Baosheng Guo1, Baoting Zhang2, Lizhen Zheng3, Tao Tang3, Jin Liu1, Heng Wu1, Zhijun Yang1, Songlin Peng4, Xiaojuan He5, Hongqi Zhang4, Kevin K M Yue4, Fuchu He6, Lingqiang Zhang6, Ling Qin3, Zhaoxiang Bian1, Weihong Tan7, Zicai Liang8, Aiping Lu9, Ge Zhang10. 1. Institute for Advancing Translational Medicine in Bone & Joint Diseases, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China; Hong Kong Baptist University Branch of State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University, Hong Kong, China; Institute of Integrated Bioinfomedicine & Translational Science, HKBU Shenzhen Research Institute and Continuing Education, Shenzhen, China; Academician CHAN Sun Chi Albert Workroom for Advancing Translational Medicine in Bone & Joint Diseases, Kunshan RNAi Institute, Kunshan Industrial Technology Research Institute, Kunshan, Jiangsu, China. 2. School of Chinese Medicine, Department of Orthopaedics & Traumatology, The Chinese University of Hong Kong, Hong Kong, China. Electronic address: zhangbaoting@cuhk.edu.hk. 3. School of Chinese Medicine, Department of Orthopaedics & Traumatology, The Chinese University of Hong Kong, Hong Kong, China. 4. Institute for Advancing Translational Medicine in Bone & Joint Diseases, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China; Hong Kong Baptist University Branch of State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University, Hong Kong, China; Institute of Integrated Bioinfomedicine & Translational Science, HKBU Shenzhen Research Institute and Continuing Education, Shenzhen, China. 5. Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Beijing 100700, China. 6. State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, China. 7. Institute for Advancing Translational Medicine in Bone & Joint Diseases, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China; Hong Kong Baptist University Branch of State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University, Hong Kong, China; Institute of Integrated Bioinfomedicine & Translational Science, HKBU Shenzhen Research Institute and Continuing Education, Shenzhen, China; Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha, China. 8. Academician CHAN Sun Chi Albert Workroom for Advancing Translational Medicine in Bone & Joint Diseases, Kunshan RNAi Institute, Kunshan Industrial Technology Research Institute, Kunshan, Jiangsu, China; Laboratory of Nucleic Acid Technology, Institute of Molecular Medicine, Peking University, Beijing, China. 9. Institute for Advancing Translational Medicine in Bone & Joint Diseases, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China; Hong Kong Baptist University Branch of State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University, Hong Kong, China; Institute of Integrated Bioinfomedicine & Translational Science, HKBU Shenzhen Research Institute and Continuing Education, Shenzhen, China; Academician CHAN Sun Chi Albert Workroom for Advancing Translational Medicine in Bone & Joint Diseases, Kunshan RNAi Institute, Kunshan Industrial Technology Research Institute, Kunshan, Jiangsu, China; Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Beijing 100700, China. Electronic address: aipinglu@hkbu.edu.hk. 10. Institute for Advancing Translational Medicine in Bone & Joint Diseases, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China; Hong Kong Baptist University Branch of State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University, Hong Kong, China; Institute of Integrated Bioinfomedicine & Translational Science, HKBU Shenzhen Research Institute and Continuing Education, Shenzhen, China; Academician CHAN Sun Chi Albert Workroom for Advancing Translational Medicine in Bone & Joint Diseases, Kunshan RNAi Institute, Kunshan Industrial Technology Research Institute, Kunshan, Jiangsu, China. Electronic address: zhangge@hkbu.edu.hk.
Abstract
OBJECTIVES: Casein kinase 2 interacting protein 1 (CKIP-1) is a newly discovered intracellular negative regulator of bone formation without affecting bone resorption. In this study, we aimed to identify a cross-species siRNA sequence targeting CKIP-1 to facilitate developing a novel RNAi-based bone anabolic drug for reversing established osteoporosis. METHODS: Eight specifically designed cross-species CKIP-1 siRNA sequences were screened in human, rhesus, rat and mouse osteoblast-like cells in vitro to identify the optimal sequence with the highest knockdown efficiency. The effect of this optimal siRNA sequence on osteogenic differentiation and matrix mineralization was further examined in osteoblast-like cells across different species, followed by an immunogenicity assessment in human peripheral blood mononuclear cells in vitro. The intra-osseous localization and silencing efficiency of the optimal siRNA were examined in vivo using a biophotonic system and real-time polymerase chain reaction, respectively. The RNAi-mediated cleavage of the CKIP-1 transcript was confirmed by rapid amplification of the 5' cDNA ends in vivo. Furthermore, the effect of the optimal siRNA sequence on osteogenic differentiation, bone turnover biomarkers, bone mass and micro-architecture parameters was investigated in healthy and osteoporotic rodents. RESULTS: The CKIP-1 siRNA sequence (si-3) was identified as the optimal sequence, which consistently maintained CKIP-1 mRNA/protein expression at the lowest level across species in vitro. The si-3 significantly increased mRNA expression levels of osteoblast phenotypic genes and matrix mineralization across species without inducing an immunostimulatory activity in vitro. The intra-osseous localization and RNAi-mediated CKIP-1 silencing with high efficiency were confirmed in vivo. Periodic intravenous injections of si-3 promoted mRNA expression of osteoblast phenotypic genes, enhanced bone formation, increased bone mass and elevated serum level of bone formation marker without raising urine level of bone resorption marker in the healthy rodents. Moreover, the si-3 treatment promoted bone formation, improved trabecular micro-architecture and reversed bone loss in the osteoporotic mice. CONCLUSIONS: The identified optimal CKIP-1 siRNA sequence (si-3) could promote osteogenic differentiation across species in vitro, stimulate bone formation in the healthy rodents and reverse bone loss in the osteoporotic mice.
OBJECTIVES:Casein kinase 2 interacting protein 1 (CKIP-1) is a newly discovered intracellular negative regulator of bone formation without affecting bone resorption. In this study, we aimed to identify a cross-species siRNA sequence targeting CKIP-1 to facilitate developing a novel RNAi-based bone anabolic drug for reversing established osteoporosis. METHODS: Eight specifically designed cross-species CKIP-1 siRNA sequences were screened in human, rhesus, rat and mouse osteoblast-like cells in vitro to identify the optimal sequence with the highest knockdown efficiency. The effect of this optimal siRNA sequence on osteogenic differentiation and matrix mineralization was further examined in osteoblast-like cells across different species, followed by an immunogenicity assessment in human peripheral blood mononuclear cells in vitro. The intra-osseous localization and silencing efficiency of the optimal siRNA were examined in vivo using a biophotonic system and real-time polymerase chain reaction, respectively. The RNAi-mediated cleavage of the CKIP-1 transcript was confirmed by rapid amplification of the 5' cDNA ends in vivo. Furthermore, the effect of the optimal siRNA sequence on osteogenic differentiation, bone turnover biomarkers, bone mass and micro-architecture parameters was investigated in healthy and osteoporotic rodents. RESULTS: The CKIP-1 siRNA sequence (si-3) was identified as the optimal sequence, which consistently maintained CKIP-1 mRNA/protein expression at the lowest level across species in vitro. The si-3 significantly increased mRNA expression levels of osteoblast phenotypic genes and matrix mineralization across species without inducing an immunostimulatory activity in vitro. The intra-osseous localization and RNAi-mediated CKIP-1 silencing with high efficiency were confirmed in vivo. Periodic intravenous injections of si-3 promoted mRNA expression of osteoblast phenotypic genes, enhanced bone formation, increased bone mass and elevated serum level of bone formation marker without raising urine level of bone resorption marker in the healthy rodents. Moreover, the si-3 treatment promoted bone formation, improved trabecular micro-architecture and reversed bone loss in the osteoporoticmice. CONCLUSIONS: The identified optimal CKIP-1 siRNA sequence (si-3) could promote osteogenic differentiation across species in vitro, stimulate bone formation in the healthy rodents and reverse bone loss in the osteoporoticmice.
Authors: Yan Deng; Jiao Chen; Yi Zhao; Xiaohui Yan; Li Zhang; Kwongwai Choy; Jun Hu; Himanshu J Sant; Bruce K Gale; Tao Tang Journal: Sci Rep Date: 2016-02-18 Impact factor: 4.379