Department of Orthopedics, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, China.
Eur Rev Med Pharmacol Sci. 2021 Jan;25(2):636-642. doi: 10.26355/eurrev_202101_24625.
The aim of this study was to investigate the influence of micro ribonucleic acid (miR)-29c-3p on rats with diabetic osteoporosis (DOP) and its underlying mechanism.
A total of 30 specific pathogen-free (SPF)-grade male Wistar rats aged 6-week-old were randomly selected and divided into three groups according to different intervention means, including: NC group (control rats only injected with normal saline), DOP group (rats with DOP induced by injection of streptozotocin), and ME group (DOP rats injected with miR-29c-3p agonist for 4 consecutive weeks). The changes in blood glucose and body weight were recorded in the rats of each group every week. Enzyme-linked immunosorbent assay (ELISA) was applied to detect the content of bone turnover markers (BTMs) in serum, such as alkaline phosphatase (ALP), osteocalcin (OC), and procollagen type I N-terminal propeptide (PINP). The variations in serum calcium (Ca) and phosphorus (P) levels in the abdominal aorta were determined using an atomic absorption spectrometer in the three groups. Meanwhile, bone mineral density (BMD) of femur and lumbar vertebra (L1-L4) were examined. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the changes in messenger RNA (mRNA) expressions of miR-29c-3p and Disheveled 2 (Dvl2) in the bone tissues of intervened rats. In addition, the staining and expression changes of Dvl2 protein in bone tissues were determined via immunohistochemistry.
The rats in NC group had normal behavioral activities, normally increased body weight, sensitive responses, as well as normal and stable blood glucose. In DOP group, the rats manifested clinical symptoms of diabetes mellitus (DM) (i.e., polydipsia, polyphagia, polyuria, and weight loss), lackluster hairs, decreased behavioral activities, slow responses, and blood glucose at a concentration higher than 16.7 mmol/L. However, the blood glucose rose first, and then, declined and it was maintained at a level higher than normal concentration in ME group. Meanwhile, the rate of weight loss significantly decreased. The results of qRT-PCR indicated that the relative expression level of miR-29c-3p in bone tissues of DOP group was remarkably lower than that in NC group (p<0.01). However, it was significantly higher in ME group than that in DOP group (p<0.05). DOP group exhibited significantly upregulated serum BTMs (ALP, CTX-1, OC, TRACP-5b, and PIPN) when compared with NC group (p<0.05) and ME group (p<0.05). Furthermore, femoral BMD decreased in DOP group (p<0.05) while increased in ME group, showing statistically significant difference between the two groups (p<0.05). Immunohistochemistry results indicated that the bone tissues of DOP rats were deeply stained, and protein expression of Dvl2 protein was significantly higher in comparison with NC group. The bone tissues were lightly stained in ME group, and the protein expression of Dvl2 was lower than that in DOP group. Besides, qRT-PCR results demonstrated that the mRNA expression changes of Dvl2 were consistent with its protein expression trends.
MiR-29c-3p reduces bone loss in rats with DOP via targeted regulation of Dvl2 expression.
本研究旨在探讨微小 RNA（miR）-29c-3p 对糖尿病性骨质疏松症（DOP）大鼠的影响及其作用机制。
将 30 只特定病原体（SPF）级雄性 Wistar 大鼠随机分为三组，根据不同的干预方法进行分组：NC 组（仅注射生理盐水的对照组）、DOP 组（注射链脲佐菌素诱导 DOP 的大鼠）和 ME 组（连续 4 周注射 miR-29c-3p 激动剂的 DOP 大鼠）。每周记录每组大鼠的血糖和体重变化。采用酶联免疫吸附试验（ELISA）检测血清骨转换标志物（BTMs）碱性磷酸酶（ALP）、骨钙素（OC）和 I 型前胶原 N 端前肽（PINP）的含量。采用原子吸收光谱仪检测三组大鼠腹主动脉血清钙（Ca）和磷（P）水平的变化。同时，检测股骨和腰椎（L1-L4）的骨密度（BMD）。采用实时定量聚合酶链反应（qRT-PCR）检测干预大鼠骨组织中 miR-29c-3p 和 Dvl2 信使 RNA（mRNA）表达的变化。此外，采用免疫组织化学法检测骨组织中 Dvl2 蛋白的染色和表达变化。
NC 组大鼠行为活动正常，体重正常增加，反应灵敏，血糖稳定。DOP 组大鼠出现糖尿病（DM）的临床症状（多饮、多食、多尿和体重减轻），毛发无光泽，行为活动减少，反应迟钝，血糖浓度高于 16.7mmol/L。然而，ME 组的血糖先升高，然后下降并维持在高于正常浓度的水平。同时，体重减轻的速度明显减慢。qRT-PCR 结果表明，DOP 组骨组织中 miR-29c-3p 的相对表达水平明显低于 NC 组（p<0.01）。然而，在 ME 组中，它明显高于 DOP 组（p<0.05）。与 NC 组相比，DOP 组血清 BTMs（ALP、CTX-1、OC、TRACP-5b 和 PIPN）显著升高（p<0.05），与 ME 组相比也显著升高（p<0.05）。此外，DOP 组股骨 BMD 降低（p<0.05），而 ME 组则升高，两组间差异有统计学意义（p<0.05）。免疫组织化学结果表明，DOP 大鼠的骨组织染色较深，Dvl2 蛋白的表达明显高于 NC 组。ME 组的骨组织染色较浅，Dvl2 蛋白的表达低于 DOP 组。此外，qRT-PCR 结果表明，Dvl2 的 mRNA 表达变化与其蛋白表达趋势一致。
miR-29c-3p 通过靶向调节 Dvl2 的表达减少 DOP 大鼠的骨丢失。
