J Xu1, Y Liu2, M Deng3, J Li3, H Cai3, Q Meng3, W Fang3, X Long4, J Ke5. 1. State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education (KLOBM), School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei Province, China. 2. Department of Radiation and Medical Oncology, Zhongnan Hospital, Wuhan University, Wuhan, Hubei Province, China. 3. Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei Province, China. 4. Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei Province, China. Electronic address: longxing_china@hotmail.com. 5. State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education (KLOBM), School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei Province, China. Electronic address: kejin@whu.edu.cn.
Abstract
OBJECTIVE: This study aimed to screen differential expression of microRNAs (miRNAs), and investigate function of the specifically selected miRNA in synovial fibroblasts from patients suffering osteoarthritis of temporomandibular joint (TMJOA). METHODS: MiRNA microarray was used to select differentially expressed miRNAs between TMJOA and normal synovial fibroblasts. The expression of screened miRNA221-3p was quantified using real-time PCR, and its specific target gene was predicted by bioinformatics. After transfection of miRNA221-3p mimics or inhibitor into synovial fibroblasts, the expression of v-Ets avian erythroblastosis virus E26 oncogene homolog 1 (Ets-1) was detected by immunohistochemistry, real-time PCR and Western blot, respectively. Dual luciferase activity was performed to identify the direct regulation of miRNA221-3p on Ets-1. Interlukin-1β (IL-1β) mimics an inflammatory situation. RESULTS: In TMJOA synovial fibroblasts, eight miRNAs were up-regulated and six miRNAs were down-regulated. MiRNA221-3p was the most down-expressed. A sequence in the 3'-untranslated (3'-UTR) of Ets-1 complementary to the seed sequence of miRNA221-3p. Elevated expression of Ets-1 associated with attenuation of miRNA221-3p. Over-expression of miRNA221-3p suppressed the activity of a reporter construct containing the 3'-UTR of Ets-1 transcript and inhibited the expression of Ets-1 as well as its downstream molecules, matrix metalloproteinase 1 (MMP1) and MMP9 in TMJOA synovial fibroblasts. IL-1β suppressed the expression of miRNA221-3p in both a dose-dependent and time-dependent manner. CONCLUSION: The reduction of miRNA221-3p in synovial fibroblasts, attributed from abundance of IL-1β in inflamed circumstance, induces Ets-1 up-regulation and then, initiates MMP1 and MMP9 secretion, thereby leading to continuously pathological development in TMJOA.
OBJECTIVE: This study aimed to screen differential expression of microRNAs (miRNAs), and investigate function of the specifically selected miRNA in synovial fibroblasts from patients suffering osteoarthritis of temporomandibular joint (TMJOA). METHODS: MiRNA microarray was used to select differentially expressed miRNAs between TMJOA and normal synovial fibroblasts. The expression of screened miRNA221-3p was quantified using real-time PCR, and its specific target gene was predicted by bioinformatics. After transfection of miRNA221-3p mimics or inhibitor into synovial fibroblasts, the expression of v-Ets avian erythroblastosis virus E26 oncogene homolog 1 (Ets-1) was detected by immunohistochemistry, real-time PCR and Western blot, respectively. Dual luciferase activity was performed to identify the direct regulation of miRNA221-3p on Ets-1. Interlukin-1β (IL-1β) mimics an inflammatory situation. RESULTS: In TMJOA synovial fibroblasts, eight miRNAs were up-regulated and six miRNAs were down-regulated. MiRNA221-3p was the most down-expressed. A sequence in the 3'-untranslated (3'-UTR) of Ets-1 complementary to the seed sequence of miRNA221-3p. Elevated expression of Ets-1 associated with attenuation of miRNA221-3p. Over-expression of miRNA221-3p suppressed the activity of a reporter construct containing the 3'-UTR of Ets-1 transcript and inhibited the expression of Ets-1 as well as its downstream molecules, matrix metalloproteinase 1 (MMP1) and MMP9 in TMJOA synovial fibroblasts. IL-1β suppressed the expression of miRNA221-3p in both a dose-dependent and time-dependent manner. CONCLUSION: The reduction of miRNA221-3p in synovial fibroblasts, attributed from abundance of IL-1β in inflamed circumstance, induces Ets-1 up-regulation and then, initiates MMP1 and MMP9 secretion, thereby leading to continuously pathological development in TMJOA.