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Prospective Lncrna-Mirna-Mrna Regulatory INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 40: 1895-1906, 2017 Prospective lncRNA-miRNA-mRNA regulatory network of long non-coding RNA LINC00968 in non-small cell lung cancer A549 cells: A miRNA microarray and bioinformatics investigation DONG-YAO LI1*, WEN-JIE CHEN1*, LEI LUO1, YONG-KUN WANG1, JUN SHANG1, YU ZHANG2, GANG CHEN2 and SHI-KANG LI1 Departments of 1Thoracic and Cardiovascular Surgery and 2Pathology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China Received December 12, 2016; Accepted October 5, 2017 DOI: 10.3892/ijmm.2017.3187 Abstract. Accumulating evidence suggests that the dysregula- adhesion. An lncRNA-miRNA-mRNA regulatory network and tion of long non-coding RNAs (lncRNAs) serves vital roles a protein-protein interaction network were then constructed. in the incidence and progression of lung cancer. However, the Eventually, a prospective lncRNA-miRNA-mRNA regulatory molecular mechanisms of LINC00968, a recently identified network of LINC00968, three miRNAs (miR-9, miR-22 and lncRNA, remain unknown. The objective of present study was miR-4536) and two genes (polo-like kinase 1 and exportin-1) to investigate the role of a prospective lncRNA-miRNA-mRNA was obtained following validation in the Cancer Genome Atlas network regulated by LINC00968 in non-small cell lung database. These results may provide novel insights to support cancer cells. Following the transfection of lentiviruses carrying future research into lncRNA in lung cancer. LINC00968 into A549 cells, the microRNA (miRNA) expres- sion profile of the cells in response to the overexpression of Introduction LINC00968 was detected using an miRNA microarray. Five differentially expressed miRNAs (DEMs) with LINC00968 Lung cancer results in the death of ~1.59 million indi- overexpression were obtained, including miR-9-3p, miR-22-5p, viduals worldwide each year, and is a leading cause of cancer miR-668-3p, miR-3675-3p and miR-4536-3p. Five target predic- mortality (1,2). According to the pathological diagnostic clas- tion algorithms and three target validation algorithms were sification, non-small cell lung cancer (NSCLC) accounts for used to obtain 1,888 prospective target genes of the five DEMs. >85% of cases of lung cancer (3). The molecular pathogenesis of The result of Gene Ontology analysis suggested that these five NSCLC has received increasing attention in recent years (4-7). DEMs were involved in complex cellular pathways, which Thus, non-coding RNAs, which serve crucial functions in included intracellular transport, organelle lumen and nucleo- numerous biological processes have become of interest. Based on tide binding. Furthermore, analysis of Kyoto Encyclopedia of their size, sequence and function, non-coding RNAs can be clas- Genes and Genomes pathways indicated that the five DEMs sified into various subclasses, the two most notable of which are were important regulators in the adherens junction and focal long-noncoding RNAs (lncRNAs) and microRNAs (miRNAs). LncRNAs are parts of non-coding RNAs with >200 nucle- otides and do not encode proteins (8). Previous studies have indicated that lncRNAs may have an important involvement in cancer biology and cellular processes (9,10). LncRNAs may Correspondence to: Professor Shi-Kang Li, Department of contribute to these processes via various mechanisms at the Thoracic and Cardiovascular Surgery, The First Affiliated Hospital post-transcriptional level, for example, by serving as precur- of Guangxi Medical University, 6 Shuangyong Road, Nanning, Guangxi Zhuang Autonomous Region 530021, P.R. China sors to miRNA and regulating mRNA by interacting with E-mail: [email protected] miRNAs (11,12). Unlike lncRNAs, miRNAs are generally 18-25 nucleotides in length, and are small non-coding protein Professor Gang Chen, Department of Pathology, The First Affiliated RNAs that are able to bind to the 3' untranslated region of target Hospital of Guangxi Medical University, 6 Shuangyong Road, mRNAs and result in the degradation and translational down- Nanning, Guangxi Zhuang Autonomous Region 530021, P.R. China E-mail: [email protected] regulation of target mRNAs (13,14). In addition, miRNAs have the ability to regulate the coding and noncoding transcriptome. *Contributed equally It is worthy of note that lncRNAs and miRNAs serve pivotal roles in numerous similar processes, including cell prolifera- Key words: lung cancer, LINC00968, miRNA microarray, tion, apoptosis and carcinogenesis. Due to the similarity of their lncRNA-miRNA-mRNA, protein-protein interaction, the Cancer functions, it may be hypothesized that an interaction between Genome Atlas lncRNAs and miRNAs occurs during the tumorigenic process. For instance, the competing endogenous RNA (ceRNA) 1896 LI et al: ROLE OF LINC00968 IN LUNG CANCER hypothesis that lncRNAs serve as molecular sponges for Target gene prediction of DEMs. In order to determine miRNAs has been proposed (15,16). Thus, the discovery of the potential association between mRNAs and miRNAs, lncRNA-miRNA-mRNA networks may lead to a more compre- the potential transcriptional targets of the DEMs were hensive understanding of the etiology and metastasis mechanism predicted using five miRNA target prediction algorithms: of cancer and to potential therapeutic targets, and such networks miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/), miRDB been studied for several types of cancer (17,18). (http://www. mirdb.org/), GeneCards (http://www.genecards. In a previous study, the present authors identified 47 l ncR NAs, org), TargetScan (http://www.Targetscan.org/) and RNA22 including LINC00968, that were differentially expressed (https://cm.Jefferson.edu/rna22/). Target genes that were between normal lung tissues and tumor samples by bioinfor- commonly predicted by at least three of these algorithms matic analysis (19). Furthermore, a significant downregulation were considered predicted target genes. Validated target of LINC00968 in lung adenocarcinoma (LUAD) tissues and genes verified by experiments were obtained from miRecords A549 cells was discovered (data not shown). These experiments (http://c1.accurascience.com/miRecords/), miRTarBase and suggest that LINC00968 may serve important roles in lung TarBase (http://diana.imis.athenainnovation.gr/DianaTools/ cancer progression. In the present study, using miRNA profiling index.php?r =tarbase/index). Target genes from any one of by microarray following the overexpression of LINC00968 in these three algorithms were listed as validated target genes. A549 cells, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the differen- GO and KEGG pathway analysis. Functional enrichment tially expressed miRNAs (DEMs) were performed. Furthermore, analysis is essential to uncover biological functions of miRNA a prospective lncRNA-miRNA-mRNA regulatory network of target genes. To gain an understanding of the biological func- LINC00968 was constructed. These results may assist in the tions of miRNA target genes, GO classification and KEGG exploration of the underlying regulatory mechanisms in the pathway enrichment analysis were performed using the bioin- progression of lung cancer. formatics software DAVID 6.7 (https://david-ncifcrf.gov/). GO classifies genes according to three categories, biological Materials and methods process, molecular function and cellular component, and presents a common descriptive framework of gene annotation Overexpression of LINC00968 in A549 cells. A549 cells and classification for the analysis of gene-set data. In addition, were obtained from the Cell Bank of the Chinese Academy of the potential involvement in biological pathways of the DEMs Sciences (Shanghai, China). Following culture in 6-well plates were detected based on analysis using the KEGG pathway overnight, the cells (2x106) were transfected with a solution database, which is a credible and informative database that of lentiviruses in F-12 culture medium containing 10% serum includes almost all biological signal pathways (20). The false (Gibco, Grand Island, NY, USA), 100 U/ml penicillin and discovery rate (FDR) <0.05 was set as the cut-off for selecting 100 µg/ml streptomycin (Hyclone, Logan, UT, USA) for 24 h. significantly enriched KEGG pathways. The solution was then removed, fresh F-12 culture medium was added and the A549 cells were incubated for another Construction of a network of lncRNA-miRNA-mRNA and 24-72 h. The conventional culture environment was 5% CO2 protein-protein interaction (PPI). Following the integration air in a humidified incubator at 37˚C. Lentiviruses encoding of the results of the predicted targets and validated targets, LINC00968 were designed and synthesized by Shanghai prospective targets of the DEMs were obtained. In order to GenePharma Co., Ltd. (Shanghai, China). An inverted fluo- demonstrate the association among lncRNAs, miRNAs and rescence microscope was applied to examine the transfection mRNAs, an lncRNA-miRNA-mRNA interaction network efficiency (data not shown). was created. The network was visualized using Cytoscape (version 3.4.0) (21). The online tool STRING (http://string-db. RNA isolation and miRNA microarray detection. The sample org/) was then used to draw a PPI network of the prospec- analysis and microarray hybridization were conducted by tive targets. The highest confidence of 0.9 was selected as the KangChen Bio-tech (Shanghai, China). Total RNA was minimum required interaction score. isolated using TRIzol (Invitrogen; Thermo Fisher
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