| BACKGROUNDSmall RNAs belong to non-coding RNA molecules (noncoding RNA, ncRNA), which regulate many functions of eukaryotic cells, including gene expression, cell cycle and individual development and so on. Small RNAs can be categorized into two groups:one is microRNA (microRNA, miRNA) and another is small interfering RNA (short interfering RNA, siRNA). MiRNA research is becoming a novel hotspot following SiRNA.MiRNAs are a class of endogenous non-coding RNA with regulatory function in eukaryotes.They have three main characteristics of structure including (1) no structure of open reading frame (ORF) and protein-coding gene, (2) around 21-25 nucleotides in size, and (3) a unique signature sequence within them. Mature miRNAs come from longer primary transcripts that are cleaved by the ribonuclease enzymes,and then assembled into the RNA induced silencing complex. The regulatory mechanism conducted by miRNA depends on the degree of complementarity between the 3'-UTR region of target mRNA and sequence of miRNA. If there is a perfect complementarity, the mRNA is cleaved by RISC. If there is a imperfect complementarity, the regulation is carried out by repression of gene translation. Studies show that miRNAs play important roles in diverse biological and pathological processes including Metabolism, cell cycle, differentiation, apoptosis, and development. In addition, miRNA is closely related with tumor development.About 50% of miRNAs are in tumor-associated chromosomal regions including LOH area, the amplified region of chromosome, fragile sites and so on. Subsequently, they are involved in regulating biological processes of tumor cell in proliferation, apoptosis, adhesion and angiogenesis,and play extensive and important roles in tumor development in the level of gene regulation. The abnormal miRNAs directly involve in tumor invasion and metastasis. Recent data clearly demonstrates that miRNAs can function as both metastasic activators and suppressors by critically regulating various stages of migration and invasion. Some miRNAs as oncogenes, tumor suppressor genes and tumor metastasis genes have been identified. All of these will help in understanding the roles of miRNAs in regulating the cancer invasion-metastasis and providing a basis for finding tumor-specific markers and new target for cancer therapy.Virus is specific intracellular pathogens, and correlate with a variety of animal and plant diseases. Replication of the virus depends on whether they can suppress the host innate immune, escape the host innate or adaptive immune scavenging, such as interferon-like response and apoptosis. To achieve these objectives, most viruses encode proteins to inactivate host cell defense. Recent studies show that virus may also produce miRNA to optimize-their own living environment by regulating it's own and host gene expression. All of these results in infection or virus-associated tumors. The miRNAs encoded by the virus cause widespread concern.EBV is the first human virus found to encode miRNAs. In 2004,Pfeffer and Neilson team firstly cloned five EBV-miRNAs from a Burkitt's lymphoma cell line latently infected with EBV. Studies show that they work in the process of virus infection. The EBV miRNAs are organized in two clusters within the EBV genome: one is in the intronic regions of the BART gene (miR-BARTl and miR-BART2) and another in the untranslated regions (UTRs) of the BHRF1 gene (miR-BHRF1-1 to miR-BHRF1-3). A year later, Pfeffer identified other thirty-three virus-encoded miRNAs through calculational and experimental study. The identification of virus-encoded miRNAs has generated new insights into molecular research of miRNA and made EBV-miRNA research hotter. All of these not only provide a new direction and new content for regulation of host and virus itself gene expression by virus miRNA, but also represent an important step in interaction between virus and host cells, In the present, the majority of viral miRNAs are identified from virus which can cause various tumors. Therefore, they are thought to be closely related with tumors. New doctrine of oncogenic virus considers that virus miRNAs transcript can complement to tumor suppressor gene of host cell. For example, EBV-miR-BHRFl has the potential binding sites with the 3 'UTR of tumor suppressor gene P53 and apoptosis regulator Bcl-2 mRNA, so it may be involved in regulation of apoptosis and cell proliferation of host cell. This may cause the loss-of-function of the host tumor suppressor gene and immune surveillance, and ultimately lead to tumor. In addition, EBV-miR-BART5 was reported to inhibit apoptosis by targeting 3 'UTR of PUMA transcript, which correlated with significantly downregulated PUMA in 60% of NPC tissues. In virus itself gene regulation, EBV-miR-BART2 is predicted to be the transcript antisense to the 3 'UTR of the BALF5 DNA polymerase. It has a perfect complementarity to the BALF5 mRNA and can inhibit lytic replication by degradating this mRNA. In addition, EBV-miR-BART2 can bind with 3'-end of BMLF1 (EB2), BBLF4 and LMP2A mRNA to regulate gene expression of virus itself; For evading host immunosurveillance, EBV-miR-BHRFl-3 can repress the expression of the IFN- inducible T-cell attracting chemokine CXCL-11.Nasopharyngeal carcinoma (NPC) is a tumor arising from the epithelial cells that cover the surface and line the nasopharynx. Its incidence is higher in southern region of China. NPC is seen primarily in middle-aged persons and few in young persons. NPC is a highly malignant tumor with typical cervical lymph node metastases frequently in its early stage. Associated etiological factor seems to follow a multi-step process in which EBV infection is more closely associated with NPC. It can infect epithelial cells and is associated with their transformation. EBV DNA is detectable in most of patients with non-keratinizing NPC and in almost all of undifferentiated NPC. Interestedly, though up to 90% of people around the world have this virus infection, only few of them can develop malignancy finally. The role of EBV in NPC pathogenesis is not entirely clear.Literature retrieval displays that there were very few reports referred to the relationship between EBV encoded miRNAs and NPC so far.In the present project, we firstly screened out the most differentially expressed EBV-miRNA between NPC and NP by miRNA array detection, and secondly established a NPC cell line CNE1 with stable overexpression of this EBV miRNA via lentivirus packaging and infecting. Thirdly, we observed the change in the proliferation, migration and invasion ability of CNE1 after this EBV-miRNA was upregulated, which was further validated by inhibiting this EBV-miRNA. Finally, RNA-seq (transcriptome) was applied to look for associated target genes. The present project may provide some new ideas and ways for further elucidating molecular pathogenesis and facilitating clinical treatment of NPC. METHODS1. The differential expression of EBV encoded miRNAs between NPC and NP was explored by miRNA array detection.2. Ebv-miR-BART7 was selected for our further study due to its highest differential expression between NPC and NP. Its expression status was validated by qRT-PCR.3. Effects of EBV-miR-BART7 on biological behaviors of CNE1 cells was explored in vitro. Lentiviral vector p1VTHM/EBV-miR-BART7 was constructed and transfected into NPC-derived cell line CNE1 was with the vector p1VTHM/ EBV-miR-BART7. After FACS sorting, CNE1-BART7, which can stably express EBV-miR-BART7, was established. Subsequently, the proliferative activity was examined by MTT and cell cycle analysis, and the invasion and migration ability was analyzed by in vitro matrigel invasion assay and transwell migration test in CNE1-mock cells and CNE1-BART7 cells.4. Inhibiting experimentEbv-miR-BART7 inhibitor was transfected into CNE1-p1VTHM/BART7 to reduce the expression of EBV-miR-BART7. The impact of EBV-miR-BART7 downregulation on invasion and migration ability was evaluated by matrigel invasion and transwell migration assay.5. Canditate target genes and pathways associated with EBV-miR-BART7 were preliminarily searched out by RNA-seq analysis combined with bioinformatic analysis.RESULTS1.The expression of some EBV-miRNAs in NPC tissues was significantly higher than that in NP tissues. Of them, EBV-miR-BART7 was the highest differentially expressed miRNA, about 30 times higher in NPC than in NP, so it was selected for our further study. 2. Quantitive real-time PCR was used to detect the expression of EBV-miR-BART7 between NPC and NP tissues, The results of 2-ΔΔCt ananlysis showed that the expression of EBV-miR-BART7 was significantly higher in NPC tissues than in NP tissues. (t=29.445,P<0.001).3. Effects of EBV-miR-BART7 overexpression on biological behaviors of CNE1 cells were analyzed.3.1 Cell proliferation assayCNE1-mock cells and CNE1-BART7 cells were detached with 0.25% trypsin-EDTA and Inhibiting seeded in 96-well plate. The cell vitality was analyzed using MTT assay. The result showed that there were no significant difference in proliferation activity between CNE1-mock and CNE1-BART7 cells (Welch/F=0.631, P=0.428).3.2 Cell cycle analyses of CNE1-mock and CNE1-BART7 cells by flow cytometryCell cycle analysis revealed there was no statistical difference in G0/G1 (t=-0.334, P=0.755), S (t=0.692,P=0.527),G2/M (t=-0.161,P=0.880) between CNE1-mock and CNE1-BART7 cells.3.3 Transwell migration testAfter 17 h incubation,cells that penetrated artificial basement membrane was more per high power field for CNE1-BART7 cells than CNE1-mock cells, the difference being statistically significant(t=16.475,P<0.001).3.4 Matrigel invasion assayIn vitro invasion assay showed that CNE1-BART7 cells had significantly enhanced cell invasion as compared with CNE1-mock cells (t= 22.088, P<0.001).The above results suggested that EBV-miR-BART7 might promote cancer cell migration and invasion.4. Inhibiting experiment 4.1 Transwell migration testTransfected cells (CNE1-BART7-inhibitor) showed a lower motility potential than negative control cells(CNE1-BART7-NC) as EBV-miR-BART7 had been downregulated (t=22.576,P<0.001).4.2 Matrigel invasion assayAfter transfected EBV-miR-BART7 inhibitor, results from in vitro Matrigel invasion assay showed that knock-down of EBV-miR-BART7 in CNE1-BART7 cells inhibited cell invasion compared with CNE1-BART7-NC (t=22.456,P<0.001).5. Prelimilary analysis of target genes and pathways associated with EBV-miR-BART7The RNA-seq method was utilized to find candidate EBV-miR-BART7 target sites.391 upregulated and 101 downregulated genes were found in NPC cell line with highly expressed EBV-miR-BART7, indicating that the main biological functions of these candidate target genes were matrix metallopeptidase activity, Oxidative phosphorylation, regulation of cytoskeleton,cell metabolism, receptor activity, cell cycle, ErbB, Wnt and TGF-beta signaling pathway, cell apoptosis, adherens factors, Focal adhesion and Calcium-binding protein. All of these biological functions were associated with traits that were the hallmarks of the malignant phenotype, with a focus on tumor-host interactions. Accordingly, the abrrent expression of EBV-miR-BART7 might lead to alternation of multiple pathways and cell regulatory mechanisms in NPC.CONCLUSION1.The results of miRNAs array and qRT-PCR revealed that the expression of EBV-miR-BART7 was significantly higher in NPC tissues than in NP tissues.2. EBV-miR-BART7 in vitro resulted in a significant enhance of cell motility and invasion, but no significant change in proliferation in NPC cell lines CNE1. 3. RNA-seq displayed that EBV-miR-BART7 might be involved in the development, including metastasis and invasion, of NPC as regulators of multiple target genes such as SL100A2, MMP1, TIMP1 etc. |
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