| MicroRNAs (miRNAs) are small noncoding RNAs that play very important roles in posttranscriptional gene regulation in animals, plants, and fungi. To date, miRNAs are also now known to be produced by viruses. To investigate whether the human papillomavirus encodes miRNAs, we take advantaged of bioinformatics approaches to predict HPV16 miRNA precursor and to analyze its mature miRNAs among its genome. Sequence analysis revealed that HPV16 encodes one pre-miRNAs candidate. We then matched deduced mature miRNA sequences from these 5 pre-miRNAs to a database of 3' untranslated sequences (UTR) from the human and HPV genome. This predicted miRNA precursor can be cleaved putatively into 3 mature miRNAs of 19 to 22 bases in length. And our subsequent searches revealed that these deduced viral miRNA (vmiRNA) sequences could potentially bind to a number of cellular and two viral target genes and in turn arrest their mRNA translation. These findings clearly suggest that viral and host cellular gene expression may be regulated by HPV16 miRNAs and also provide a foundation for the further experimental researches to test the regulatory functions of these predicted HPV16 miRNAs.To study the HPV16E6 specific shRNA expressed by U6 plasmid to inhibit the expression of HPV oncogene and the growth of cervical cancer cell, we selected 4 target mRNA sequences to the exon and interon of HPV16E6 mRNA, and synthesized dsDNA to construct the recombinant pSilencerl. 0-U6 vector which expresses HPV16E6 short hair-pin dsRNA. After transfected into HPV16 DNA positive cervical cancer cell line CaSki, then observe the expression level of HPV16E6 E7 genes and their proteins as well as the inhibition effects of cell growth. Our results that the cellular growth velocity was down-regulated both by 4 HPV16E6 specific siRNA in 0-96 h after recombinant pSilencerl. 0-U6 vector transfected into Caski cell. The abundance of HPV16E6 E7 mRNA inside CaSki cells were also reduced by the 4 HPV16E6 specific siRNA respectively, while one HPV16E6 specific siRNA targetting to the interon sequence of E6 gene inhibited the HPV16E6 mRNA only. HPV16E6 protein was undetectable with Western blot assay 72 h later after 4 HPV16E6 specific siRNA transfected into CaSki cell. It suggested that the growth of cervical cancer cells was inhibited by HPV16E6 specific siRNA. The HPV16E6 specific siRNA targeting to the interon of E6mRNA can inhibit the expression of HPV16E6 gene only ,while the HPV16E6 specific siRNAs targeting to the exon of E6mRNA can inhibit the expression of both E6 and E7 genes, which can be used as a good candidates of cancer therapeutical siRNA drug.Bone formation proceeds by the precise coordination of many factors. An osteoblast transcription factor, CBFA1, is a critical transcriptional regulator of several bone-specific genes, such as osteopontin and osteocalcin, and by controlling bone extracellular matrix deposition. Deregulation of one essential transcriptional factor, Cbfa1, has been shown to lead to metabolic bone disease, including osteoporosis and osteopetrosis. Retinoblastoma protein (pRb) was found to function as a direct transcriptional coactivator of Cbfa1 promoting osteoblast differentiation and was required for Cbfa1-mediated osteogenesis. We recently reported that one interferon-inducible p204 protein is also associated with Cbfa1 and enhance osteogenesis. Here we further demonstrated that induced expression of p204 in osteogenesis is required for the Cbfa1-dependent gene activation, because that low expression of p204 controlled through adenovirus encoding antisense p204 abolished the osteoblast-specific gene activation by Cbfa1 in the osteogenesis assay with a pluripotent mesenchymal cell line C2C12. Using multiple proteins co-immunoprecipitation and GST-pull-down assay, we revealed that p204, Rb and Cbfa1 form a ternary protein complex via Rb as a linker. In addition, this p204/Rb/bfal transcriptional complex is detectable in the promoter of osteocalcin gene, as experiented by chromatin immunoprecipitation. These results suggest that Rb and p204 play synergistical roles in enhancing Cbfa1- mediated osteogenesis in responding to BMP-2 signal, and that Rb may be necessary for p204 binding Cbfa1 and enhancing the osteogenesis.Cbfa1 is a major target of the bone morphogenetic protein (BMP) pathway. It is a global regulator of osteogenesis and is crucial for regulating the expression of bone-specific genes such as osteopontin and osteocalcin. Runx2 and Inhibitor of differentiation (Id) proteins were also BMP induced proteins and inhibitors of the differentiation of C2C12 cells to osteoblast. We wanted to know whether Ids bind to Cbfa1 and thereafter inhibit the activity of Cbfa1. Id1, 2, 3 can bind to Cbfa1 in vivo and during the BMP-2 induced the C2C12 cells by CoIP and GST pulldown assay. Id2 binds to Cbfa1 by its HLH domain, and Cbfa1 binds to Id2 through its fragment of 222-280aa. Id2 could inhibit the Cbfa1-mediated osteogenesis of C2C12 cells to osteoblasts by decreasing the ALP activity and OCL level in the media. By chromatin immunoprecipitation assay, Id can inhibit the sequence-specific binding to OCL promoter DNA of Cbfa1. By immunofluorescent cell stain, Id2 translocates under the stimulation of BMP-2 protein. Western Blot results showed that Id2 can decrese the p204 and Cbfa1 protein level during the induction of BMP-2 to C2C12 cells. It suggested that endogenous Id proteins inhibited the differentiation of C2C12 cells to osteoblast. This was in consequence of the binding of Id1, Id2, or Id3 protein to the Cbfa1 proteins and the resulting inhibitions (i) of the binding of this transcription factor to DNA and (ii) of their synergistic transactivation of the expression of p204 and Cbfa1 itself. It needs further research to show that whether translocation of Id is associated with the de-inhibition of Cbfa1 activity by Id protein.Id protein inhibits the Cbfa1-mediated transactivation activity of OCL gene during the BMP-2 induced osteogenesis that represses the osteoblastic differentiation. Interferon induced protein p204 is required for the differentiation of murine C2C12 embryonal stem cells to beating osteoblast cell, and p204 protein in the differentiating C2C12 cells enhance Cbfa1-mediated transactivation activity. Since p204 binds both Ids and Cbfa1 and enhance the differentiation during the myogenesis and cardiac myogenesis, we want to know whether p204 has the same effect in osteogenesis as it does in those two differentiation. p204 can bind to Id from the complex of Cbfa1 and Id2 and have a high affluency to Id2 by the GST pull-down assay. p204 could also overcome the demonstrated inhibition by Id2 of C2C12 cells to osteoblasts by increasing the ALP activity and OCL level in the media. By chromatin immunoprecipitation assay, p204 can overcome the inhibition of the sequence-specific binding to OCL promoter DNA of Cbfa1 by the Id proteins. It suggested that p204 overcame this inhibition by Id proteins in consequence of (i) binding and sequestering Id proteins, release Cbfa1 protein (ii) decreasing the level of Id proteins in the nucleus by increasing their translocation from the nucleus to the cytoplasm, and (iii) accelerating their degradation by proteasomes, (iv) overcome the inhibition of Id to Cbfa1 binding the OCL promoter. This loop arose in consequence of it that p204 overcame the inhibition of the synergistic activity of Cbfa1 by the Id proteins and activate the osteogenesis.In brief, this paper shows the regulation of gene expression in cervical cancer by miRNA and siRNA, as well as the interaction of p204, Rb, Cbfa1, Ids in the osteogenesis,...
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