Role And Molecular Mechanism Of MiR-362 In Non-Small-Cell Lung Cancer Metastasis

Systems biology is a subject which studies the composition of all components(genes,mRNAs,proteins,etc.)in biological systems,and the interrelationships between these components under specific conditions.Our research is to study the overall changes of the upstream and downstream molecules in the biological system after changing a certain gene.Lung cancer represents one of the most frequently malignant diseases over the world.The latest statistical results show that the mortality rate of lung cancer ranks the first among malignant tumors,while the incidence and mortality of lung cancer are still rising.Non-small-cell lung cancer(NSCLC)accounts for approximately 85%of lung cancer cases.In recent years,with the comprehensive application of surgical resection,chemotherapy,radiotherapy and biological therapy,the life quality and survival of NSCLC patients have increased significantly,but the 5-year survival rate of patients is still lower.The cause is due to the tumor metastasis,approximately 40%of phase I and 60%of phase II patients with NSCLC die of distant metastasis.Therefore,studying the molecular mechanism of invasion and migration of NSCLC and elucidating its mechanism will not only help to understand the molecular changes of NSCLC in disease progression,but also help to improve the survival of patients.Metastasis of the malignant tumor is the result of a combination of multiple levels(genes,proteins,etc.)and multiple molecules.With the completion of genome sequencing,the role of non-coding genes in the development of tumors get more and more attention.MicroRNA(miRNA)is a class of endogenous microRNA molecules which regulates the expression of proteins through post-transcriptional level.According to new studies,miRNAs play important roles in the occurrence and development of tumors.Our previous study showed that miR-362 is highly expressed in NSCLC tissues.However,the function and mechanism of miR-362 in NSCLC remains to be elucidated.The research in this paper is part of the complex biological system and is an important supplement to the research of the whole system theory.The differences between the normal group and the miR-362 knockout group were analyzed by t test in this study.Combined with analysing multiple groups of data from experiments in vitro through one-way analysis of variance method,we explored the role and molecular mechnism of miR-362 in NSCLC metastasis.Objective Function and mechanism of miR-362 in NSCLC metastasis.Methods We construct miR-362 knockout A549/95-D cells by using CRISPR/Cas9 genome engineering technology.The effect of miR-362 on the ability of the migration and invasion is determined by transwell assay.The effect of miR-362 on the ability of cell proliferation is studied by colony formation assay.The effect of miR-362 on cell cycle and apoptosis rate are detected by flow cytometry.A subcutaneous tumor model in nude mice is established to detect NSCLC tumor growth in vivo.MiR-362 candidate target genes are identified by bioinformatics methods combined with molecular biology methods and we assess the biological effect of the target gene on NSCLC.MiR-362 target gene expression in NSCLC and adjacent tissues are determined by immunohistochemical staining and analyzed the correlation with miR-362 expression level.Results 1.The level of miR-362 expression was higher in NSCLC tissues and cells.The level of miR-362 expression was higher in NSCLC tissues than in adjacent normal tissues(p = 0.0498).After surgical resection miR-362 expression was closely related to tumor recurrence time(p = 0.0454).The level of miR-362 expression was also elevated in five NSCLC cell lines(A549,95-D,NCI-H1299,NCI-H292 and NCI-H460).2.miR-362 promoted NSCLC cell invasion,migration and colony formation in vitro and tumor formation in ivivo.Successfully construct miR-362 knockout A549 and 95-D cell lines.Compared with A549 wild type cells,the expression of miR-362 in A549KO cells was significantly down-regulated,the invasion and migration ability decreased by 48.68%(p?0.0003)and 51.56%(p = 0.0005),the colony formation ability decreased by 47.78%(p= 0.0005).Compared with 95-D wild type cells,the expression of miR-362 in 95-DKO cells was significantly down-regulated,the invasion and migration ability decreased by 30.45%(p P 0.0001)and 33.23%(p = 0.0002),the colony formation ability decreased by 30.56%(p = 0.0153).The tumor growth ability was significantly decreased in vivo in tumor formation experiment(p<0.0001)when miR-362 was absent.There was no significant difference in cell cycle and apoptosis rate.3.The miR-362 direct target gene SEMA3A was found and identified,we explored the function of SEMA3A.Bioinformatics combined with molecular biology analysis showed that SEMA3A is a direct target gene of miR-362.Invasion and migration ability decreased by 61.64%(p ?0.0006)and 75.90%(p = 0.0002)after transfection of A549 cells with the vector containing SEMA3A ORF,the colony formation ability decreased by 56.77%(p ?0.0016).Invasion and migration ability decreased by 61.29%(p = 0.0005)and 70.29%(p=0.0023)after transfection of 95-D cells with with the vector containing SEMA3A ORF,the colony formation ability decreased by 63.39%(p<0.0001).4.Immunohistochemical staining showed that the expression level of SEMA3A was significantly increased in adjacent normal lung tissues.This upregulated was strongly correlated with the downregulation expression of miR-362.Conclusion The miR-362 knockout lung cancer cell line(A549KO and 95-DKO cells)has been successfully constructed by using CRISPR/Cas9 system.The expression of miR-362 is higher in NSCLC,and miR-362 promotes NSCLC cell invasion,migration and colony formation in vitro and tumor formation in vivo.MiR-362 can promote NSCLC cell metastasis through the downregulation of SEMA3A,which is a target of miR-362 and inhibit NSCLC cell metastasis.MiR-362/SEMA3A axis elucidate the molecular mechanism of NSCLC invasion and migration and could lead to a potential therapeutic target in NSCLC treatment.The innovative results achieved in this study are follows:1.This study used CRISPR/Cas9 system-mediated gene editing technology to construct firstly miR-362 knockout A549 and 95-D cell lines with Cre/LoxP system,which also provided a reference for further establishment of other gene knockout cell lines of A549/95-D.2.Furthermore,miR-362 promoted NSCLC cell invasion,migration and colony formation in vitro.miR-362 knockout promoted the sensitivity of chemotherapy drugs of inhibiting tumor proliferation in vivo,which sets a foundation for further study of the mechanism and function of miR-362 in NSCLC.3.The miR-362 direct target gene SEMA3A was found and identified.4.miR-362/SEMA3A axis elucidates the one molecular mechanism of NSCLC invasion and migration,which could be a potential therapeutic target in NSCLC treatment.To sum up,using a variety of mathematical statistics methods,this study found that miR-362 is highly expressed in NSCLC tissues,which promotes the metastasis of NSCLC by down-regulating the expression of SEMA3A.This study provides a new mechanism for elucidating NSCLC metastasis and provides a new potential therapeutic target for NSCLC therapy.