Effects of ginkgolide B on proliferation and osteogenic differentiation of human perimembrane stem cells by regulating miR-24-3p

doi:10.3877/cma.j.issn.2095-1221.2024.04.005

Abstract

Abstract:

Objective

To investigate the effect of ginkgolide B (GB) regulating miR-24-3p on human peripheral stem cells' proliferation and osteogenic differentiation.

Methods

Human periodontal stem cells were isolated and cultured in vitro. The cells were treated with 10, 25, 50 μmol/L GB. Anti-miR-NC and anti-miR-24-3p were transfected into cells by liposome method, and miR-NC and miR-24-3p were transfected into cells and treated with 50 μmol/L GB. MTT detected cell proliferation; After osteogenic induction of cells with different treatments, the ALP activity was detected by the kit; the protein expressions of PCNA, Osx, OPN and OCN were detected by Western blot; and RT-qPCR detected the expression level of miR-24-3p.

Results

GB increased the proliferative activity (48 h, 72 h) [ (0.52 ± 0.05, 0.64 ± 0.05, 0.80 ± 0.08 vs 0.33 ± 0.05), (0.72 ± 0.05, 0.85 ± 0.04, 1.08 ± 0.11 vs 0.51 ± 0.06) ], ALP activity, PCNA (0.45 ± 0.06, 0.75 ± 0.08, 1.12 ± 0.08 vs 0.27 ± 0.05), PCNA (0.41 ± 0.05, 0.57 ± 0.06, 0.78 ± 0.06 vs 0.26 ± 0.04), Osx (0.45 ± 0.06, 0.68 ± 0.05, 0.79 ± 0.03 vs 0.33 ± 0.04 ), OPN (0.56 ± 0.06, 0.75 ± 0.05, 0.92 ± 0.06 vs 0.42 ± 0.05), OCN (0.48 ± 0.05, 0.59 ± 0.04, 0.76 ± 0.08 vs 0.30 ± 0.06) protein expression of human periodontal ligament stem cells in a dose-dependent manner, and decreased the expression level of miR-24-3p (0.35 ± 0.04, 0.52 ± 0.06, 0.87 ± 0.06 vs 1.00 ± 0.09 ) (P < 0.05). Interfering with miR-24-3p increased the proliferative activity [48 h: (0.57 ± 0.06 vs 0.35 ± 0.04) ; 72 h (0.90 ± 0.10 vs 0.47 ± 0.06) ], ALP activity (0.68 ± 0.05 vs 0.30 ± 0.04 ), PCNA (0.47 ± 0.05 vs 0.29 ± 0.04), Osx (0.50 ± 0.04 vs 0.35 ± 0.04), OPN (0.62 ± 0.05 vs 0.39 ± 0.03 ), OCN (0.75 ± 0.07 vs 0.42 ± 0.06) protein expressions of human periodontal ligament stem cells (all P < 0.05). Compared with GB+miR-NC group, the OD value [48 h: (0.67 ± 0.05 vs 0.82 ± 0.07) ; 72 h: (0.84 ± 0.05 vs 1.10 ± 0.09) ], ALP activity (0.56 ± 0.04 vs 1.15 ± 0.15), PCNA (0.46 ± 0.06 vs 0.79 ± 0.07), Osx (0.52 ± 0.03 vs 0.80 ± 0.05), OPN (0.63 ± 0.04 vs 0.91 ± 0.09 ), OCN (0.45 ± 0.06 vs 0.78 ± 0.08) protein expression of GB+miR-24-3p group decreased (all P < 0.05) .

Conclusion

GB may promote proliferation and osteogenic differentiation of human periodontal stem cells by decreasing miR-24-3p.

Key words: Ginkgolide B, miR-24-3p, Human peripheral membrane cells, Proliferation, Osteogenic differentiation

Yanjiao Li, Lei Liang, Fang Jin, Zhiwei Wang. Effects of ginkgolide B on proliferation and osteogenic differentiation of human perimembrane stem cells by regulating miR-24-3p[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2024, 14(04): 229-235.

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URL: https://zhxbygxbzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.2095-1221.2024.04.005

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Figures/Tables 12

References 23

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基因	序列（5’→3’）	预期扩增产物长度（bp）
miR-24-3p	上游TTTGGCTCAGTTCAGCAG	80
	下游TTTGGCACTAGCACATT
miR-150-5p	上游TCGGCGTCTCCCAACCCTTGTAC	158
	下游GTCGTATCCAGTGCAGGGTCCGAGGT
miR-299-5p	上游ACACTCCAGCTGGGTGGTTTACCGTCCCAC	235
	下游CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGATGTATGT
miR-21	上游GCCGCTAGCTTATCAGACT	92
	下游AGTGCAGGGTCCGAGGTA
miR-106a-5p	上游AAAAGTGCTTACAGTGCAGGTAG	126
	下游GAAAAGTGCTTACAGTGCAGGT
U6（内参）	上游CGGGTTTGTTTTGCATTTCT	126
	下游AGTCCCAGCATGAACAGCTT

基因	序列（5’→3’）	预期扩增产物长度（bp）
miR-24-3p	上游TTTGGCTCAGTTCAGCAG	80
	下游TTTGGCACTAGCACATT
miR-150-5p	上游TCGGCGTCTCCCAACCCTTGTAC	158
	下游GTCGTATCCAGTGCAGGGTCCGAGGT
miR-299-5p	上游ACACTCCAGCTGGGTGGTTTACCGTCCCAC	235
	下游CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGATGTATGT
miR-21	上游GCCGCTAGCTTATCAGACT	92
	下游AGTGCAGGGTCCGAGGTA
miR-106a-5p	上游AAAAGTGCTTACAGTGCAGGTAG	126
	下游GAAAAGTGCTTACAGTGCAGGT
U6（内参）	上游CGGGTTTGTTTTGCATTTCT	126
	下游AGTCCCAGCATGAACAGCTT

分组	实验次数	OD（490 nm）			PCNA
分组	实验次数	24 h	48 h	72 h	PCNA
对照	3	0.25 ± 0.03	0.33 ± 0.05	0.51 ± 0.06	0.26 ± 0.04
10 μmol/L GB	3	0.21 ± 0.05	0.52 ± 0.05^a	0.72 ± 0.05^a	0.41 ± 0.05^a
25 μmol/L GB	3	0.24 ± 0.05	0.64 ± 0.05^ab	0.85 ± 0.04^ab	0.57 ± 0.06^ab
50 μmol/L GB	3	0.22 ± 0.06	0.80 ± 0.08^abc	1.08 ± 0.11^abc	0.78 ± 0.06^abc
F值		0.421	33.921	34.545	52.708
P值		0.743	< 0.001	< 0.001	< 0.001

分组	实验次数	OD（490 nm）			PCNA
分组	实验次数	24 h	48 h	72 h	PCNA
对照	3	0.25 ± 0.03	0.33 ± 0.05	0.51 ± 0.06	0.26 ± 0.04
10 μmol/L GB	3	0.21 ± 0.05	0.52 ± 0.05^a	0.72 ± 0.05^a	0.41 ± 0.05^a
25 μmol/L GB	3	0.24 ± 0.05	0.64 ± 0.05^ab	0.85 ± 0.04^ab	0.57 ± 0.06^ab
50 μmol/L GB	3	0.22 ± 0.06	0.80 ± 0.08^abc	1.08 ± 0.11^abc	0.78 ± 0.06^abc
F值		0.421	33.921	34.545	52.708
P值		0.743	< 0.001	< 0.001	< 0.001

分组	实验次数	ALP活性	Osx	OPN	OCN
对照	3	0.27 ± 0.05	0.33 ± 0.04	0.42 ± 0.05	0.30 ± 0.06
10 μmol/L GB	3	0.45 ± 0.06^a	0.45 ± 0.06^a	0.56 ± 0.06^a	0.48 ± 0.05^a
25 μmol/L GB	3	0.75 ± 0.08^ab	0.68 ± 0.05^ab	0.75 ± 0.05^ab	0.59 ± 0.04^a
50 μmol/L GB	3	1.12 ± 0.08^abc	0.79 ± 0.03^abc	0.92 ± 0.06^abc	0.76 ± 0.08^abc
F值		87.889	61.523	46.975	31.738
P值		< 0.001	< 0.001	< 0.001	< 0.001

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