Dynamic MicroRNA Expression Profiles And Involvement Of MiR-18a In Carcinogenesis Of Nasopharyngeal Carcinoma Through Dicer1

MicroRNAs,(or 'miRNAs', which are small noncoding RNA molecules), can bind to the complementary sequences in the3'UTR of multiple target mRNAs by regulating mRNA stability and translation through the action of the RNA-induced silencing complex (RISC), miRNAs have been a hotspot in research for their involvement in biological processes and tumour development."MicroRNomics" is to describe a novel subdiscipline of genomics that studies including the identification, expression, structure, expression regulation, targets, and biological functions of miRNAs. The tumorigenesis and development of Nasopharyngeal Carcinoma (NPC) is a multistep process involving multiple genetic and environment factors. However, there has been limited research on microRNomics and the interaction of miRNAs in NPC. In this study, we are aiming to elucidate the spatiotemporal roles of miRNAs in different clinical stages of NPC and related regulatory mechanism on the microRNomics scale. We are also going to study the regulatory roles of miR-18a on the microRNomics of NPC.[The dynamic expression of miRNomics in Multi-stages of Nasopharyngeal Carcinoma]Laser capture microdissection (LCM) was used to separate the cancer tissues from the normal tissues. Illumina's miRNA expression arrays were used for searching the differentially expressed miRNAs between the NPC and control samples.48miRNAs with significant change were obtained by the MultiClassDif statistical software. The differentially expressed miRNAs were clustered into the six expression models by the biology software cluster3.0software based on their similar expression patterns,3of which were divided into down-regulated patterns in Multi-stages and Lymphoid node metastasis of Nasopharyngeal Carcinoma; and3of which were divided into up-regulated models. The targetScan software was used for searching and predicting the target genes of these differentially expressed miRNAs. As more than thousands target genes were predicted and predicted target gene lists were then narrowed down by searching the data from ftp://ftp.ncbi.nih.gov/repository/UniGene/to find the nasopharyngeal-specific gene. We further performed data reduction strategy to overlay these nasopharyngeal-specific target genes with cDNA expression data (GEO:GSE12452). As the results, several representative target genes were selected, some of which were verified by real-time PCR in different clinical stages samples. The selected target genes were analyzed in the Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway. The most enriched GO terms in the predicted target genes of up-regulated miRNAs were cell adhesion, apoptosis and cell death; in the predicted target genes of down-regulated miRNAs the enriched GO terms were focused on cell proliferation, death and apoptosis. The target genes of up-regulated miRNAs were mainly involoved in the adherens junction, Focal adhesion, which were related with cancer metastasis pathway. The target genes of down-regulated miRNAs were mostly implicated in pathways in cancer. Based on the inverse correlation of miRNAs and their target genes, we built miRNAs-Genes interactions into a bipartite network. The expression of miRNAs is directly regulated not only by Drosha and Dicer1, but also by transcription factors (TF). We then used UCSC Genome database to analyze the promoter regions of miRNA genes for the transcription factor (TF) binding sites. We then constructed TF-miRNA networks. We further investigated the TF-miRNAs-target genes feedback loop by integrating the TF-miRNA networks and miRNA-target genes networks. We found that some TFs such as ETS2could regulate the expression of miRNA expression and the miRNAs in turn suppress the expression of TF, forming feedback loops. These works will provide new ideas for the research in nasopharyngeal carcinoma.[MiR-18a promotes nasopharyngeal carcinoma cell growth, migration and invasion]MiR-18a was found significantly highly expressed in multistages and lymphoid node metastasis of NPC by using miRNAs array. The differential expression of miR-18a was verified by real-time PCR in the NPC samples and cancer cell lines. The in situ hybridization revealed that the expression of miR-18a was correlated with the clinical stages, the lymph node metastasis, EBV infection and prognosis of NPC patients. MTT, wound healing and transwell with matrix assays proved that miR-18a promoted HK1,5-8F and6-10B cell proliferation, mobility, invasion and metastasis ability, while inhibiting the expression of miR-18a decreased the ability of NPC cell to proliferate, migrate, invade and metastase. We then constructed the miR-18a overexpressed and knockdown cell lines by lentivirus based vectors. The efficency of transfection was verified by real-time PCR. In vivo roles of miR-18a in the cell growth and migration were assessed by the tumor formation following subcutaneous or intravenous injection into nude mice. The nude mice formed bigger subcutaneous tumors in mice which the cells overexpressed with miR-18a compared to the control group. The mobility and metastasis of cells in vivo were examined at different time points by using the IVIS imaging system (xenogen).[The carcinogenic mechanism of miR-18a mediated by Dicerl in Nasopharyngeal Carcinoma]We used three TargetScan to search for miRNA binding sites in the3'UTR of Dicerl. The luciferase assay, real-time PCR and western blot were performed to confirm that miR-18a binds to the3'UTR of Dicerl, suppressing the endogenous expression of Dicerl in NPC cell lines HK1and5-8F. MiRNA array assay were performed to investigate the miRNA profiles change caused by miR-18a. The overexpression of miR-18a caused78%globally downregulated expression of miRNAs and the global downregulation of miRNAs were restored by overexpression of Dicer1. MiR-200families and miR-143were shown significantly suppressed by miR-18a in the miRNA array. The oncogenic roles of miR-18a through targeting Dicer1were verified in the Dicer1overexpressed and knockdown cell. Real-time PCR and western blot assay showed that miR-18a can regulate the expression miR-200families and the EMT biomarkers. It also showed that miR-18a can regulate the expression of miR-143and its target gene K-Ras expression, accelerating the development of NPC.As described aboved, we obtained a group of miRNAs that may play important roles on development of NPC by using the data reduction strategies and we also revealed the dynamic expression profiles of these miRNAs. The selected target genes were analyzed in the GO biological process and KEGG biological pathway. We further investigated the mechanisms of miRNAs expression regulation by analyzing the TF binding sites of miRNA genes using UCSC Genome database. We also investigated the miRNAs expression regulation by miR-18a. MiR-18a showed obvious promoting effect on tumorigenesis of NPC by targeting Dicerl, in turn impairing the biogenesis of microRNome. In this paper, we elucidate the relationship of miR-18a and miR-200families and miR-143. The clinical features of NPC such as clinical stages of NPC, EB virus infection were also shown correlated to the expression of miR-18a.