Preliminary Study Of MiR-155Function In Nasopharyngeal Carcinoma

Background:Nasopharyngeal carcinoma (NPC) is a malignant tumor originating from nasopharyngeal mucosa. NPC has the property of high degree of malignancy and cervical lymph node metastasis in the early stage. Studies have suggested that the Epstein-Barr virus (EBV) playes important roles in NPC initiation, progression and metastasis. MicroRNAs (miRNAs) are genes involved in cancer initiation, progression, metastasis, angiogenesis, and prognosis, etc.miRNAs are a class of endogenous small non-coding RNAs found in eukaryotes with regulatory functions, composed by21-25nucleotides. It is characterized by having no open reading frame (ORF) and protein coding genes, but unique feature of sequence, which mainly regulate the gene expression in the post-transcriptional level. Lee et al. firstly discovered miRNA lin-4in C. Elegans, while Reinhart and his teams found the second non-coding of miRNA let-7. miRNAs play important roles in regulating a series of important life activities, such as organism metabolism, cell cycle, cell proliferation, cell differentiation, cell apoptosis, individual development, and so on. miRNAs are genes involved in cancer initiation, progression, metastasis and angiogenesis. Until now, many miRNAs have been found with functions as proto-oncogene or tumor suppressor gene, such as let-7, miR-21, miR-155, miR-17, miR-143and miR-145, miR-372and miR-373, and miR-26a, etc.miR-155located on human chromosome21and the third exon of B-cell integration of the cluster (Bic) gene containing no open reading frame (ORF). Researches have indicated that miR-155plays roles in hematopoietic cells generation, immune response, inflammatory response, etc. miR-155is overexpressed in B-cell lymphoma and leukemia, and in a wide range of human solid tumors, including breast, lung, liver, colon, pancreatic, cervical and prostate cancers, etc. The miR-155gene induced the development of lymphomas and leukemia in mice, while the enforced expression of miR-155gene promoted the cell proliferation of breast cancer, lung cancer and hepatocellular carcinoma, suggesting the oncogenic function of miR-155. EBV-induced miR-155is also up-regulated in NPC, suggesting certain functions of miR-155in pathogenesis of NPC, which remains to be fully demonstrated in the future. The above-mentioned information encourages us to explore the functions of miR-155in NPC.Methods:1. Detection of expression profile of miR-155in NPC cell lines and tissuesqRT-PCR was used to detect the expression of miR-155in NPC-derived cell lines CNE1, CNE2,5-8F,6-10B, HONE1, HNE1, SUNE1and C666-1and in immortalized nasopharyngeal epithelial cell strain NP69.miR-155expression in15human NPC tissues and10human chronic nasopharyngitis tissues was detected by qRT-PCR.2. Generation of NPC cell lines overexpressing miR-155transgene Lentiviruses carrying miR-155and enhancand green fluorescent protein (EGFP) genes were produced, followed by infecting NPC-derived cell line CNE1, CNE2, HONE1and SUNE1using lentivirus supernatant. Finally, NPC cell lines overexpressing miR-155transgene were confirmed to be generated by EGFP assay under inverted fluorescence microscope and qRT-PCR used to detect the expression of miR-155transgene in CNE1, CNE2, HONE1and SUNE1.3. The effects of miR-155overexpression on NPC cell proliferation, migration and epithelial-mesenchymal transition (EMT)The ability of cell proliferation was detected by CCK8aasay, cell cycle and colony formation assay, while the in vitro migration ability of NPC cells was analyzed by transwell chamber assay. The expression of EMT-related gene, such as E-cadherin, α-catenin, fibronectin, N-caherin and vimentin, was assayed by qRT-PCR, Western blot and immunofluorescence in NPC cell lines overexpressed miR-155and control.4. The effects of down-regulated expression of endogenous miR-155by miR-155inhibitor on NPC cell proliferation, migration and EMTmiR-155inhibitor was transfected into NPC cell lines HONE1and SUNE1to down-regulate the endogenous miR-155expression. The influences of miR-155downregulation on proliferation ability and migration were evaluated by cell proliferation assay CCK8and transwell migration assay, respectively.Results:1. Expression profile of miR-155in NPC cell lines and tissuesqRT-PCR was used to detect the expression of miR-155among different NPC cell lines and human NPC tissues. The results indicated that the levels of miR-155expression was significantly higher in NPC cell lines (such as CNE1, CNE2,5-8F, 6-1OB, C666-1, HNE1, SUNE1and HONE1) than in NP69cells. qRT-PCR results showed that the levels of the miR-155expression in NPC tissues and chronic nasopharyngitis tissues are no significant difference.2. Generation of NPC cell lines stably overexpressing miR-155transgeneNPC cell lines (i.e., CNE1, CNE2, HONE1and SUNE1) stably overexpressing miR-155transgene were confirmed to be successfully generated by EGFP assay under inverted fluorescence microscope and qRT-PCR used to detect the expression of miR-155transgene in CNE1, CNE2, HONE1and SUNE1.3. miR-155overexpression in CNE2and HONE1cells induced cell proliferation, cell migration and EMTThe results from CCk8assay, cell cycle and colony formation assay strongly supported that miR-155overexpression increased cell growth in miR-155-expressing CNE2and HONE1cells. Additionally, in this study, the stably enforced expression of miR-155in human NPC cells (i.e., CNE2and HONE1) induced an epithelial-mesenchymal transition (EMT), as shown by downregulation of epithelial protein E-cadherin and a-catenin, and upregulation of mesenchymal proteins vimentin, fibronectin1and N-cadherin, and promoted cell motility, as indicated by Transwell Migration Assay.4. The down-regulated miR-155expression in NPC cells inhibited cell proliferation, cell migration and EMTThe results from CCk8assay indicated that down-regulated miR-155expression in SUN1and HONE1cells inhibited cell proliferation. Moreover, the down-regulated miR-155expression in SUN1and HONE1cells blocked an epithelial-mesenchymal transition (EMT), as shown by up-regulation of epithelial protein E-cadherin, and down-regulation of mesenchymal proteins vimentin and N-cadherin, and inhibited cell motility, as indicated by Transwell Migration Assay.Conclusion:Taken together, these findings demonstrate that miR-155can promote cell proliferation and cell motility, and trigger EMT in NPC cells.