed centrifugation and detected by transmission electron microscope,NTA and western blot.The expression levels of exosomal mi R-338-3p in BEAS-2B cells,A549 cells and SK-MES-1 cells wereexplored.BEAS-2B cells were co-cultured with A549 cells and SK-MES-1 cells and the expression levels of exosomal mi R-338-3p in NSCLC cells were explored.GW4869 was used to suppress the section of exosomes from BEAS-2B cells.Exosomes derived from BEAS-2B cells were collected and co-cultured with NSCLC cells.Then exosomes derived from BEAS-2B cells transfected with mi R-338-3p mimics and mi R-338-3p mimics-NC were collected and co-cultured with NSCLC cells.The expression levels of CHL1 gene protein in different tumor cells were detected by western blot.MTS assay was used to explore the growth of tumor cells with different treatments.Tumor cells with different treatments were dyed by Annexin V-APC and 7-AAD and then detected by flow cytometry.The downstream molecules of MAPK signaling pathway in tumor cells with different treatments were explored by western blot.In the second part of this study,the dual luciferase assay,q RT-PCR and western blot assays were used to demonstrate that mi R-338-3p could specifically bind to CHL1 gene to inhibit the expression level of CHL1 gene.The expression levels of CHL1 gene protein in tumor cells in different groups were detected by western blot.The MTS rescue assay was used to detect the growth rates of tumor cells with different treatments.Tumor cells with different treatments in the rescue assay were dyed by Annexin V-APC and 7-AAD and then detected by flow cytometry.We explored the association between the expression level of CHL1 gene and the OS(Overall Survival)of NSCLC patients from TCGA data via R software.THPA database was used to explore the distribution of CHL1 protein in lung adenocarcinoma tissues and lung squamous carcinoma tissues.GSEA was utilized to explore the pathway of CHL1 gene in NSCLC.The expression levels of CHL1 gene in A549 cells and SK-MES-1 cells were regulated with different treatments.MTS assay was used to explore the growth of tumor cells with different treatments.Tumor cells with different treatments were dyed by Annexin V-APC and 7-AAD and then detected by flow cytometry.The downstream molecules of MAPK signaling pathway in different groups were explored by western blot.The third part of this study screened the site of CHL1 gene by Haplo Reg and Hap Map database.We enrolled 618 NSCLC patients and 632 healthy controls fromthree hospitals.Logistic regression analysis was used to analyze the association between CHL1 gene polymorphism and the risks of NSCLC,LUAD(Lung Adenocarcinoma)and LUSC(Lung Squamous Carcinoma).The cross-analysis and additive interaction were used to explore the interaction between CHL1 gene polymorphism and environmental risk factors.Results: The results of the first section of this study showed that a total of 182 differently expressed exosomal mi RNAs was obtained by sequencing analysis,including 71 up-regulated exosomal mi RNAs and 111 down-regulated exosomal mi RNAs.exosomal mi R-338-3p was down-regulated in NSCLC patients.The pathway analysis showed that mi R-33-3p may be enriched in MAPK pathway.Compared to NSCLC cells,exosomal mi R-338-3p was over-expressed in BEAS-2B cells.After coculture with BEAS-2B cells,the expression levels of exosomal mi R-338-3p was upregulated in NSCLC cells.After the treatment of GW4869,the expression level of mi R-338-3p in BEAS-2B cells was increased,conversely,the expression level of mi R-338-3p was decreased in BEAS-2B cells-derived exosomes.After co-culture with exosomes derived from BEAS-2B cells,the expression levels of exosomal mi R-338-3p in NSCLC cells were up regulated.Then exosomes from BEAS-2B cells transfected with mi R-338-3p mimics and mi R-338-3p mimics-NC were collected and co-cultured with NSCLC cells.The results of q RT-PCR assays verified that the expression levels of mi R-338-3p in NSCLC cells increased more and the expression levels of CHL1 gene in NSCLC cells decreased more in the meantime.The upregulation of exosomal mi R-338-3p inhibited the growth of tumor cells compared to negative control.The results of apoptotic assays demonstrated that the upregulation of exosomal mi R-338-3p promoted the apoptotic rates of tumor cells compared to negative control.The results of western blot assays proved that the activity of MAPK pathway was suppressed in tumor cells with the upregulation of exosomal mi R-338-3p.The results of the second section of this study showed that mi R-338-3p could directly bind to CHL1 gene 3’UTR region to inhibit the expression of CHL1 gene.In the rescue assays,CHL1 gene could partly rescue the effect of mi R-338-3p on the growth and apoptotic rates of tumor cells.The upregulation of CHL1 gene was associated with the OS of NSCLC from TCGA data.The results from THPA showed that CHL1 gene protein was up-regulated in NSCLC tissues.GSEA results predicted that CHL1 gene may be enriched in MAPK pathway.CHL1 gene was over-expressed in NSCLC cells.The downregulation of CHL1 gene inhibited the growth of tumor cells and the upregulation of CHL1 gene promoted the growth of tumor cells.The results of apoptotic assays demonstrated that downregulation of CHL1 gene promoted the apoptotic rates of tumor cells and the upregulation of CHL1 gene inhibited the apoptotic rates.The results of western blot assays verified that the activity of MAPK pathway was inactivated in tumor cells with downregulation of CHL1 gene and activated in cells with upregulation of CHL1 gene.The results of the third section of this study showed that there was no significant association between rs425366 polymorphism and the risk of patients with NSCLC.And there was also no significant relationship between rs425366 polymorphism and LUAD and LUSC.No significant additive interactions between rs425366 polymorphism and smoking and cooking oil fume exposure were observed.Conclusion: 1.exosomal mi R-338-3p was delivered to tumor cells to regulate the expression of CHL1 gene to mediate cells proliferation and apoptosis by MAPK pathway.2.mi R-338-3p could directly bind to CHL1 gene and mediate the growth and apoptosis of tumor cells by inhibiting the expression of CHL1 gene.3.The relationships between CHL1 gene polymorphism and the risk of NSCLC,LUAD and LUSC are not statistically significant,and the additive interactions between CHL1 gene polymorphism and smoking and cooking oil fume exposure are either not statistically significant.