| BackgroundLung cancer(LC)remains the first cause of cancer-related mortality and ranks the second in incidence in the world,but which ranks the first both in incidence and mortality in China.Lung adenocarcinoma(LUAD)is the most common histological subtype of lung cancer,accounting for approximately 40%of all cases of lung cancer.Despite recent progress in diagnose and therapy have made major progress in the improving prognosis of LUAD,the overall five-years survival rate for LUAD barely reaches approximately 20%.Thus,there is a growing need to characterize the molecular pathogenesis and identify new clinical biomarkers for LUAD in order to better understand the underlying mechanisms and to improve treatment outcomes of this malignancy.Micro RNAs(miRNAs)can downregulate genes expression by repressing or degrading messenger RNAs(m RNAs)targets through complete or incomplete combining with 3?-untranslated regions(UTRs)of targeted m RNAs.Regulating the expression of targeted m RNAs,miRNAs involve in multiple pathological processes,such as tumor,inflammation,diabetes and hypertension.The miRNAs play vital roles in the process of proliferation,apoptosis,migration,invasion,drug resistance and immune escape of tumor cells.Besides,miRNAs,particular the circulating-miRNAs,have been becoming a hotspot for clinical oncology due to the values of clinical biomarkers for disease screening,early detection,predicting prognosis and evaluating efficacy of anticancer drugs.Therefore,in this study,we firstly identified miR-539-5p based on small RNA sequencing(s RNA-seq)and bioinformatics analysis,and then explored the mechanisms of miR-539-5p regulate the malignant behaviors in lung adenocarcinoma cells.Methods(1)Construction of the Co-DEmiRNA-DEm RNA regulatory networkThrough s RNA-seq,we obtained the plasma miRNAs data from clinical cohort(GSE151963),and then identified the plasma differentially expressed miRNAs(plasma?DEmiRNAs)by comparing LUAD patients and healthy controls.Meanwhile,we downloaded the miRNAs and m RNAs sequencing datas from LUAD and normal controls tissue in the Cancer Genome Atlas(TCGA)database,and then identified the tissue differentially expressed miRNAs(tissue?DEmiRNAs)and the tissue differentially expressed m RNAs(tissue?DEm RNAs)by comparing LUAD and normal controls.The isoform expressions of the tissue?DEmiRNAs were identified using the R package miRNAme Converter.By overlapping the plasma?DEmiRNAs and tissue?DEmiRNAs,we identified the consistently common differentially expressed miRNAs(Co-DEmiRNAs)from GSE151963 and TCGA?LUAD datasets.The targeted m RNAs of the Co-DEmiRNAs were predicted using miRDB,Target Scan(version 7.2)and miRWalk databases.The target genes were identied by DEm RNAs,and then were confirmed as the common differentially expressed m RNAs(Co-DEm RNAs).Based on Co-DEmiRNAs and Co-DEm RNAs,we constructed the Co-DEmiRNA-DEm RNA regulatory network using the Cytoscape software version3.7.0.The top five miRNAs(degree>100)were selected as hub miRNAs in the network.The biological functions of the targeted m RNAs of these hub miRNAs were evaluated using the Gene Ontology(GO)and the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses with the R package Cluster Profiler using p<0.05 statistically significant.(2)Identification of miR-539-5p expression in LUAD patients and LUAD cellsGiven degrees of hub miRNAs in the Co-DEmiRNA-DEm RNA network,we confirmed miR-539-5p as our study subject.In order to identify the miR-539-5p expression in LUAD,we analyzed the expressions level in plasma and tissue samples from LUAD patients from GSE151963 and TCGA?LUAD dataset,and compared the LUAD cell lines(A549,NCI-H1975,and SPC-A1)with human bronchial epithelial cell line(BEAS-2B)using the Quantitative real-time PCR(q RT-PCR).(3)Evaluation of biological significance of miR-539-5p in LUAD cellsWith lipo6000?,miR-539-5p mimic,mimic negative control(mimic NC),miR-539-5p inhibitor and inhibitor negative control(inhibitor NC)were transfected into A549 cell line to alter the miR-539-5p expression.Applying CCK-8 assay,colony formation assays,scratch assays,transwell assays,flow cytometric analysis,and western-blot,we determined that the altered expression of miR-539-5p that was able to affect cell proliferation,migration,invasion,cell cycle,apoptosis,and Bcl-2/Bax after transfection.(4)Screening and identifying the targeting m RNA of miR-539-5p in LUADDifferentially expressed genes(DEGs)were identified by four LUAD datasets(GSE10072,GSE19188,GSE7670,and GSE31210)with p<0.05 and|log2 FC|>1,and common differentially expressed genes(Co-DEGs)were obtained.In addition,we confirmed hub targets of miR-539-5p in LUAD with overlapping from Co-DEGs and the targeting m RNAs of miR-539-5p in Co-DEmiRNA-DEm RNA network.Through GEPIA,we explored the clinical values of hub targets and identified direct target of miR-539-5p in LUAD.Applying the Kaplan Meier plotter and cbioportal,we confirmed the clinical values of the expression and gene alterations of direct target of miR-539-5p in LUAD.Owing to the above analysis,ribonucleotide reductase M2(RRM2)was confirmed as the targeting m RNA of miR-539-5p in LUAD.Using q RT-PCR,we detected the expression of RRM2 in LUAD cell lines(A549,NCI-H1975,and SPC-A1)and human bronchial epithelial cell line(BEAS-2B).After miR-539-5p mimic,mimic NC,inhibitor and inhibitor NC transfection,we detected the m RNA and protein levels of RRM2 in A549 cell line.Target sequences of miR-539-5p in the 3?-UTRs of RRM2 was confirmed by bioinformatics analysis,and pmiR-RB-Report?-h-RRM2-3?UTR wild type(WT)and pmiR-RB-Report?-h-RRM2-3?UTR mutant(Mut)were designed and chemically synthesized.Mi R-539-5p mimic or mimic NC was transfected into A549 cell line using lipo6000?,together with pmiR-RB-Report?-h-RRM2-3?UTR WT or pmiR-RB-Report?-h-RRM2-3?UTR Mut.After being incubated at 37?with 5%CO2,the luciferase activities were examined using a Dual-Luciferase Reporter assay system.(5)Confirmation of the biological effect of miR-539-5p/RRM2 in LUAD cellsTo assess the biological significance of RRM2 in LUAD cells,firstly,A549 cell line with high and low RRM2 expression were screened with lentiviral transfection(LV-RRM2 and LV-RRM2-RNAi),and then the stable overexpression and knockdown RRM2(OE?RRM2 and sh RRM2)LUAD cell line were constructed,respectively.Second,using CCK-8 assay,colony formation assays,scratch assays,transwell assays,flow cytometric analysis,and WB analysis,we determined that the altered expression of RRM2 was able to affect cell proliferation,migration,invasion,cell cycle,apoptosis,and expressions of Bcl-2/Bax after transfection.Finally,combining with bioinformatics analysis,PCR array and western-blot methods were used to evaluate the key factors(TP53?CDC25A?CDC25C?RAD1 and PPM1D)in the p53 and cell cycle pathways in vitro.To investigate whether or not miR-539-5p exerts its biological effects in LUAD cells by regulation of RRM2,a series of rescue experiments was carried out.Firstly,the stable overexpression RRM2(OE?RRM2)and negative control(NC)A549 cell line were constructed via lentiviral transfection(LV-RRM2),and then were transfected with miR-539-5p mimic or miR-539-5p mimic NC.Via CCK-8 assays,colony formation assays,transwell chambers assays,flow cytometric analysis,and western-blot analysis,we analyzed that overexpressed RRM2 could resist the effects of miR-539-5p in LUAD cell in vitro.Results(1)Applying|log2 FC|>1 and p<0.05,30 Co-DEmiRNAs,including 19consistent Co-DEmiRNAs(6 upregulated and 13 downregulated)and 11 inconsistent Co-DEmiRNAs(2 upregulated and 9 downregulated from GSE151963 and 9upregulated and 2 downregulated from TCGA?LUAD),were identified through an intersection analysis between the TCGA?LUAD and GSE151963 datasets.In total,5479 target m RNAs from 19 consistently Co-DEmiRNAs were identified.Next,760Co-DEm RNAs(152 upregulated and 608 downregulated)were identified through the intersection analysis from DEm RNAs in TCGA?LUAD and target m RNAs.The Co-DEmiRNA-DEm RNA regulatory network consists of 19 Co-DEmiRNAs and 760Co-DEm RNAs.Furthermore,top five miRNAs were identified as hub miRNAs,including miR-539-5p(216 degrees),miR-656-3p(209 degrees),let-7b-5p(135degrees),miR-2110(125 degrees)and miR-92b-3p(117 degrees).And then,677Co-DEm RNAs from five hub miRNAs targets were used to identify the potential functions of hub miRNAs using GO and KEGG pathway enrichment analyses,and836 results in the GO analysis and 48 signaling pathways emerged from the KEGG pathways analysis were obtained.(2)Owing to the Co-DEmiRNA-DEm RNA network,we confirmed the miR-539-5p as our study subject.Compared to the LUAD cell lines(A549,NCI-H1975,and SPC-A1),the q RT-PCR data showed that the miR-539-5p was evidently upregulated in human bronchial epithelial cell line(BEAS-2B),which was harmonized with the results from TCGA?LUAD and GSE151963 datasets.(3)After transfection via miR-539-5p mimic,mimic NC,miR-539-5p inhibitor and inhibitor NC,the q RT-PCR analysis data were verified that the miR-539-5p was evidently unregulated or downregulated in A549 cell.Overexpression of miR-539-5p was able to inhibit cell proliferation,colony formation,migration and invasion,induce cell cycle arrest and apoptosis,and increase Bax/Bcl-2.While downregulation of miR-539-5p was able to promote cell proliferation,colony formation,migration and invasion,inhibit cell apoptosis,and decrease Bax/Bcl-2.(4)Using bioinformatics analysis from GEO and the Co-DEmiRNA-DEm RNA network,RRM2 was identified as the direct target of miR-539-5p.The data showed that RRM2 was unregulated in LUAD tissues and positively associated with pathological stage.In addition,high expression of RRM2 was related with poor overall survival(OS)and disease free survival(DFS).Besides,the status of RRM2alterations was not associated with LUAD prognosis.Compared with human bronchial epithelial cell line(BEAS-2B)using q RT-PCR,RRM2 was markedly overexpressed in LUAD cell lines(A549,NCI-H1975,and SPC-A1).The m RNA and protein levels of RRM2 were decreased after overexpressing miR-539-5p.Co-transfected A549 cell using miR-539-5p mimic or mimic NC along with a luciferase reporter construct containing the WT or Mut miR-539-5p binding sequence in the 3?-UTRs of RRM2,overexpression of miR-539-5p significantly decreased RRM2 WT reporter luciferase activities but not affect the RRM2 Mut reporter luciferase activities.(5)After transfection,the q RT-PCR and western-blot results were verified that the RRM2 was evidently unregulated or downregulated in A549 cell,and then sh RRM2 and OE?RRM2 A549 cell were successful constructed,respectively.Downregulation of RRM2 was able to inhibit the cell proliferation,colony formation,migration and invasion,induce cell cycle arrest and apoptosis,and increase Bax/Bcl-2.While the overexpression of RRM2 was able to promote cell proliferation,colony formation,migration and invasion,inhibit cell apoptosis,and decrease Bax/Bcl-2.Next,the downregulation of RRM2 was able to inhibit the expression of CDC25A?CDC25C?RAD1 and PPM1D,but enhance TP53 level.(6)In vitro,a series of rescue experiments showed that overexpressed RRM2was able to resists the miR-539-5p effects of inhibit cell proliferation,colony formation,migration and invasion,induce cell cycle arrest and apoptosis,and increase Bcl-2/Bax,and these results were associated with the alteration of CDC25A,CDC25C,RAD1,PPM1D and TP53 levels in p53 and cell cycle pathways.ConclusionThe miR-539-5p is a“suppressor”miRNA in occurrence and development of LUAD,and its biological effects can inhibit the cell proliferation,colony formation,migration and invasion,induce cell cycle arrest and promote cell apoptosis owing to regulating RRM2 expression.These findings contribute to identifying the miR-539-5p/RRM2 axis as new markers and therapeutic targets for LUAD patients in clinical settings. |
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