Comparison of expression of miR-145-5p in osteogenic differentiation of bone marrow mesenchymal stem cells derived from normal versus type 2 diabetic rats
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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 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 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)
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 -
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