Chinese Journal of Tissue Engineering Research ›› 2018, Vol. 22 ›› Issue (24): 3831-3836.doi: 10.3969/j.issn.2095-4344.0799

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Validation of miRNA-140 targeting autophagy-related gene uncoordinated 51 like kinase-1

Lu Yan-yan1, Yao Nan2, Xu Xue-meng1, Liu Wen-gang1, Cai Da-ke2, Huang Dan-e2, Zhao Chuan-xi1, Chen Guo-cai1   

  1. 1Department of Orthopedics, Guangdong Second Traditional Chinese Medicine Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510095, Guangdong Province, China; 2Guangdong Province Engineering Technology Research Institute of TCM/Guangdong Provincial Key Laboratory of Research and Development in Traditional Chinese Medicine, Guangzhou 510095, Guangdong Province, China
  • Received:2017-12-25
  • Contact: Liu Wen-gang, M.D., Chief physician, Department of Orthopedics, Guangdong Second Traditional Chinese Medicine Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510095, Guangdong Province, China
  • About author:Lu Yan-yan, Doctoral candidate, Department of Orthopedics, Guangdong Second Traditional Chinese Medicine Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510095, Guangdong Province, China
  • Supported by:

    the Key Subject Construction Project of State Administration of Traditional Chinese Medicine of China, No. [2012]7; the Natural Science Foundation of Guangdong Province, No. 2014A030310128; the Chinese Medicine Dominant Disease Breakthrough Project of Guangdong Province, No. [2015]19; the Scientific Research Project of Traditional Chinese Medicine Bureau of Guangdong Province, No. 20164002, 20172008 and 20171023

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Abstract:

BACKGROUND: Autophagy can alleviate the development of knee osteoarthritis by inhibiting cell apoptosis. However, it is still unclear that whether miR-140 can regulate the key gene uncoordinated 51 like kinase-1 (ULK1) in autophagy.
OBJECTIVE: To explore the regulatory role of miR-140 on the ULK1 gene.
METHODS: The microRNA website was used to predict whether the ULK1 was the target gene of miR-140. Firstly, the full-length sequence of 3'-untranslated region of ULK1 was embed in the downstream of the renilla luciferase gene. Secondly, the mutant sequence of binding sites was constructed to obtain wild type vector and mutant vector. Thirdly, the expression activity of luciferase was detected after transfecting 293T cells with these luciferase vectors. Lastly, 293T cells were treated with miRNA-140 inhibitor, and the expression level of ULK1 protein was detected by western blot assay.
RESULTS AND CONCLUSION: The expression activity of luciferase was significantly decreased in 293T cells transfected by wild type vector and miR-140 mimics compared with the cells transfected by wild type vector alone (P < 0.01). In addition, the expression of ULK1 protein was significantly increased when miR-140 was inhibited (P < 0.01). These results imply that miR-140 can regulate the expression of ULK1 gene by binding with its gene sequence.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Tissue Engineering, Osteoarthritis, Apoptosis

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