Screening Of Differentially Expressed MiRNA And CircRNA Of Non-small Cell Lung Cancer And Preliminary Verification Of MiR-582-3p Function

Background and purposeGlobally,lung cancer（LC）is one of the malignant tumors with very high morbidity and mortality.There are many types of lung cancer,among which non-small cell lung cancer（NSCLC）is relatively common type.NSCLC has become one of the most malignant tumors that threaten people’s health and lives,and is a disease that seriously affects the function of organs.Although the cure rate of early NSCLC is high,when patients enter advanced NSCLC,their prognosis and survival rate are very poor.At present,molecular targeted therapy technology has been applied to the treatment of advanced NSCLC,which means that NSCLC has entered the era of precision targeted therapy.Therefore,determining the target of NSCLC treatment is of great significance for improving the cure rate of patients.MicroRNA（microRNA,miRNA）,can play a regulatory role in genes by combining with messengerRNA（mRNA）and mediate different biological functions.It is a smallRNA that is not protein-encoded.CircularRNA（circularRNA,circRNA）is abundantly expressed in the cytoplasm.It can act as a competitive endogenousRNA（ceRNA）or miRNA sponge to play a role.It is a kind of non-coding smallRNA.In addition,miRNA and circRNA combine to form the smallest ceRNA network.ceRNA shares the recognition unit of miRNA,increasing the complexity of miRNA-based regulatory networks.Moreover,the classification of circRNAs in exosomes is related to changes in miRNAs,and plays a role by participating in multiple networks.In the ceRNA network,transcription factors（TF）can also directly regulate the expression of miRNA and regulate the production of proteins.The above shows that miRNAs play a vital role in the complex regulatory network that promotes the occurrence and development of cancer.Studies have reported that ceRNA networks have shown great potential in cancer diagnosis and targeted therapy,but their role in NSCLC is still unclear.Therefore,our research aims to construct a circRNA-miRNA-mRNA interaction ceRNA network and a TF network,so as to screen out the key differentially expressed miRNAs and circRNAs,and explore the mechanisms by which differentially expressed miRNAs regulate the occurrence and development of NSCLC.Method1.RNA sequencingThe subjects of the study were 38 patients with non-small cell lung cancer admitted to the First Hospital of Jilin University from January 2016 to January 2017 and 23 patients with age-matched normal persons.18 NSCLC patients and 18age-matched patients were finally included.The matched normal human peripheral blood samples were subjected toRNA sequencing.Construct a smallRNA-seq library by using Tru Seq^TMRNA Small Sample Preparation Kits.Then the reverse transcription reaction is performed.Complementary DNA（c DNA）was amplified by PCR,and next-generation sequencing was performed on the library on the Illumina Hiseq 2000 platform.We use EpicenterRibo-Zero TM kit（Illumina）to perform circRNA sequencing,and useR’s limma package method to screen for significantly differentially expressed genes between the NSCLC group and the control group.In NSCLC patients,we set the threshold to 2 times the difference value Above,the p value is less than 0.05.2.Functional analysis of differentially expressed genes and construction of ceRNA and TF networksAccording to the records in the miRNA regulatory target gene database,we have constructed a significantly differentially expressed miRNA regulatory target gene network.Searched and downloaded the conserved miRNA target genes recorded in the miRNecords and the MiRWalk database,and required miRNA-regulated target genes to be recorded in at least 3 databases of miRanda,Mir Target2,Pic Tar,PITA,and Target Scan.We construct miRNA regulatory network by integrating the miRNA-target gene data of the above two parts.Then,use the clusterprofiler package to perform Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway enrichment analysis on target genes.In addition,in order to analyze the TF regulatory network,we extracted the differentially expressed TF and target gene transcription regulation interaction relationship from the transcriptionalRegulatoryRelationships Unraveled by Sentense-based Text ming（TRRUST）database,based on the interaction of miRNA target genes（Including miRNA-circRNA and miRNA-mRNA interaction）,TF regulatory network is established by connecting miRNA,TF,circRNA and differentially expressed genes.3.Preliminary verification of the function of differentially expressed genesFirst,the differentially expressed miRNA,circRNA should be detected by quantitative reverse transcription polymerase chain reaction（qRT-PCR）.After sequencing,the remaining 20 NSCLC patient serum samples and 5 normal human serum samplesRNA were used for qRT-PCR analysis,and PCR amplification was performed on Applied Biosystems and Vii A^TM7 PCR system（Foster City,CA,USA）.Calculate the expression of miRNA and circRNA by the 2-ΔΔCT method related to GAPDH.In addition,40 surgical specimens（NSCLC tissues and adjacent tissues）of lung cancer patients provided by the Clinical MedicalResearch Center of our hospital have detected the expression level of miR-582-3p in NSCLC tissues and adjacent tissues of NSCLC.Then,in order to further verify the role of differentially expressed genes in NSCLC,we conducted cell experiments.Through routine cell culture of NSCLC cell line A549 and human normal lung epithelial cell line BEAS-2B,the expression levels of miR-582-3p in A549 and BEAS-2B cells were detected respectively,and then the cells were transfected to construct miR-582-3p mimic and miR-582-3p ihibitor plasmids were transiently transfected.The transfection efficiency of miR-582-3p was verified by qRT-PCR,and then further cell function experiments were performed.Under the conditions of miR-582-3p mimic or miR-582-3p inhibitor transfection,cell proliferation was detected by CCK8,cell cycle and apoptosis were detected by flow cytometry,cell migration and invasion were detected by Transwell,and clone formation Detect changes in cell growth.Then,we used qRT-PCR and Western blot experiments to detect the expression of related genes in the TGF-β and Wnt signaling pathways at the mRNA and protein levels,respectively.Then,the expression levels of related pathway proteins were verified by immunohistochemistry experiments.Finally,the tumor formation experiment in nude mice further verified the effect of miR-582-3p on tumor formation.We chose Blank as a control,transfected human NSCLC cells A549 with miR-582-3p mimic and miR-582-3p inhibitor,and cultured them on a large scale to prepare cell suspensions.We randomly divided the nude mice into Blank group,miR-582-3p mimic group and miR-582-3p inhibitor group,and then injected the prepared cell suspension subcutaneously into the nude mice.Modeling Week 5（Day 35）At the time,the nude mice were sacrificed,the tumors were peeled off from the nude mice,the tumors were weighed,and photographs were taken.Then,HE staining was used to identify the morphological characteristics of mouse tumors under the condition of miR-582-3p mimic or miR-582-3p inhibitor transfected.Results1.circRNA and miRNA sequencing results and related bioinformatics analysis resultsBetween the NSCLC group and the healthy control group,we used the analysis of the limma package to screen 59 significantly differentially expressed miRNAs,including 16 up-regulated miRNAs and 43 down-regulated miRNAs.In addition,we screened a total of 292 circRNAs with significant differential expression,including137 circRNAs with up-regulated expression and 155 circRNAs with down-regulated expression.We screened a total of 232 lncRNAs with significant differential expression,including 9 lncRNAs with up-regulated expression and 223 lncRNAs with down-regulated expression.2.KEGG pathway and GO analyze the functions of differentially expressed genes（miRNA and circRNA）and construct ceRNA and TF networksFirst,the results of functional analysis of differentially expressed miRNAs showed that four pathways were up-regulated:Prion diseases,Fructose and mannose metabolism,Toxoplasmosis And Central carbon metabolism in cancer.The 16down-regulated pathways,for example,the pathways of olfactory transduction（Olfactory transduction,p = 6.90E-35）and neuroactive ligand receptors,respectively Interaction（Neuroactive ligand-receptor interaction,p = 5.22E-27）,Nicotine addiction（p = 5.02E-16）and Drug metabolism-cytochrome P450（Drug metabolism-cytochrome P450,p = 2.24E-13）)In the pathway.We constructed a ceRNA network（circRNA-miRNA-mRNA）based on the results predicted by miRanda and Targetscan: there are interactions between 206 differentially expressed circRNAs and 309 differentially expressed miRNAs.We found 5 important miRNA nodes that are highly connected,including miR-582-5p,miR-665,miR-1197,miR-657 and miR-582-3p;3 important circRNAs,including circRNA4046,circRNA4406 and circRNA4882.In addition,we used the KEGG pathway to enrich and analyze the pathways related to significantly differentially expressed miRNAs,and found that miR-582-3p and miR-665 are closely related to cancer pathways,miR-582-3p and miR-582-5p Enriched in the Wnt pathway.Among other important pathways,miR-665 is related to MAPK pathway and adhesion junction,and miR-582-3p is related to TGF-βsignaling pathway.Based on the differentially expressed miRNAs,through the TRRUST database,we found that the regulatory relationship of TF is related to the genes targeted by miRNAs that are differentially expressed.A TF network was constructed with this.In the TF network,we obtained 8 significant TFs from 47 down-regulated genes（including DLX5（distal deletion gene 5）,E2F8（E2F transcription factor 8）,ETV1（ETS variant 1））,FOXG1（forkhead box G1）,FOXP2（forkhead box P2）,KLF12（Kruppel-like factor 12）,ONECUT2（one cut homeobox 2）and WT1（Wilms tumor1）.As miRNA targets,the TF regulatory network includes Three circRNAs（circRNA4046,circRNA4882,and circRNA4406）.In addition,we also found that in the TF regulatory network,miR-582-3p can target and regulate the expression of circRNA4046,CTNND2 andRBFOX1;miR-657 targets WT1 and miR-582-5p targets ETV1 while regulating androgen receptor（AR）)Gene.3.Preliminary verification of the function of differentially expressed genesFirst,we verified 5 differentially expressed miRNAs（miR-1197,miR-665,miR-657,miR-582-3p and miR-582-）in blood samples of NSCLC patients and healthy controls by qRT-PCR analysis.5p)expression level.The results showed that,compared with the control group,the expression level of miR-1197 in the NSCLC group was lower,but not significant;the expression levels of miR-582-5p and miR-657 showed the same trend.Compared with the control group,miR The expression levels of miR-582-5p and miR-657 were reduced;in addition,the expression levels of miR-665 and miR-582-3p in the NSCLC group were increased compared with the control group.Among them,compared with the control group,the expression levels of miR-665 and miR-582-3p were increased in the NSCLC group.The expression level of miR-582-3p was significantly increased in（p <0.05）.Secondly,in order to further explore the role of miR-582-3p in lung cancer,with BEAS-2B cells as a control,through qRT-PCR experiments,we detected the expression of miR-582-3p in A549 cells,which is similar to that of BEAS-2B.In comparison,the expression of miR-582-3p in A549 cells was significantly increased（p <0.01）.Taking Blank as a control,we transfected A549 cells with miR-582-3p mimic and miR-582-3p inhibitor,and then tested the behavioral characteristics of tumor cells.We found that compared with the Blank group,miR-582-3p mimic was able to Promote cell proliferation,cell migration,invasion and clone formation,and reduce the proportion of cell apoptosis;compared with the Blank group,miR-582-3p inhibitor can inhibit cell proliferation,cell migration,invasion and clone formation.In addition,we tested the mRNA and protein expression levels of genes in the Wnt and TGF-β signaling pathways.Compared with Blank,the expression of wnt-5a and Axin in the miR-582-3p mimic group was significantly reduced（p <0.01,p <0.05）,the expression of SMAD3 was significantly increased（p <0.01）;the expression level of SMAD3 was further verified by immunohistochemistry,and the expression level of SMAD3 in NSCLC tissues was higher than that in adjacent tissues.Finally,with Blank as a control,A549 cells were transfected with miR-582-3p mimic and miR-582-3p inhibitor,and the above three types of cells were inoculated in nude mice to observe the tumorigenic ability of nude mice.The experimental results showed that the tumor volume gradually increased over time.Compared with the Blank group,the tumor volume of the miR-582-3p mimic group increased significantly（p <0.05）;the miR-582-3p inhibitor group compared with the Blank group,the tumor The volume was significantly reduced（p <0.05）.It shows that the miR-582-3p mimic group promotes tumor growth,and the miR-582-3p inhibitor group inhibits tumor growth.In addition,the results of HE staining experiments showed that in the control group,tumor cells were large and uneven in size,with a large nucleoplasm ratio,deep staining of nuclei,and frequent mitoses.In the miR-582-3p overexpression group,the tumor cell volume increased and the mitotic phase increased;and after inhibiting the expression level of miR-582-3p,we observed that the tumor cell volume decreased and the nucleoplasm ratio decreased significantly.Conclusions1.Through bioinformatics analysis and construction of a ceRNA network,we screened and obtained 5 miRNAs significantly related to NSCLC,namely miR-582-3p,miR-582-5p,miR-665,miR-1197 and miR-657.2.Pathway enrichment analysis found miR-582-3p was associated with Wnt signaling pathway and TGF-β signaling pathway.3.In the TF network,three circRNAs related to NSCLC were found,including circRNA4046,circRNA4406 and circRNA4882.circRNA4046 may be involved in NSCLC tumorigenesis through miR-582-3p targeting to regulate CTNND2 andRBFOX1 genes.4.miR-582-3p is highly expressed in NSCLC tissues and lung cancer cells.Inhibiting the expression of miR-582-3p can inhibit the growth and migration of lung cancer cells;5.miR-582-3p may regulate the growth of lung cancer through Wnt and TGF signaling pathways;6.When inhibiting the expression level of miR-582-3p,it can inhibit the formation of tumors in mice.