Glycyrrhiza extract inhibits the proliferation, migration and invasion of thyroid tumor cells by regulating the expression of miR-212-5p/BCL2L2 gene

doi:10.3877/cma.j.issn.2095-1221.2021.01.004

Abstract

Abstract:

Objective

To investigate the effects and molecular mechanism of Glycyrrhiza extract (GL-1) on the proliferation, migration and invasion of thyroid tumor cells.

Methods

Thyroid tumor cells CAL-62 cells were treated with 10, 20, 30 μg/mL GL-1, or transfected with miR-212- 5p mimics, anti-miR-212-5p, si-BCL2L2, pcDNA-BCL2L2. Among them, cells transfected with pcDNA-BCL2L2 were treated with 30 μg/mL GL-1. The proliferation rate of CAL-62 cells was determined by MTT colorimetry, the migration and invasion rate of CAL-62 cells were detected by Transwell chamber method, the expression of miR-212-5p in CAL-62 cells was detected by real-time quantitative PCR (RT-qPCR) , and the expression levels of related protein BCL2L2, Cyclin D1 and Matrix metalloprotease 2 (MMP-2) were analyzed by Western blot. Biological information predicted the downstream target gene of miR-212-5p, which was further verified by double luciferase gene reporting experiments. The data were compared by one-way analysis of variance, Tukey's post-test and t test, and P < 0.05 was considered statistically significant.

Results

Compared with control group, the cell viability was significantly reduced in a dose-and time-dependent manner after treated by GL-1 at 10 μg/mL、20 μg/mL、30 μg/mL concentrations for 24 h, 48 h and 72 h respectively (P < 0.05) . In addition, compared with control group, after treated with GL-1, the number of invasion cells (143.56±14.22, 100.32±10.23, 68.23±6.49 vs 189.65±15.23, respectively) , migration cells (198.56±14.35, 141.35±12.58, 89.56±8.95 vs 295.36±17.56, respectively) and expression level of BCL2L2 protein (0.76±0.08, 0.51±0.06, 0.24±0.02 vs 1.00±0.12, respectively) were significantly reduced, while the level of miR-212-5p (1.61±0.11, 1.99±0.13, 2.28±0.15 vs 1.00±0.07, respectively) were increased in CAL-62 cells (P < 0.05) , and the data were showed in a dose-dependent manner. The viability CAL-62 cells, migration rate, invasion rate and the expression levels of Cyclin D1, MMP-2 proteins were significantly decreased after overexpression of miR-212- 5p and silencing expression of BCL2L2 at 24 h, 48 h and 72 h (P < 0.05) . Biological information prediction and dual luciferase gene report experiment confirmed BCL2L2 was the target gene of miR-212- 5p. Overexpression of miR-212-5p significantly inhibited the expression level of BCL2L2 protein, however, silencing miR-212-5p significantly increased this protein lever (P < 0.05) . Overexpression of BCL2L2 reversed the inhibitory effect of Glycyrrhiza extract on the proliferation, migration, invasion and expression of Cyclin D1 and MMP-2 in CAL-62 cells.

Conclusion

Glycyrrhiza extract could inhibit the proliferation, migration and invasion of thyroid tumor cells by regulating the expression of miR-212-5p/BCL2L2.

Key words: Glycyrrhiza, miR-212-5p, BCL2L2, Thyroid tumor, Proliferation

Zhongqin Wang, Qing Hu, Jinfeng Zhang. Glycyrrhiza extract inhibits the proliferation, migration and invasion of thyroid tumor cells by regulating the expression of miR-212-5p/BCL2L2 gene[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2021, 11(01): 25-33.

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URL: https://zhxbygxbzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.2095-1221.2021.01.004

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Figures/Tables 16

References 24

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基因	序列（5，→3，）	预期扩增产物长度（bp）
miR-212-5p	上游CCTCGACTGGGGGTGTAAACAT	223
	下游GTGGAGTCGATTGCGTGTC
U6	上游GCTTCGGCAGCACATATACTAAAAT	185
	下游CGCTTCAGAATTTGCGTGTCAT

基因	序列（5，→3，）	预期扩增产物长度（bp）
miR-212-5p	上游CCTCGACTGGGGGTGTAAACAT	223
	下游GTGGAGTCGATTGCGTGTC
U6	上游GCTTCGGCAGCACATATACTAAAAT	185
	下游CGCTTCAGAATTTGCGTGTCAT

分组	实验次数	细胞活力（h）			细胞迁移数（个）	细胞侵袭数（个）
分组	实验次数	24	48	72	细胞迁移数（个）	细胞侵袭数（个）
对照组	3	0.67±0.09	1.24±0.11	1.68±0.12	295.36±17.56	189.65±15.23
10 μg/mL GL-1	3	0.51±0.07^a	0.78±0.11^a	1.14±0.13^a	198.56±14.35^a	143.56±14.22^a
20 μg/mL GL-1	3	0.42±0.07^a	0.59±0.09^ab	0.86±0.14^ab	141.35±12.58^ab	100.32±10.23^ab
30 μg/mL GL-1	3	0.31±0.05^ab	0.44±0.10^abc	0.62±0.12^abc	89.56±8.95^abc	68.23±6.49^abc
F值		13.623	34.239	38.193	123.937	57.530
P值		< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

分组	实验次数	细胞活力（h）			细胞迁移数（个）	细胞侵袭数（个）
分组	实验次数	24	48	72	细胞迁移数（个）	细胞侵袭数（个）
对照组	3	0.67±0.09	1.24±0.11	1.68±0.12	295.36±17.56	189.65±15.23
10 μg/mL GL-1	3	0.51±0.07^a	0.78±0.11^a	1.14±0.13^a	198.56±14.35^a	143.56±14.22^a
20 μg/mL GL-1	3	0.42±0.07^a	0.59±0.09^ab	0.86±0.14^ab	141.35±12.58^ab	100.32±10.23^ab
30 μg/mL GL-1	3	0.31±0.05^ab	0.44±0.10^abc	0.62±0.12^abc	89.56±8.95^abc	68.23±6.49^abc
F值		13.623	34.239	38.193	123.937	57.530
P值		< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

分组	实验次数	miR-212-5p	BCL2L2蛋白
对照组	3	1.00±0.07	1.00±0.18
10 μg/mL GL-1	3	1.61±0.11^a	0.71±0.09^a
20 μg/mL GL-1	3	1.99±0.13^ab	0.45±0.05^ab
30 μg/mL GL-1	3	2.28±0.15^abc	0.18±0.01^abc
F值		65.035	34.35
P值		< 0.001	< 0.001

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