Lin Sanfu, Chen Shoubo, Fang Kaibin, Shi Jinnan, Wu Wenhua, Wang Wenhuai
Department of Orthopaedics, the Second Affiliated Hospital of Fujian Medical University, Quanzhou Fujian, 362000, P. R. China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2023 May 15;37(5):615-621. doi: 10.7507/1002-1892.202211037.
To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1).
The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture.
The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased ( <0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day ( <0.05). There was no significant difference in p-CREB1/CREB1 between different time points ( >0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity ( <0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased ( <0.05). There was no significant difference in p-CREB1/CREB1 between the two groups ( >0.05).
Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.
通过调节环磷腺苷反应元件结合蛋白1（CREB1），研究miR-26a-5p对脂肪来源间充质干细胞（ADSCs）成骨分化的调控作用。
取4只3-4周龄雌性C57BL/6小鼠的脂肪组织，采用消化分离法分离并培养细胞。经形态学观察及流式细胞术鉴定后，对第3代细胞进行成骨分化诱导。在成骨分化诱导后的0、3、7和14天，通过茜素红染色观察钙沉积情况，检测碱性磷酸酶（ALP）活性，采用实时荧光定量PCR检测miR-26a-5p和CREB1 mRNA表达，采用蛋白质免疫印迹法检测CREB1蛋白及其磷酸化（磷酸化CREB1，p-CREB1）水平。通过starBase数据库预测miR-26a-5p与CREB1之间的结合位点后，利用人胚肾细胞（HEK）-293T细胞进行双荧光素酶报告基因实验以验证靶向关系（以培养48小时后的荧光素酶活性表示）。最后，将miR-26a-p抑制剂（实验组）和相应的阴性对照（对照组）转染至ADSCs。在成骨诱导培养7和14天后，进行茜素红染色、ALP活性检测、实时荧光定量PCR（miR-26a-5p）及蛋白质免疫印迹法[CREB1、p-CREB1、 runt相关转录因子2（RUNX2）和骨钙素（OCN）]检测。
培养的细胞被鉴定为ADSCs。随着成骨诱导培养时间的延长，钙化结节数量和ALP活性显著增加（P<0.05）。细胞中miR-26a-5p的相对表达逐渐降低，而CREB1 mRNA和蛋白的相对表达以及p-CREB1蛋白的相对表达增加。7、14天与0天之间差异显著（P<0.05）。不同时间点之间p-CREB1/CREB1无显著差异（P>0.05）。starBase数据库预测miR-26a-5p与CREB1具有靶向结合序列，双荧光素酶报告基因实验显示，miR-26a-5p过表达显著抑制CREB1野生型荧光素酶活性（P<0.05）。成骨诱导7和14天后，与对照组相比，实验组的钙化结节数量、ALP活性以及CREB1、p-CREB1、OCN和RUNX2蛋白的相对表达显著增加（P<0.05）。两组之间p-CREB1/CREB1无显著差异（P>0.05）。
敲低miR-26a-5p通过上调CREB1及其磷酸化促进ADSCs的成骨分化。
