Upregulation of Wnt-1 gene expression by soybean isoflavones to promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

doi:10.3877/cma.j.issn.2095-1221.2021.05.006

Abstract

Abstract:

Objective

To investigate the mechanism of the effect of soybean isoflavone (ISO) on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells by regulating Wnt-1 gene expression.

Methods

Primary marrow mesenchymal stem cells were isolated and cultured, and treated with ISO at different concentrations (40, 60, 80 μg/ μL) (ISO-L group, ISO-M group, ISO-H group) . Liposome-based transfection technology was used to transfect pcDNA and pcDNA-Wnt-1 into bone marrow mesenchymal stem cells (pcDNA group and pcDNA-Wnt-1 group) , respectively, and to transfect si-NC and si-Wnt-1 into bone marrow mesenchymal stem cells, followed by the treatment with ISO (ISO+si-NC group and ISO+si-Wnt-1 group) . MTT was used to detect cell proliferation. The effects of ISO on Wnt-1 expression were detected by RT-qPCR and Western blot, respectively. RT-qPCR and Western blot were used to detect the expression levels of ALP, OPN, and Runx2, respectively. The independent sample t test was used for the comparison between two groups, and the one-way analysis of variance and pair LSD-t test were used for the comparison between multiple groups.

Results

Compared with the control group, the cell viability of ISO-L group, ISO-M group and ISO-H group was significantly increased [24 h: (0.17±0.02) vs (0.21±0.02) , (0.25±0.02) , (0.29±0.03) , 48 h: (0.26±0.03) vs (0.39±0.03) , (0.48±0.04) , (0.53±0.05) ; 72 h: (0.37±0.03) vs (0.54±0.04) , (0.65±0.06) , (0.78±0.07) ], mRNA and protein levels of ALP, OPN, Runx2 and Wnt-1 in bone marrow mesenchymal stem cells were also significantly increased [ALP mRNA: (1.00±0.06) vs (1.74±0.16) , (2.53±0.21) , (3.06±0.29) ; ALP protein: (0.41±0.04) vs (0.62±0.06) , (0.74±0.05) , (0.83±0.07) . OPN mRNA: (1.01±0.08) vs (1.52±0.15) , (2.11±0.19) , (2.74±0.25) ; OPN protein: (0.34±0.03) vs (0.51±0.05) , (0.63±0.06) , (0.76±0.07) . Runx2 mRNA: (1.01±0.08) vs (1.52±0.15) , (2.11±0.19) , (2.74±0.25) ; Runx2 protein: (0.34±0.03) vs (0.51±0.05) , (0.63±0.06) , (0.76±0.07) ], and they all showed a gradually increasing trend (P < 0.05) . Compared with the pcDNA group, the cell viability of the pcDNA-Wnt-1 group [24 h: (0.18±0.02) vs (0.24±0.02) ; 48h: (0.25±0.03) vs (0.51±0.05) ; 72h: (0.36±0.03) vs (0.72±0.06) ], ALP mRNA [ (1.01±0.08) vs (2.89±0.27) ] and protein [ (0.40±0.04) vs (0.79±0.07) ], OPN mRNA [ (0.99±0.07) vs (2.53±0.25) ] and protein [ (0.33±0.03) to (0.71± 0.06) ], Runx2 mRNA [ (1.00±0.06) vs (2.14±0.21) ] and protein [ (0.21±0.02) vs (0.62±0.06) ] expression levels all showed a gradual increase trend, and the difference was statistically significant (all P < 0.05) . Compared with the ISO+si-NC group, the cell viability of the ISO+si-Wnt-1 group [24 h: (0.25±0.03) vs (0.19±0.02) ; 48 h: (0.56±0.05) vs (0.31±0.03) ; 72 h: (0.82±0.08) vs (0.49±0.04) ], ALP mRNA [ (3.14±0.31) vs (1.67±0.16) ] and protein [ (0.87±0.08) vs (0.51±0.05) ], OPN mRNA [ (2.79±0.26) vs (1.43±0.13) ]and protein [ ( 0.79±0.08) vs (0.43±0.04) ], Runx2 mRNA [ (2.36±0.24) vs (1.35±0.13) ]and protein [ (0.68±0.07) vs (0.34±0.03) ] expression levels all showed a gradual decrease trend, and the differences were statistically significant (all P < 0.05) .

Conclusion

ISO can enhance the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells by regulating the expression of Wnt-1.

Key words: Soy bean isoflavone, Wnt-1, Bone marrow mesenchymal stem cells, Proliferation, Osteogenic differentiation

Yidong Liu, Yufeng Jia, Yan Wang, Yang Liu, Jinyue Ning. Upregulation of Wnt-1 gene expression by soybean isoflavones to promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2021, 11(05): 292-297.

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Figures/Tables 12

References 16

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基因	引物序列（5'→3'）	预期扩增长度（bp）
Wnt-1	上游AACCACAGTCGTCAGAACCG	187
	下游AGGTTCGTGGAGGAGGCTAT
ALP	上游CTTCATAAGCAGGCGGGGG	242
	下游TGCCGATGGCCAGTACTAAA
OPN	上游CAGGACAACAACGGAAAGGG	114
	下游ATCCGACTGATCGGCACTCT
Runx2	上游CATCCCCATCCATCCACTCC	126
	下游TCACTGTGCTGAAGAGGCTG
GAPDH	上游GGAGCGAGATCCCTCCAAAAT	197
	下游GGCTGTTGTCATACTTCTCATGG

基因	引物序列（5'→3'）	预期扩增长度（bp）
Wnt-1	上游AACCACAGTCGTCAGAACCG	187
	下游AGGTTCGTGGAGGAGGCTAT
ALP	上游CTTCATAAGCAGGCGGGGG	242
	下游TGCCGATGGCCAGTACTAAA
OPN	上游CAGGACAACAACGGAAAGGG	114
	下游ATCCGACTGATCGGCACTCT
Runx2	上游CATCCCCATCCATCCACTCC	126
	下游TCACTGTGCTGAAGAGGCTG
GAPDH	上游GGAGCGAGATCCCTCCAAAAT	197
	下游GGCTGTTGTCATACTTCTCATGG

分组	实验次数	OD值（490 nm）
分组	实验次数	24 h	48 h	72 h
对照	3	0.17±0.02	0.26±0.03	0.37±0.03
ISO-L	3	0.21±0.02^a	0.39±0.03^a	0.54±0.04^a
ISO-M	3	0.25±0.02^ab	0.48±0.04^ab	0.65±0.06^ab
ISO-H	3	0.29±0.03^abc	0.53±0.05^abc	0.78±0.07^abc
F值		15.238	28.542	32.909
P值		< 0.001	< 0.001	< 0.001

分组	实验次数	OD值（490 nm）
分组	实验次数	24 h	48 h	72 h
对照	3	0.17±0.02	0.26±0.03	0.37±0.03
ISO-L	3	0.21±0.02^a	0.39±0.03^a	0.54±0.04^a
ISO-M	3	0.25±0.02^ab	0.48±0.04^ab	0.65±0.06^ab
ISO-H	3	0.29±0.03^abc	0.53±0.05^abc	0.78±0.07^abc
F值		15.238	28.542	32.909
P值		< 0.001	< 0.001	< 0.001

分组	实验次数	mRNA			蛋白
分组	实验次数	ALP	OPN	Runx2	ALP	OPN	Runx2
对照	3	1.00±0.06	1.01±0.08	0.98±0.07	0.41±0.04	0.34±0.03	0.23±0.02
ISO-L	3	1.74±0.16^a	1.52±0.15^a	1.36±0.11^a	0.62±0.06^a	0.51±0.05^a	0.39±0.03^a
ISO-M	3	2.53±0.21^ab	2.11±0.19^ab	1.84±0.17^ab	0.74±0.05^ab	0.63±0.06^ab	0.54±0.05^ab
ISO-H	3	3.06±0.29^abc	2.74±0.25^abc	2.31±0.21^abc	0.83±0.07^abc	0.76±0.07^abc	0.67±0.06^abc
F值		62.132	52.521	44.519	31.429	32.202	58.527
P值		< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

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