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      Characterization of the essential role of bone morphogenetic protein 9 (BMP9) in osteogenic differentiation of mesenchymal stem cells (MSCs) through RNA interference


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          Mesenchymal stem cells (MSCs) are multipotent stem cells and capable of differentiating into multiple cell types including osteoblastic, chondrogenic and adipogenic lineages. We previously identified BMP9 as one of the most potent BMPs that induce osteoblastic differentiation of MSCs although exact molecular mechanism through which BMP9 regulates osteogenic differentiation remains to be fully understood. Here, we seek to develop a recombinant adenovirus system to optimally silence mouse BMP9 and then characterize the important role of BMP9 in osteogenic differentiation of MSCs. Using two different siRNA bioinformatic prediction programs, we design five siRNAs targeting mouse BMP9 (or simB9), which are expressed under the control of the converging H1 and U6 promoters in recombinant adenovirus vectors. We demonstrate that two of the five siRNAs, simB9-4 and simB9-7, exhibit the highest efficiency on silencing exogenous mouse BMP9 in MSCs. Furthermore, simB9-4 and simB9-7 act synergistically in inhibiting BMP9-induced expression of osteogenic markers, matrix mineralization and ectopic bone formation from MSCs. Thus, our findings demonstrate the important role of BMP9 in osteogenic differentiation of MSCs. The characterized simB9 siRNAs may be used as an important tool to investigate the molecular mechanism behind BMP9 osteogenic signaling. Our results also indicate that recombinant adenovirus-mediated expression of siRNAs is efficient and sustained, and thus may be used as an effective delivery vehicle of siRNA therapeutics.

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          Most cited references82

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          Small silencing RNAs: an expanding universe.

          Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNA classes have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, their modes of target regulation and in the biological pathways they regulate. There is a growing realization that, despite their differences, these distinct small RNA pathways are interconnected, and that small RNA pathways compete and collaborate as they regulate genes and protect the genome from external and internal threats.
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            CRISPR/Cas9 in Genome Editing and Beyond

            The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.
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              A simplified system for generating recombinant adenoviruses.

              Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We report herein a strategy that simplifies the generation and production of such viruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, using homologous recombination in bacteria rather than in eukaryotic cells. After transfections of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of green fluorescent protein, encoded by a gene incorporated into the viral backbone. Homogeneous viruses can be obtained from this procedure without plaque purification. This system should expedite the process of generating and testing recombinant adenoviruses for a variety of purposes.

                Author and article information

                Genes Dis
                Genes Dis
                Genes & Diseases
                Chongqing Medical University
                27 April 2018
                June 2018
                27 April 2018
                : 5
                : 2
                : 172-184
                [a ]Ministry of Education Key Laboratory of Diagnostic Medicine and School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
                [b ]Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
                [c ]The School of Pharmacy and the Affiliated Hospitals of Chongqing Medical University, Chongqing 400016, China
                [d ]Key Laboratory of Orthopaedic Surgery of Gansu Province and the Department of Orthopaedic Surgery, The Second Hospital of Lanzhou University, Lanzhou, 730030, China
                [e ]Department of Biochemistry and Molecular Biology, China Three Gorges University School of Medicine, Yichang 443002, China
                [f ]Department of Surgery, The Affiliated Zhongnan Hospital of Wuhan University, Wuhan 430071, China
                [g ]Department of Orthopaedic Surgery, Chongqing Hospital of Traditional Chinese Medicine, Chongqing 400021, China
                [h ]Department of Orthopaedic Surgery, Xiangya Second Hospital of Central South University, Changsha 410011, China
                [i ]Department of Surgery, Laboratory of Craniofacial Biology and Development, Section of Plastic Surgery, The University of Chicago Medical Center, Chicago, IL 60637, USA
                Author notes
                [] Corresponding author. School of Clinical Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.Fax: +86 23 6848 5658. yaguangweng@ 123456126.com
                © 2018 Chongqing Medical University. Production and hosting by Elsevier B.V.

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                : 6 April 2018
                : 17 April 2018

                bmp9,bone formation,mesenchymal stem cells,osteogenic differentiation,rna interference,recombinant adenovirus,sirna


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