| Part Ⅰ The diagnostic value of microRNA-486 in non-small cell lung cancerAims:The diagnostic value of microRNA-486(miR-486)in non-small cell lung cancer(NSCLC)is still unaddressed.This section aims to evaluate the clinical applicability of miR-486 as a biomarker for the diagnosis of NSCLC by using meta-analysis.Methods:Keywords "microRNA-486","miRNA-486","miR-486","lung cancer","lung carcinoma","lung neoplasms","NSCLC" and their combinations were searched in PubMed,Web of Science and EMBASE databases for all the studies on the diagnosis of non-small cell lung cancer related to miR-486 published before December 24,2020.Then the studies that meet the inclusion and exclusion criteria were used for meta-analysis.Results:After screening,14 related studies were finally included.The pooled sensitivity and specificity of these 14 studies were 0.78(95%CI:0.72-0.83)and 0.81(95%CI:0.70-0.89),respectively.The positive likelihood ratio(PLR),negative likelihood ratio(NLR)and diagnostic odds ratio(DOR)were 4.1(95%CI:2.4-6.8),0.27(95%CI:0.200.36)and 15(95%CI):7-32).Subgroup analysis showed that miR-486 has better diagnostic value in Asian population,with sensitivity of 0.80(95%CI:0.73-0.85),specificity of 0.85(95%CI:0.59-0.96)and AUC of 0.84(95%CI:0.80-0.87).Plasma sample study showed higher diagnostic accuracy,with a sensitivity of 0.78(95%CI:0.73-0.83),a specificity of 0.88(95%CI:0.74-0.95),and an AUC of 0.84(95%CI:0.81)-0.87).Regression analysis showed that race(P>0.05),sample size(P>0.05),or sample source(P>0.05)were not sources of potential heterogeneity in sensitivity or specificity.Sensitivity analysis and publication bias showed that the meta-analysis of the diagnostic value of miR-486 in NSCLC in this study was highly robust.In addition,compared with a single miR-486,the combination of biomarkers had better diagnostic performance in NSCLC,with a sensitivity of 0.83(95%CI:0.79-0.87),a specificity of 0.91(95%CI:0.87-0.94),and an AUC of 0.92(95%CI:0.90-0.94).Conclusions:This part of the research suggests that miR-486 may be used as a biomarker for early diagnosis of NSCLC.And the combination of biomarkers based on miR486 is more accurate than a single miR-486 in diagnosing NSCLC.Part Ⅱ Bioinformatics mining of the function of microRNA-486 in nonsmall cell lung cancerAims:This part of the research aims to explore the biological functions of miR-486 and reveal its possible mechanism in the occurrence and development of NSCLC through integrated bioinformatics analysis.Methods:First,miRTarBase database and experimentally verified miRNA-target gene regulatory information were used to identify target genes of miR-486.Then,the DAVID database(http://david.ncifcrf.gov)was used to conduct gene ontology(GO)and eyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis on the target genes.Next,a protein-protein interaction(PPI)was constructed by using Cytoscape software and node genes were identified using Cytoscape software plug-in CytoNCA.Finally,the Gene expression profiling interactive analysis(GEPIA)database was used to further evaluate the prognostic value of nodal genes in NSCLC.Results:GO functional enrichment analysis showed that at the biological process level,the target genes of mir-486 were mainly enriched in negative transcriptional regulation,mesenchymal to epithelial transformation,cell proliferation and transforming growth factorβ(TGF-β)receptor signaling pathway etc.The target genes of mir-486 were mainly enriched in transcriptional disorders in cancer,central carbon metabolism in cancer,HIF-1 signaling pathway,p53 signaling pathway,proteoglycan in cancer,NSCLC,FOXO signaling pathway,glucagon signaling pathway,insulin signaling pathway,insulin resistance,cancer pathway and ErbB signaling pathway which was shown by KEGG pathway enrichment analysis.PPI network construction and node gene screening showed that the top 10 node genes were CDKN1A,erbB2,ITGB3,PIK3R1,PTEN,rafl,Smad2,Snai1,Syk and UBB.The node genes were mainly enriched in FOXO signaling pathway,PI3K Akt signaling pathway,cancer pathway,central carbon metabolism pathway in cancer,proteoglycan pathway in cancer,erbB signaling pathway,microRNAs pathway in cancer,NSCLC pathway and B-cell receptor signaling pathway.The prognostic value evaluation showed that compared with the lowly expressed genes,the prognosis of patients with high expression of CDKN1A(P=0.04),PTEN(P=0.005)and Snai1(P=2.1e-05)was worse,while the prognosis of patients with high expression of PIK3R1(P=0.04)was better.However,the expression levels of ERBB2(P=0.43),ITGB3(P=0.66),rafl(P=0.91),Smad2(P=0.13),Syk(P=0.73)and UBB(P=0.33)were not related to the prognosis of OS in patients with NSCLC.The results of network module screening and analysis showed that the genes of the most important modules in PPI network were highly correlated with the following key signal pathways:cancer pathway,FOXO signal pathway,p53 signal pathway,cell cycle,microRNA in cancer,PI3K Akt signal pathway and NSCLC.Conclusions:microRNA-486 may participate in the occurrence and development of NSCLC by regulating some key genes(CDKN1A,erbB2,ITGB3,PIK3R1,PTEN,rafl,Smad2,Snai1,Syk and UBB)and signal pathways(non-small cell lung cancer pathway).It is worthwhile to perform further cell biology and molecular biology research.Part Ⅲ Functional verification of miR-486 target genes and their downstream target genes in NSCLCAims:This part aims to verify the regulatory effect of miR-486 on the malignant biological behavior of NSCLC at the tissue,cell,and molecular level,to clarify the target genes of miR-486 and its function in process of NSCLC using biological experimental methods.Methods:The expression of miR-486-5p in 30 samples of cancerous tissue and adjacent tissues of NSCLC patients was detected by RT-PCR.Then the NSCLC cell lines with overexpression and inhibition of miR-486-5p was established.Migration,invasion and proliferation capabilities of these cell lines were detected by using cell scratch test,trans well test and CCK-8 cell proliferation test.On the basis of the biological information analysis in part 2,the RT-qPCR method and dual luciferase reporter gene system are used to further verify the target genes of miR-486-5p.The expression of key miR-486-5p target genes in NSCLC cancer tissues were detected using immunohistochemical staining.Finally,the NSCLC cell line with miR-486-5p key target gene knockout was constructed,and the cell scratch test,trans well test and CCK-8 cell proliferation test were used to detect the miR486-5p key target gene’ s migration,invasion,and proliferation of NSCLC cells with miR486-5p key target gene knockout.Results:The expression of miR-486-5p in adjacent tissues was about 1.3 times of that in cancer tissues,and the expression level of miR-486-5p in cancer tissues was significantly lower than that in the control group shown by RT-PCR results.Cell scratch experiments showed that overexpression of miR-486-5p significantly inhibited the migration of NSCLC cells,while knockdown of miR-486-5p promoted the migration of NSCLC cells.Trans well experimental results showed that overexpression of miR-486-5p inhibited the invasion of NSCLC cells,but knockdown of miR-486-5p promoted the invasion of NSCLC cells.CCK-8 cell proliferation experiments showed that overexpression of miR-486-5p significantly inhibited the proliferation of NSCLC cells,and knockdown of miR-486-5p significantly promoted the proliferation of NSCLC cells.RT-qPCR and dual luciferase reporter gene system verified that SMAD2 is the target gene of miR-486-5p.At the same time,immunohistochemical staining showed that SMAD2 was highly expressed in lung cancer tissues,which was significantly higher than that in adjacent tissues,and the expression of SMAD2 was positively correlated with pathological grades.The expression of SMAD2 was higher in lung cancer patients with high pathological grades.Cell scratch experiment showed that knockdown of SMAD2 significantly inhibited the migration of NSCLC cells.Trans well experiment showed that knockdown of SMAD2 effectively inhibited the invasion of NSCLC cells,and this trend was consistent with the biological effect of overexpression of miR-486-5p.CCK-8 cell proliferation experiments showed that knockdown of SMAD2 and overexpression of miR-486-5p had similar functions to inhibit the proliferation of NSCLC cells.Conclusions:This part of the study suggests that the expression of miR-486-5p in cancer tissues is significantly down-regulated,miR-486-5p can regulate the migration,invasion and proliferation of NSCLC cells,and may be related to the regulation of the expression of its target gene SMAD2. |
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