| Objective: Lung cancer is the most common malignant tumor in China,the incidence of which ranks the first among male patients in China and the second among female patients.With a high mortality rate,lung cancer is one of the most important cancer deaths in China and the world.Due to the absence of obvious clinical signs and the lack of effective early screening methods in the majority of patients with lung cancer at the early stage,the majority of patients were diagnosed with lung cancer at a later stage,with a 5-year survival rate of less than 18%.Therefore,it is of great significance to study the mechanism of lung cancer,biomarkers of early cancer screening and therapeutic targets.Micro RNAs are current cancer research is the most widely in the field of non-coding RNA,a lot of evidence indicates that micro RNAs play an important role in development of tumor,the miRNA can not only directly regulating tumor cell proliferation,apoptosis,and can also help tumor cells inhibit the body’s immune microenvironment and tumor immune escape,in addition,the miRNA by adjusting the EMT signaling pathways,such as promote the invasion and metastasis of tumor cells,and also play an important role in the drug resistance of tumor.Mi RNA also has certain value in the early diagnosis and screening of tumor,prognosis and evaluation of response to treatment.Free miRNAs in the circulatory system are susceptible to ribonuclease,PH and other factors,and their physicochemical properties are not stable.Mi RNAs in exosomes are more stable than free miRNAs in circulation because they are protected by the outer membrane.The important role of exosomal miRNA in pathogenesis and development of lung cancer was found.Method: To investigate the effect of exosomal miRNAs on NSCLC,we performed transcriptome analysis of exosomal miRNAs in the blood of patients with lung cancer using miRNA microarray technology.A total of6 samples were included in this study,all aged between 50 and 60 years,among which 4 were male patients with non-small cell lung cancer(NSCLC),2 were metastatic NSCLC,2 were primary NSCLC and 2 were healthy males.GSE118370 was used for integration and the co expressed m RNA was analyzed by gene ontology analysis,signal pathway analysis and cytohubba analysis.RNA interference technology,western blot technology,cell proliferation and migration experiments were used to verify the mechanism of miRNA / m RNA in lung adenocarcinoma.Results: The results showed that miR-4436 a and miR-4687-5p were significantly up-regulated miRNAs in the metastatic group compared with the non-metastatic group.Among the differentially down-regulated genes,miR-22-3p,miR-3666,miR-4449,miR-4448,miR-6751-5p and miR-92a-3p in the metastasis group were significantly down-regulated compared with the non-metastasis group,while miR-3160-5p and miR-4448 in the metastasis group were significantly down-regulated compared with the healthy control group.There were 5 up-regulated miRNA differences between the non-metastatic group and the control group,including miR-22-3p,miR-3666,miR-4449,miR-6751-5p and miR-92a-3p in the non-metastatic group and the healthy control group.To further evaluate the role of these miRNAs in NSCLC,we performed a meta-analysis of sequencing results and GSE118370,a microarray dataset in the GEO database that includes six lung adenocarcinoma tissues and six adjacent normal lung tissues.Lung adenocarcinoma is a non-small cell lung cancer.The results showed that RRM2,AURKA,NEK2,CHEK1,BIRC5,CENPF,MCM10,UBE2D1,CDH1,HMMR and other top 10 gene sequences were consistent.These target genes are involved in the p53 signaling pathway and play an important role in tumors.RRM2 was located at the top of the differential genes,and hsa-miR-3160-5p,hsa-miR-4448,hsa-miR-1254,hsa-miR-4324,hsa-miR-4687-5p,hsa-miR-4436 a had binding regions with RRM2.But compared with non metastasis group and control group,only hsa-mir-4448 was down regulated in metastasis group,so we focused on the potential mechanism of miR-4448 / RRM2 in lung cancer.In TCGA database,RRM2 expression in lung cancer tissues was higher than that in normal tissues.Overexpression of RRM2 is associated with low survival of lung cancer.These miRNAs may be involved in oncogenesis,proliferation,migration, and invasion of NSCLC.In particular,miR-4448 may target RRM and are expected to be biomarkers for NSCLC.Combined with these results,we suggest that miR-4448 may promote proliferation and metastasis of NSCLC by regulating RRM2 expression.To verify our results,we first constructed a mir-4448 overexpressed and down-regulated A549 cell line.After overexpression of miR-4448,RRM2 expression was down-regulated from the level of RNA and protein.Consistent with this result,the down-regulated expression of miR-4448 increased the expression of RRM2 from the level of RNA and protein.Our results preliminarily indicated that miR-4448 may have a regulatory relationship.To further verify our results,we performed a double luciferase reporter assay,which showed that miR-4448 directly and specifically binds to the3’utr of RRM2 and down-regulates RRM2 gene expression at the post-transcriptional level.The down-regulated expression of miRNA-4448 accelerated the proliferation and clone formation of A549 cells,and showed higher invasiveness.These results showed that miRNA-4448 could inhibit the proliferation and metastasis of lung cancer by inhibiting the expression of RRM2.Low expression of miRNA-4448 in lung cancer patients may be associated with malignancy of the tumor and poor prognosis.Conclusion: Mir-4448/RRM2 can regulate the proliferation and metastasis of lung adenocarcinoma,which may be used as the early diagnosis and treatment marker of lung adenocarcinoma and a new drug target... |
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