摘要
背景:酪蛋白激酶2结合蛋白1(casein kinase 2-interaction protein-1,CKIP-1)是一种重要的骨形成负调控基因,其敲除鼠骨质显著增强、骨形成和骨密度也显著提高。而miRNA作为较早发现的小分子调控物,对大多数编码基因具有调控作用,在成骨分化中发挥重要作用。目的:探讨miRNA/CKIP-1对骨质疏松患者骨髓间充质干细胞成骨分化的影响及其分子机制。方法:采用miRNA-Seq技术检测2022年3-6月在开封市中心医院骨外科就诊32例骨质疏松患者及同期体检中心健康人群骨髓间充质干细胞中miRNA的变化情况;利用Targetscan网站预测靶向调控CKIP-1的miRNA,利用荧光素酶报告基因实验检测miRNA与CKIP-1启动子区DNA的结合;在骨髓间充质干细胞中转染miR-192-5p类似物(miR-192-5p mimics)/阴性对照(NC mimics)或miR-192-5p抑制剂(miR-192-5p inhibitor)/阴性对照(NC inhibitor),成骨诱导后第7,14天,通过实时荧光定量PCR技术及茜素红染色检测成骨标志基因Runt相关转录因子2(Runx2)、骨钙素、抗骨桥蛋白、骨唾液蛋白及CKIP-1的表达水平和骨髓间充质干细胞向成骨细胞分化的情况;采用蛋白质免疫印迹实验及茜素红染色检测miR-192-5p/CKIP-1/轴对细胞成骨分化的的调控作用。结果与结论:与健康组相比,骨质疏松组有16个miRNA表达明显升高,53个miRNA表达明显降低(P<0.05);利用Targetscan网站预测,并通过荧光素酶报告基因实验验证,发现miR-192-5p与CKIP-1有互补的核苷酸序列(P<0.05);过表达miR-192-5p,Runx2、骨钙素、骨桥素和骨唾液蛋白的表达水平显著升高(P<0.05),抑制miR-192-5p,Runx2、骨钙素、骨桥素和骨唾液蛋白的表达水平显著降低(P<0.05),而沉默CKIP-1的表达后,Runx2、骨钙素及骨桥素的蛋白水平增加(P<0.05),逆转了敲低miR-192-5p对细胞成骨分化的抑制作用。上述结果证实,miR-192-5p在骨质疏松症中表达降低;miR-192-5p通过靶向抑制CKIP-1的表达,促进骨髓间充质干细胞成骨分化。
BACKGROUND:Casein kinase 2 binding protein 1(CKIP-1)is an important negative control gene in bone formation.After the deletion of this gene,the overall bone of mice was significantly enhanced,and bone formation and bone density were significantly increased.microRNA(miRNA)as the early found small molecular regulator has a regulatory effect on most of the coding genes and plays an important role in osteogenic differentiation.OBJECTIVE:To investigate the effect and molecular mechanism of miRNA/CKIP-1 axis on osteogenic differentiation of bone marrow mesenchymal stem cells from osteoporosis patients.METHODS:The miRNA-Seq technology was used to detect the changes of miRNA in bone marrow mesenchymal stem cells in 32 patients with osteoporosis treated in the Department of Orthopedics,Kaifeng Central Hospital from March to June 2022 and healthy people in the physical examination center during the same period.The Targetscan website was used to predict the miRNA targeted to regulate CKIP-1.Luciferase reporter gene assay was used to detect the binding of miRNA and CKIP-1 promoter DNA.miR-192-5p analogs(miR-192-5p mimics)/negative control(NC mimics)or miR-192-5p inhibitors(miR-192-5p inhibitor)/negative control(NC inhibitor)were transfected in bone marrow mesenchymal stem cells.On days 7 and 14 after osteogenic induction,the expression levels of Runt-related transcription factor 2(Runx2),osteocalcin,anti-osteopontin,bone sialoprotein,and CKIP-1,and the differentiation of bone marrow mesenchymal stem cells into osteoblasts were detected by real-time fluorescence quantitative PCR and alizarin red staining.The regulatory effect of miR-192-5p/CKIP-1/axis on osteogenic differentiation of cells was detected by western blot assay and alizarin red staining.RESULTS AND CONCLUSION:Compared with the healthy group,the expression levels of 16 miRNAs were significantly up-regulated and those of 53 miRNAs were significantly down-regulated in the osteoporosis group(P<0.05).Using Targetscan website and verified by luciferase reporter gene experiment,it was found that miR-192-5p and CKIP-1 had complementary nucleotide sequences(P<0.05).Overexpression of miR-192-5p significantly increased the expression levels of Runx2,osteocalcin,osteopontin,and bone sialoprotein(P<0.05),and inhibition of miR-192-5p significantly decreased the expression levels of Runx2,osteocalcin,osteopontin,and bone sialoprotein(P<0.05).After silencing the expression of CKIP-1,the protein levels of Runx2,osteocalcin,and osteopontin increased(P<0.05);the inhibitory effect of knockdown of miR-192-5p on osteogenic differentiation of cells was reversed.Above results confirm that the expression of miR-192-5p is decreased in osteoporosis.miR-192-5p promotes osteogenic differentiation of bone marrow mesenchymal stem cells by targeting the inhibition of CKIP-1 expression.
作者
鄂正康
辛红伟
于清波
张允帅
E Zhengkang;Xin Hongwei;Yu Qingbo;Zhang Yunshuai(Sixth Department of Orthopedics,Kaifeng Central Hospital(Affiliated People’s Hospital,Henan University),Kaifeng 475000,Henan Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2025年第13期2641-2647,共7页
Chinese Journal of Tissue Engineering Research
基金
河南省医学科技攻关计划项目(LHGJ20200842)。
