Comparison of expression of miR-145-5p in osteogenic differentiation of bone marrow mesenchymal stem cells derived from normal versus type 2 diabetic rats
-
摘要:
背景 糖尿病导致骨髓间充质干细胞(bone marrow mesenchymal stem cells，BMMSCs) 成骨分化能力降低， miRNAs在此过程中发挥重要作用，其中miR-145-5p对细胞成骨成软骨分化有重要的调节作用。然而miR-145-5p对糖尿病源BMMSCs成骨分化的影响尚不清楚。 目的 比较正常和2型糖尿病大鼠BMMSCs成骨分化能力，初步探讨在成骨分化中miR-145-5p及其靶基因SEMA3A和Wnt通路关键蛋白β-catenin表达的差异。 方法 12只GK大鼠采用高糖高脂饲料喂养构建2型糖尿病大鼠模型，12只Wistar大鼠常规饲养作为对照组。无菌条件下分离大鼠股骨，全骨髓培养法培养正常和2型糖尿病大鼠BMMSCs(分别对应WT-BMMSCs和GK-BMMSCs)；CCK-8检测细胞增殖能力；结晶紫染色检测细胞集落形成能力；碱性磷酸酶染色及半定量分析检测碱性磷酸酶活性；茜素红染色检测矿化基质形成；qRT-PCR和Western blot检测成骨相关标志物、miRNA-145-5p、SEMA3A和β-catenin的表达。 结果 干细胞培养4-9 d时，GK-BMMSCs的增殖低于WT-BMMSCs，差异有统计学意义(P＜0.01)；10 d时，其集落形成率显著低于WT-BMMSCs(P＜0.01)；成骨诱导7 d时，GK-BMMSCs碱性磷酸酶(alkaline phosphatase，ALP)活性(P＜0.001)、成骨相关标志物碱性磷酸酶基因 (P＜0.01)和蛋白(P＜0.001)表达、骨钙素(osteocalcin，OCN) 基因(P＜0.001)和蛋白(P＜0.001)表达均显著低于WT-BMMSCs，1型胶原蛋白(type 1 collagen，COL1)(P＜0.01)基因的表达显著低于WT-BMMSCs，而Runt相关转录因子2(Runt-related transcription factor-2，Runx2)(P＜0.001)蛋白表达显著高于WT-BMMSCs。在WT-BMMSCs成骨分化中，miR-145-5p表达下调，SEMA3A表达上调，而在GK-BMMSCs成骨分化中，miR-145-5p和SEMA3A表达均上调，β-catenin(P＜0.001)在GK-BMMSCs中的表达显著降低。成骨诱导21 d时，WT-BMMSCs矿化基质染色较深。 结论 2型糖尿病大鼠BMMSCs细胞增殖、集落形成及成骨分化能力降低，推测在正常大鼠BMMSCs成骨分化中miR-145-5p起抑制作用，在2型糖尿病大鼠BMMSCs成骨分化中miR-145-5p起促进作用。 -
关键词:
- 2型糖尿病 /
- microRNA-145-5p /
- 骨髓间充质干细胞 /
- 成骨分化 /
- 大鼠
Abstract:Background Diabetes reduces the osteogenic differentiation ability of bone marrow mesenchymal stem cells (BMMSCs), and miRNAs play an essential role in this process, during which miR-145-5p plays a vital role in osteogenic and chondrogenic differentiation of cells. However, the effect of miR-145-5p on the osteogenic differentiation of diabetic BMMSCs remains unclear. Objective To compare the osteogenic differentiation ability of BMMSCs derived from normal and type 2 diabetic rats respectively, and preliminarily explore the differences in the expression of miR-145-5p, its potential target gene SEMA3A and the key protein β-catenin of Wnt pathway in osteogenic differentiation. Methods Twelve GK rats were fed on a high-fat and high-sugar diet to establish type 2 diabetes rat models as the experimental group. Twelve Wistar rats were fed a standard diet and served as the control group. Rat femur was isolated under aseptic condition. BMMSCs derived from normal and type 2 diabetic rats (WT-BMMSCs and GK-BMMSCs) were cultured by whole bone marrow culture method. CCK-8 was used to detect the cell proliferation. Colony formation ability was detected by the crystal violet stain assay. Alkaline phosphatase activity was detected by alkaline phosphatase staining and semi-quantitative analysis. Alizarin red staining was used to detect the formation of the mineralized matrix. The expression levels of bone-related markers, miRNA-145-5p, SEMA3A and β-catenin were detected by qRT-PCR and Western blot. Results When the stem cells were cultured for 4-9 days, the proliferation of GK-BMMSCs was significantly lower than that of WT-BMMSCs (P<0.01). On day 10, colony formation rate was significantly lower than that of WT-BMMSCs (P<0.01). At 7 days of osteogenic induction, alkaline phosphatase activity of GK-BMMSCs (P<0.001), osteogenesis-related markers alkaline phosphatase (ALP) gene (P<0.01) and protein (P<0.001) expressions, osteocalcin (OCN) gene (P<0.001) and protein (P<0.001) expressions were significantly lower than those of WT-BMMSCs, type 1 collagen (COL1) gene expression level was significantly lower than that of WT-BMMSCs (P<0.01), and Runt-related transcription factor-2 (RUNX2) protein expression level was significantly higher than that of WT-BMMSCs (P<0.001). While miR-145-5p and SEMA3A were up-regulated during the osteogenic differentiation of WT-BMMSCs, miR-145-5p and SEMA3A were down-regulated during the osteogenic differentiation of GK-BMMSCs, and the expression of β-catenin (P<0.001) was considerably reduced in GK-BMMSCs. At 21 days of osteogenesis induction, WT BMMSCs had darker staining of the mineralized matrix. Conclusion The proliferation, colony formation and osteogenic differentiation of BMMSCs decrease in type 2 diabetic rats. It is speculated that miR-145-5p plays an inhibitory role in osteogenic differentiation of BMMSCs in normal rats while a promoting role in osteogenic differentiation of BMMSCs in type 2 diabetic rats. -
图 1 8-13周Wistar与GK大鼠体重
(n=12) aP＜0.001
Figure 1. Body weight of Wistar and GK rats at 8-13 weeks (n=12) aP＜0.001
图 2 8-13周Wistar与GK大鼠血糖
(n=12) aP＜0.001
Figure 2. Blood glucose of Wistar and GK rats at 8-13 weeks (n=12) aP＜0.001
图 3 倒置显微镜观察原代(p0)与第三代(p3)正常和糖尿病大鼠BMMSCs (标尺 100 μm)
Figure 3. Primary and the third generation BMMSCs derived from normal and type 2 diabetic rats (scale bar=100 μm)
图 4 流式细胞学鉴定BMMSCs表面抗原
Figure 4. Identification of BMMSCs surface antigen by flow cytometry
图 5 CCK8试剂盒检测正常和糖尿病大鼠股骨BMMSCs生长曲线(n=6) aP＜0.01
Figure 5. Detection of growth curve of BMMSCs derived from normal and type 2 diabetic rats by CCK8 kit (n=6) aP＜0.01
图 6 正常和糖尿病大鼠股骨BMMSCs集落形成实验
Figure 6. Colony formation experiment of femoral BMMSCs in normal rats and type 2 diabetic rats
图 7 正常与2型糖尿病大鼠BMMSCs成骨分化能力比较
A：成骨诱导7 d碱性磷酸酶染色(标尺 100 μm)；B：成骨诱导7d ALP半定量分析 (n=6)；C：成骨诱导21天茜素红染色显示钙化结节形成(标尺 100 μm)
Figure 7. Comparison of osteogenic differentiation capacity of BMMSCs derived from normal rats and type 2 diabetic rats
A: ALP staining was performed on day 7 after osteogenic differentiation(scale bar=100μm); B: Quantification of ALP activity was shown(n=6); C：ARS staining was performed on day 21 after osteogenic differentiation to show the formation of calcified nodules(scale bar=100μm)
图 8 成骨诱导7d正常与2型糖尿病大鼠BMMSCs ALP、OCN 、RUNX2、COL1基因表达(n=3)
Figure 8. Expression of the osteoblast differentiation-related genes ALP, OCN, RUNX2, COL1 on day 7 after osteogenic differentiation by real-time PCR (n=3)
图 9 成骨诱导7d正常与2型糖尿病大鼠BMMSCs ALP、OCN、RUNX2蛋白水平的表达
A： ALP、OCN、RUNX2蛋白的表达； B：ALP、OCN 、RUNX2蛋白水平表达的定量分析(n=3)
Figure 9. Western blot analysis of ALP, OCN, RUNX2 on day 7 after osteogenic differentiation
A：Western blotting of ALP, OCN, RUNX2；B：Protein levels of ALP, OCN, RUNX2 quantified by densitometry(n=3)
图 10 成骨诱导7d正常与2型糖尿病大鼠BMMSCs miR-145-5p、SEMA3A、β-catenin的表达(n=3)
Figure 10. Expression of miR-145-5p, SEMA3A, β-catenin on day 7 after osteogenic differentiation by real-time PCR (n=3)
图 11 成骨诱导7天正常与2型糖尿病大鼠BMMSCs SEMA3A的蛋白表达
A：SEMA3A蛋白的表达 B：SEMA3A蛋白水平表达的定量分析(n=3)
Figure 11. Western blot analysis of SEMA3A on day 7 after osteogenic differentiation
A: Western blotting of SEMA3A; B: Protein levels of SEMA3A quantified by densitometry(n=3)
表 1 引物序列
Table 1. Primer sequences
基因 引物序列 SEMA3A F-CTTGCTCGGGACCCTTATTG R-AGGCTCTCTGTGACTTCGGACT β-catenin F-TGCCATCTGTGCTCTTCGTC R-CAATCCAACAGTTGCCTTTATCAG ALP F-TGGTGAGTGACACGGACAAGAA R-GCCTGGTAGTTGTTGTGAGCAT COL1 F-CGTGGAAACCTGATGTATGCTTG R-CCTATGACTTCTGCGTCTGGTGA OCN F-TGACAAAGCCTTCATGTCCAA R-CTCCAAGTCCATTGTTGAGGTAG RUNX2 F-TACCCAGGCGTATTTCAGATGAT R-TGTAAGTGAAGGTGGCTGGATAGT GAPDH F-CTGGAGAAACCTGCCAAGTATG R-GGTGGAAGAATGGGAGTTGCT U6 F-CTCGCTTCGGCAGCACA R-AACGCTTCACGAATTTGCGT 
下载: 导出CSV
-
[1] Sun H,Saeedi P,Karuranga S,et al. IDF Diabetes Atlas:global,regional and country-level diabetes prevalence estimates for 2021 and projections for 2045[J]. Diabetes Res Clin Pract,2022,183: 109119. doi: 10.1016/j.diabres.2021.109119 [2] Ribot J,Denoeud C,Frescaline G,et al. Experimental type 2 diabetes differently impacts on the select functions of bone marrow-derived multipotent stromal cells[J]. Cells,2021,10(2): 268. doi: 10.3390/cells10020268 [3] Hua YW,Bi RY,Zhang Y,et al. Different bone sites-specific response to diabetes rat models:bone density,histology and microarchitecture[J]. PLoS One,2018,13(10): e0205503. doi: 10.1371/journal.pone.0205503 [4] Mohsin S,Baniyas MM,AlDarmaki RS,et al. An update on therapies for the treatment of diabetes-induced osteoporosis[J]. Expert Opin Biol Ther,2019,19(9): 937-948. doi: 10.1080/14712598.2019.1618266 [5] Xing Q, Feng JY, Zhang XL. Semaphorin3B promotes proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells in a high-glucose microenvironment[J/OL].https: //doi.org/10.1155/2021/6637176. [6] Zhang P,Zhang HG,Lin JL,et al. Insulin impedes osteogenesis of BMSCs by inhibiting autophagy and promoting premature senescence via the TGF-β1 pathway[J]. Aging (Albany NY),2020,12(3): 2084-2100. doi: 10.18632/aging.102723 [7] Iaquinta MR,Lanzillotti C,Mazziotta C,et al. The role of microRNAs in the osteogenic and chondrogenic differentiation of mesenchymal stem cells and bone pathologies[J]. Theranostics,2021,11(13): 6573-6591. doi: 10.7150/thno.55664 [8] Zhu J,Fu QW,Shao JH,et al. Regulating effect of Circ_ATRNL1 on the promotion of SOX9 expression to promote chondrogenic differentiation of hAMSCs mediated by miR-145-5p[J]. J Tissue Eng Regen Med,2021,15(5): 487-502. doi: 10.1002/term.3189 [9] Liu HM,Liu P. Kartogenin promotes the BMSCs chondrogenic differentiation in osteoarthritis by down-regulation of miR-145-5p targeting Smad4 pathway[J]. Tissue Eng Regen Med,2021,18(6): 989-1000. doi: 10.1007/s13770-021-00390-9 [10] Liu XD,Zhu WZ,Wang L,et al. miR-145-5p suppresses osteogenic differentiation of adipose-derived stem cells by targeting semaphorin 3A[J]. In Vitro Cell Dev Biol Anim,2019,55(3): 189-202. doi: 10.1007/s11626-019-00318-7 [11] 黎娅,范培云,马晓雨,等. 长期稳定的SD大鼠2型糖尿病模型制备方法[J]. 中国实验动物学报,2020,28(3): 364-369. [12] 李强强,谢亚东,张怀斌,等. SD大鼠骨髓间充质干细胞分离培养及鉴定的实验研究[J]. 现代生物医学进展,2021,21(13): 2410-2414. [13] 陆玖青,吕佳姝,谢亚佳,等. 糖尿病大鼠颌骨结构及颌骨骨髓间充质干细胞成骨分化的研究[J]. 口腔颌面外科杂志,2020,30(3): 144-149. [14] Cortet B,Lucas S,Legroux-Gerot I,et al. Bone disorders associated with diabetes mellitus and its treatments[J]. Joint Bone Spine,2019,86(3): 315-320. doi: 10.1016/j.jbspin.2018.08.002 [15] 胡巍,刘娜,王译凡,等. 2型糖尿病对大鼠长骨结构与骨髓间充质干细胞增殖分化的影响[J]. 中华老年口腔医学杂志,2019,17(1): 1-8. [16] 李强强,谢亚东,杨国清,等. 骨髓间充质干细胞成骨分化的研究进展[J]. 医学综述,2022,28(3): 434-438. [17] Liang C,Liu X,Liu CY,et al. Integrin α10 regulates adhesion,migration,and osteogenic differentiation of alveolar bone marrow mesenchymal stem cells in type 2 diabetic patients who underwent dental implant surgery[J]. Bioengineered,2022,13(5): 13252-13268. doi: 10.1080/21655979.2022.2079254 [18] Hock JM,Krishnan V,Onyia JE,et al. Osteoblast apoptosis and bone turnover[J]. J Bone Miner Res,2001,16(6): 975-984. doi: 10.1359/jbmr.2001.16.6.975 [19] Yu LJ,Qu H,Yu YF,et al. LncRNA-PCAT1 targeting miR-145-5p promotes TLR4-associated osteogenic differentiation of adipose-derived stem cells[J]. J Cell Mol Med,2018,22(12): 6134-6147. doi: 10.1111/jcmm.13892 -
下载:
点击查看大图
计量
- 文章访问数: 10
- HTML全文浏览量: 10
- PDF下载量: 0
- 被引次数: 0
