抗肿瘤药物依托泊苷促进间充质干细胞成骨分化的研究

doi:10.3969/j.issn.1674-8115.2021.07.002

摘要/Abstract

摘要：

目的·研究抗肿瘤药物依托泊苷对间充质干细胞（mesenchymal stem cell，MSC）成骨分化的促进作用及可能的机制。方法·体外分离C57BL/6J小鼠骨髓和人骨髓来源MSC。给予小鼠MSC不同浓度（0~12.50 μmol/L）的依托泊苷12、24、36、48和60 h后，采用CCK-8法检测MSC的增殖活性。采用凋亡检测试剂盒检测依托泊苷（0、0.001、0.01、0.1、1 μmol/L）对大鼠成骨性骨肉瘤细胞系UMR-106凋亡的影响。分别以依托泊苷（0、0.001、0.01、0.1、1 μmol/L）处理小鼠和人MSC（同时诱导成骨分化）、UMR-106细胞，通过碱性磷酸酶（alkaline phosphatase，ALP）染色、茜素红染色分析细胞成骨分化情况。通过实时荧光定量PCR技术检测依托泊苷对小鼠MSC成骨分化标志物Alp、骨钙素、Ⅰ型胶原α1链mRNA水平的调控。将0.001 μmol/L依托泊苷与UMR-106细胞共孵育后，通过Illumina Xten高通量转录组测序和生物信息学分析，寻找依托泊苷上调的成骨分化相关靶基因，并通过PCR验证。Western blotting检测依托泊苷（0、0.001、0.01、0.1、1 μmol/L）处理的小鼠MSC中成骨特异性转录因子（osterix，OSX）、矮小相关转录因子2（runt-related transcription factor 2，RUNX2）及DNA结合抑制因子1（inhibitor of DNA binding 1，ID1）的表达变化。结果·依托泊苷对小鼠MSC增殖的影响呈现浓度依赖性；当浓度≥0.20 μmol/L时，依托泊苷开始抑制MSC的增殖，48 h的半数抑制浓度（half-maximal inhibitory concentration，IC₅₀）为2.192 μmol/L，60 h的IC₅₀为1.399 μmol/L。凋亡检测结果显示依托泊苷浓度为0.001~1 μmol/L时UMR-106细胞的凋亡率为16.137%~28.300%。ALP染色、茜素红染色及荧光定量PCR结果显示0.001~0.1 μmol/L依托泊苷对小鼠和人MSC以及UMR-106细胞具有促成骨分化的作用。RNA测序分析表明，依托泊苷可上调UMR-106细胞结缔组织生长因子基因表达。Western blotting分析结果显示，0.001、0.01 μmol/L依托泊苷显著增加小鼠MSC OSX、RUNX2及ID1的表达。结论·抗肿瘤药物依托泊苷可促进MSC成骨分化，该作用可能与其上调ID1、RUNX2、OSX表达有关。

关键词: 依托泊苷, 间充质干细胞, 成骨分化, 骨肿瘤, 骨重建

Abstract:

Objective·To study the effect and possible mechanism of the anti-tumor drug etoposide in promoting osteogenic differentiation of mesenchymal stem cells (MSCs).

Methods·Primary cultured MSCs were isolated from the bone marrow of C57BL/6J mice and humans. After the mouse MSCs were given different concentrations (0?12.50 μmol/L) of etoposide for 12, 24, 36, 48 and 60 h, the cell proliferation ability was detected by CCK-8 kit assay. The apoptosis detection kit was performed to detect apoptosis of osteoblast/osteosarcoma cell line UMR-106 treated by etoposide (0, 0.001, 0.01, 0.1, 1 μmol/L). The mouse and human MSCs (simultaneously inducing osteogenic differentiation), and UMR-106 cells were treated with etoposide (0, 0.001, 0.01, 0.1, 1 μmol/L), whose osteogenic differentiation was analyzed by alkaline phosphatase (ALP) staining and alizarin red staining. The mRNA levels of osteogenic differentiation markers i.e., Alp, osteocalcin, and collagen type Ⅰ α 1 chain were analyzed by real-time quantitative PCR. After the UMR-106 cells were treated with etoposide (0.001 μmol/L), Illumina Xten′s high-throughput transcriptome sequencing and bioinformatics analysis were used to find the target gene related with osteogenic differentiation, which was then verified by PCR. Western blotting was used to detect the expression of osterix (OSX), runt-related transcription factor 2 (RUNX2) and DNA binding inhibitor 1 (ID1) in the etoposide (0, 0.001, 0.01, 0.1, 1 μmol/L) treated mouse MSCs.

Results·The effect of etoposide on the proliferation of mouse MSCs was concentration-dependent. When the concentration was ≥0.20 μmol/L, etoposide inhibited the proliferation of MSCs. The half-maximal inhibitory concentrations (IC₅₀) for 48 h and 60 h were 2.192 μmol/L and 1.399 μmol/L, respectively. Apoptosis detection results showed that the apoptotic rates of UMR-106 cells treated with 0.001?1 μmol/L etoposide were 16.137%?28.300%. The results of ALP staining, alizarin red staining and PCR showed that etoposide at 0.001?0.1 μmol/L significantly promoted the osteogenic differentiation of mouse and human MSCs and UMR-106 cells. RNA sequencing analysis showed that etoposide up-regulated connective tissue growth factor expression in the UMR-106 cells. Western blotting results showed that 0.001 and 0.01 μmol/L etoposide significantly increased the protein expression of OSX, RUNX2 and ID1 in the mouse MSCs.

Conclusion·The anti-tumor drug etoposide can promote the osteogenic differentiation of MSCs, which may be related to the up-regulation of ID1, RUNX2 and OSX expressions.

Key words: etoposide (VP-16), mesenchymal stem cell (MSC), osteogenic differentiation, bone tumor, bone remodeling

中图分类号:

R966

陆艳青, 周兴, 李姣, 彭建平, 王传东, 张晓玲. 抗肿瘤药物依托泊苷促进间充质干细胞成骨分化的研究[J]. 上海交通大学学报(医学版), 2021, 41(7): 849-857.

Yan-qing LU, Xing ZHOU, Jiao LI, Jian-ping PENG, Chuan-dong WANG, Xiao-ling ZHANG. Promotive effect of antitumor drug etoposide on osteogenic differentiation of mesenchymal stem cells[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(7): 849-857.

图/表 7

图1 依托泊苷对MSC增殖和UMR-106凋亡的影响Note： A. Proliferation activity of MSC after treatment with etoposide (0?12.5 μmol·L-1) for 12?60 h. B. Apoptosis conditions of UMR-106 cells after treatment with etoposide (0?1 μmol·L-1) for 3 d. ①P=0.044, ②P=0.022, ③P=0.000, compared with the control (0 μmol·L-1) group..

Fig 1 Effects of etoposide on MSC proliferation and UMR-106 apoptosis

图2 ALP染色检测依托泊苷对细胞ALP活性的影响Note： A. The human MSC treated with etoposide (0?1 μmol·L-1) for 7 d. B. The mouse MSC treated with etoposide (0?1 μmol·L-1) for 7 d. C. The UMR-106 cells treated with etoposide (0?1 μmol·L-1) for 3 d. ①P=0.000, ②P=0.025, ③P=0.001, ④P=0.004, ⑤P=0.006, compared with the control (0 μmol·L-1) group.

Fig 2 Effect of etoposide on the ALP activity of the cells detected by ALP staining

图3 茜素红染色检测依托泊苷对MSC钙结节含量的影响Note： A. The human MSC treated with etoposide (0?1 μmol·L-1) for 14 d. B. The mouse MSC treated with etoposide (0?1 μmol·L-1) for 14 d. ①P=0.000, compared with the control (0 μmol·L-1) group.

Fig 3 Effect of etoposide on the content of calcium nodules in MSC detected by alizarin red staining

图4 实时荧光定量PCR检测依托泊苷对小鼠MSC成骨相关基因表达的影响Note： Changes of Alp (A), Ocn (B)and Col1α1 (C) mRNA expressions after 5 d of etoposide (0?1 μmol·L-1) treatment. ①P=0.004, ②P=0.000, ③P=0.015, ④P=0.045, ⑤P=0.006, ⑥P=0.013, compared with the control (0 μmol·L-1) group treated with osteogenic medium.

Fig 4 Effect of etoposide on the expression of osteogenic-related genes in mouse MSC by real-time qPCR

图5 RNA测序和PCR检测依托泊苷对UMR-106细胞mRNA表达的影响Note： A?C. Venn diagram (A), heatmap diagram (B) and scatter diagram (C) of the differential expressed mRNAs between the UMR-106 cells treated with 0.001 μmol·L-1 etoposide for 3 d and the control (0 μmol·L-1) group. D. Changes of Ctgf mRNA expression in the UMR-106 cells treated with etoposide (0?0.1 μmol·L-1) for 3 d detected by PCR. ①P=0.001, compared with the control (0 μmol·L-1) group.

Fig 5 Effect of etoposide on the expression of mRNAs in the UMR-106 cells by RNA sequencing and PCR

表1 依托泊苷促进UMR-106细胞表达上调倍数最高的前13个基因

Tab 1 Top up-regulated 13 genes promoted by etoposide in the UMR-106 cells detected by RNA sequencing

Gene	Full name	log₂ fold change	P value
Clta	Clathrin， light chain A	17.509	4.70×10^－3
Rab1a	RAB1A， member RAS oncogene family	11.153	4.23×10^－3
Taf1	TATA-box binding protein associated factor 1	8.726	5.54×10^－4
Clasp2	Cytoplasmic linker associated protein 2	6.243	3.36×10^－4
Ehmt2	Euchromatic histone lysine methyltransferase 2	5.865	1.49×10^－14
Tpm1	Tropomyosin 1， alpha	5.728	1.44×10^－17
Rell1	RELT-like 1	4.845	1.68×10^－44
Tpm1	Tropomyosin 1， alpha	3.859	1.69×10^－8
Gpbp1l2	GC-rich promoter binding protein 1-like 2	3.799	8.20×10^－31
Uck2	Uridine-cytidine kinase 2	3.299	1.54×10^－4
Polr3gl	RNA polymerase Ⅲ subunit G like	3.202	1.75×10^－3
Akap11	A-kinase anchoring protein 11	3.064	1.11×10^－24
Ctgf	Connective tissue growth factor	2.982	1.06×10^－5

图6 Western blotting检测依托泊苷对小鼠MSC成骨相关蛋白表达的影响Note： A. The protein bands of RUNX2, OSX and ID1 in the MSCs treated with etoposide (0?1 μmol·L-1) for 7 d in the osteogenic medium or not. B. The relative level of RUNX2 protein. C. The relative level of OSX protein. D. The relative level of ID1 protein. ①P=0.014, ②P=0.001, compared with the control (0 μmol·L-1) group treated with osteogenic medium.

Fig 6 Effect of etoposide on the expression of osteogenic related proteins in mouse MSC by Western blotting

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