The Establishment Of NPC Cell Line Overexpress MiR-302s And Characterization Of Its Biological Behaviors

microRNAs(miRNAs) are a recently discovered family of small non-coding RNAs, which exist in eukaryotes and regulate gene expression post-transcriptionally by binding to complementary sites on target mRNAs,miRNAs play important roles in regulation of cell proliferation,development,apoptosis and cell differentiation.The mature miRNAs are 17-24 bp single-stranded RNA molecules.Each miRNA seems to be able to regulate multiple genes.It was shown recently that the expression of certain genes is more dependent on the level of regulatory miRNAs than on the level of mRNAs that encode the proteins(Johnson et al.2005).Recently,a set of miRNAs was described to be ESC-specific in mouse,with their expression being repressed during ESC differentiation and undetectable in adult mouse organs.This set of miRNAs consists of miR-290,miR-291a-3p,miR-292-3p, miR-293,miR-294 and miR-295(miR-290 family),and miR-302a,miR-302b, miR-302c and miR-302d(miR-302 family).MiRBase database show that miR-302 was first cloned from mouse embryonic stem cells.Human miR-302 was predicted based on homology with mouse and later confirmed by cloning in human embryonic stem cells,miR-302a is located in a cluster with the related miR-302b,miR-302c,miR-302d,and miR-367 on human chromosome 4.Results of SHI-LUNG LIN found that mir-302s expressed most abtmdantly in slow-growing human ES cells and quickly decreased after cell differentiation and proliferation.Therefore,mir-302s was investigated as one of the key factors essential for maintenance of ES cell self-renewal and pluripotency.When transgenically transfect the mir-302s into several human cancer cell lines,The mir-302-transfected cells(we also called mirPS cells) not only expressed many key ES cell markers,such as Oct3/4,SSEA-3,SSEA-4,Sox2,and Nanog,but also had a highly demethylated genome similar to a reprogrammed zygotic genome.Microarray analyses further revealed that genomewide gene expression pattems between the mirPS and human ES H1 and H9 cells shared over 86%similarity.Using molecular guidance in vitro,these mirPS cells could differentiate into distinct tissue cell types, such as neuron-,chondrocyte-,fibroblast- and spermatogonia-like primordial cells.Each member of the mir-302 family is able to simultaneously regulate over 445 cellular genes and they all share almost the same target genes.Many of the mir-302 targeted genes are active developmental signals involved in initiation or facilitation of lineage-specific cell differentiation during early embryonic development.By suppressing these developmental genes,mir-302s may reprogram cells into a more ES-like state.Alternatively,mir-302s may also attenuate the expression of their predicted target transcription factors,such as SP3,HMG-box,forkhead-box,and LIM-homeobox gene families,to provide the cell reprogramming effect.Thus,the function of mir-302s is more likely to attenuate the global production of developmental signals and/or transcription factors rather than to directly create transcriptional stimulation on certain embryonic signaling pathways.Nevertheless, mir-302s may not be the only miRNA family involved in this mechanism because their target genes are also redundantly silenced by the group of mir-93,mir-367, mir-371,mir-372,mir-373,and mir-520 in human ES cells,as predicted by the TARGETSCAN program.Learning why these target genes must be simultaneously silenced during the reprogramming process of cancer-ES cell transformation may shed light on the mechanism underlying this miRNA-mediated effect on ES cell maintenance and self-renewal.Deborah showed that Oct4 and Sox2 bound to a conserved promoter region of miR-302.The expression of miR-302a is dependent on Oct4/Sox2 in human ESCs (hESCs),and miR-302a is expressed at the same developmental stages and in the same tissues as Oct4 during embryogenesis,miR-302a is predicted to target many cell cycle regulators,and the expression of miR-302a in primary and transformed cell lines promotes an increase in S-phase and a decrease in G1-phase cells. Correspondingly,the inhibition of miR-302 causes hESCs to accumulate in G1 phase. Moreover,they showed that miR-302a repressed the productive translation of an important G1 regulator,cyclin D1,in hESCs.The transcriptional activation of miR-302 and the translational repression of its targets,such as cyclin D1,may provide a link between Oct4/Sox2 and cell cycle regulation in pluripotent cells.So many studies about mir-302 have focused on the ESC or the target genes which upstream or downstream it.We just know mir-302s expressed abundantly in ESC,but when we transfected mir-302s into nasopharyngeal carcinoma cells what would happen,it's not clear.In this research,the role of mir-302s in NPC will be studied.1 Transfection and identification of the lentiviral expression vector of human miR-302s precursorLentivector-based microRNA precursor constructs were confirmed by DNA sequencing.The viral supernatant was harvested from 293FT cells which were co-transfected with expression vectors and packaging plasmids.CNE2 cells were infected with the viral supernatant and the cells with stable miR-302s expression were separated by fluorescence activated cell sorter(FASC). In situ hybridization was used to detect the expression of miR-302s in CNE2 cells,CNE2-PMIRNA1 and CNE2-PMIRH/302abcd cells.The results indicated there were no positive signals in CNE2 cells,however,strongly positive signals were manifested in the CNE2-PMIRH/302abcd cells transfected with miR-302s precursor.And the TaqMan real-time RT-PCR also confirmed this results.2 Effects of miR-302s overexpression on biological behaviors of CNE2 cellsThe successfully transfected cells were used in vitro experiments.MTT assay showed CNE2-PMIRH/302abcd cells have a significantly enhanced proliferation compared with CNE2 and CNE2- pMIRNA1 cells(P＜0.001 ).However,the cell cycle distribution detected by flow cytometry showed that CNE2-PMIRH/302abcd cells have an increase in S-phase and a decrease in G1-phase cells.These results indicated overexpression of miR-302s partially enhanced proliferatin of CNE2 cells.The results of in vitro motility assay showed that CNE2-PMIRH/302abcd cells had significantly enhanced migration as compared with CNE2 and CNE2-pMIRNA1 cells(P＜0.001).This showed over-expression of miR-302s partially led to a up-regulated migration of CNE2 cells.Flat colony forming experiment proved that CNE2-PMIRH/302abcd cells formed more clonies than CNE2 and CNE2- pMIRNA1 cells(P＜0.05).This also indicates that the cells after transfection with miR-302s had a enhanced tumorigenicity.In vivo experiments don't have significant changes perhaps correlate with the less cases or we should observe longer time for these mouses.The expression of CD133 remained invariable when transfected the mir-302s also can't exclude the possibility of dedifferentiation from cancer cells to cancer stem cells. Conclusion1 Successfully transfected and separated the cells which over-expression the miR-302a precursors and detected by in situ hybridization and TaqMan real-time RT-PCR.2 Overexpression of miR-302s in vitro results in an enhanced of cell proliferation and motility in NPC-derived cell lines CNE2.but the expression of CD133 remains invariable which detected by flow cytometry.