Epigenetic regulation of collagen Ⅹ in human mesenchymal stem cells during chondrogenic differentiation

doi:10.3877/cma.j.issn.1674-134X.2020.02.007

Abstract

Abstract:

Objective

To evaluate the relationship between collagen Ⅹ and the methylation status of its promoter during the initial period of human mesenchymal stem cells (hMSCs) chondrogenic differentiation. ]

Methods

The hMSCs were collected from six patients who underwent the intervertebral fusion and informed the ethical agreement. The pellet was cultured by centrifugal precipitation, within 2% fetal bovine serum (FBS) high-glucose Dulbecco's modified Eagle medium containing TGF-β3. Quantitative PCR was performed 3 d after differentiation to detect the expression of cartilage -related genes and methylation changes in the collagen Ⅹ promoter region. The data were analyzed by variance analysis and Kruskal-Wallis test.

Results

Bioinformatics indicated that there might be CpG-rich regions in the promoter region of collagenⅩ(F=37.526, P<0.001). During the process of chondrogenic differentiation, the expression of collagen Ⅹgradually increased, and the difference was statistically significant compared with the control group. Compared with the control group, the methylation level of the collagen Ⅹ promoter region gradually decreased, and the difference was also statistically significant(F=11.066, P<0.01).

Conclusion

In the process of the hMSCs′ chondrogenic differentiation in vitro, the results would suggest that the methylation status of the collagen Ⅹ promoter may involve and enhance the differentiation ability; which implies that epigenetic regulation would affect stem cell differentiation.

Key words: Epigenetic repression, Human mesenchymal stem cells, Chondrocytes, Collagen typeⅩ

Xinle Luo, Weimin Zhu, Hao Zhang, Yawei Hu, Shaochu Chen, Ming Gong. Epigenetic regulation of collagen Ⅹ in human mesenchymal stem cells during chondrogenic differentiation[J]. Chinese Journal of Joint Surgery(Electronic Edition), 2020, 14(02): 167-172.

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Figures/Tables 5

References 20

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基因名称	正向引物	反向引物
DNMT3A	5’-GGGACGGAAAGAGAGAGACA-3’ (F)	5’-CCTGCTGCTAACTGGGTTCT-3’ (R)
DNMT3B	5’-GAGCGGGCGGCAACG-3’ (F)	5’-CCCTTCATGCTTTCCTGCCG-3’ (R)
DNMT1	5’-GGAGGGCTACCTGGCTAAAG-3’ (F)	5’-TACGGGCTTCACTTCTTGCT-3’ (R)
蛋白聚糖	5’-GCTGCAGTGATCTCAGAAGAAG-3’ (F)	5’-GATGGTGAGGGAAGACCCTA-3’ (R)
胶原蛋白Ⅱa1(col-Ⅱa1)	5’-CCAGGATGCCCGAAAATTAG-3’ (F)	5’-TTCTCCCTTGTCACCACGAT-3’ (R)
胶原蛋白Xa1(col-Xa1)	5’-GCAGCATTACGACCCAAGAT-3’ (F)	5’-CATGATTGCACTCCCTGAAG-3’ (R)
内参18s RNA	5’-CCTGGATACCGCAGCTAGGA-3’ (F)	5’-GCGGCGCAATACGAATGCCCC-3’ (R)

基因名称	正向引物	反向引物
DNMT3A	5’-GGGACGGAAAGAGAGAGACA-3’ (F)	5’-CCTGCTGCTAACTGGGTTCT-3’ (R)
DNMT3B	5’-GAGCGGGCGGCAACG-3’ (F)	5’-CCCTTCATGCTTTCCTGCCG-3’ (R)
DNMT1	5’-GGAGGGCTACCTGGCTAAAG-3’ (F)	5’-TACGGGCTTCACTTCTTGCT-3’ (R)
蛋白聚糖	5’-GCTGCAGTGATCTCAGAAGAAG-3’ (F)	5’-GATGGTGAGGGAAGACCCTA-3’ (R)
胶原蛋白Ⅱa1(col-Ⅱa1)	5’-CCAGGATGCCCGAAAATTAG-3’ (F)	5’-TTCTCCCTTGTCACCACGAT-3’ (R)
胶原蛋白Xa1(col-Xa1)	5’-GCAGCATTACGACCCAAGAT-3’ (F)	5’-CATGATTGCACTCCCTGAAG-3’ (R)
内参18s RNA	5’-CCTGGATACCGCAGCTAGGA-3’ (F)	5’-GCGGCGCAATACGAATGCCCC-3’ (R)

分组	样本	DNMT1	DNMT3A	DNMT3B
对照组	3	0.78±0.14	0.82±0.13	0.75±0.05
诱导组	3	0.73±0.09	0.94±0.21	1.27±0.25^a
诱导＋5’-AZA组	3	0.61±0.14	0.59±0.04	0.67±0.13^b
F值		1.447	4.485	11.329
P值		>0.05	>0.05	<0.05

分组	样本	DNMT1	DNMT3A	DNMT3B
对照组	3	0.78±0.14	0.82±0.13	0.75±0.05
诱导组	3	0.73±0.09	0.94±0.21	1.27±0.25^a
诱导＋5’-AZA组	3	0.61±0.14	0.59±0.04	0.67±0.13^b
F值		1.447	4.485	11.329
P值		>0.05	>0.05	<0.05

分组	样本	蛋白聚糖	胶原蛋白IIa1	胶原蛋白Xa1
对照组	3	1.01±0.11	5.65±0.18	1.49±0.22
诱导组	3	0.98±0.05	2.22±0.21^a	1.23±0.13^a
诱导＋5’-AZA组	3	1.11±0.24	2.25±0.66^b	2.85±0.34^b
F值		0.528	64.722	37.526
P值		>0.05	<0.05	<0.05

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