沉默促癌基因Yap1联合丹参酮ⅡA对肝癌细胞Huh-7的影响

洪莹晖; 叶明亮; 罗杰; 王纯; 刘佳良; 任超; 蓝偲瑜; 赵秋; 常莹

doi:10.3969/j.issn.1001-5256.2021.02.020

沉默促癌基因Yap1联合丹参酮ⅡA对肝癌细胞Huh-7的影响

DOI: 10.3969/j.issn.1001-5256.2021.02.020

洪莹晖^{1, 2, 3},
叶明亮^{1, 2, 3},
罗杰^{1, 2, 3},
王纯^{1, 2, 3},
刘佳良^{1, 2, 3},
任超^{1, 2, 3},
蓝偲瑜^{1, 2, 3},
赵秋^{1, 2, 3},
常莹^{1, 2, 3, ,}

1.
武汉大学中南医院消化内科，武汉 430071
2.
湖北省肠病医学临床研究中心，武汉 430071
3.
肠病湖北省重点实验室，武汉 430071

基金项目:

国家自然科学基金项目 (81670554);

湖北省自然科学基金项目 (2017CFA068)

利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突，特此声明。

作者贡献声明：洪莹晖、叶明亮负责课题设计，实验操作以及资料分析，撰写论文；罗杰、王纯、任超、蓝偲瑜、刘佳良参与修改论文；洪莹晖、赵秋、常莹负责拟定写作思路，撰写文章并最后定稿；赵秋、常莹提供资金支持。

详细信息

作者简介:
洪莹晖(1996—)，女，主要从事肝脏肿瘤的病理机制和临床研究

通信作者:
常莹，changying@whu.edu.cn

中图分类号: R735.7
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- 文章访问数: 387
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- 被引次数: 0
出版历程
- 收稿日期: 2020-08-10
- 录用日期: 2020-09-04
- 出版日期: 2021-02-20

Effect of oncogene Yap1 silencing combined with tanshinone ⅡA on Huh-7 hepatoma cells

Yinghui HONG^{1, 2, 3},
Mingliang YE^{1, 2, 3},
Jie LUO^{1, 2, 3},
Chun WANG^{1, 2, 3},
Jialiang LIU^{1, 2, 3},
Chao REN^{1, 2, 3},
Siyu LAN^{1, 2, 3},
Qiu ZHAO^{1, 2, 3},
Ying CHANG^{1, 2, 3
, ,}

1.
Department of Gastroenterology, Zhongnan Hospital of Wuhan University, Wuhan 430071, Chin
2.
Hubei Clinical Center of Intestinal and Colorectal Diseases, Wuhan 430071, China
3.
Hubei Key Laboratory of Intestinal and Colorectal Diseases, Wuhan 430071, China

摘要

摘要: 目的研究基因Yap1与丹参酮ⅡA对肝癌细胞Huh-7增殖、迁移侵袭的影响。方法选用武汉大学中南医院10对人肝细胞癌及癌旁组织标本，收集时间2019年6月1日—2019年12月1日。实时荧光定量法和蛋白质印迹法用于检测基因Yap1的表达以及表型相关分子的变化。采用MTT细胞增殖检测试剂检测不同浓度丹参酮ⅡA处理后细胞的增殖抑制率。蛋白质印迹实验检测不同干预下细胞凋亡、迁移相关标志物表达变化。流式细胞检测和Transwell实验分别检测细胞凋亡和细胞迁移以及侵袭。计量资料两组间比较采用t检验；计数资料两组间比较采用χ²检验。结果 10对人肝癌组织标本中有8对Yap1的表达明显高于癌旁组织。相对于人正常肝上皮细胞L02，Yap1在肝癌细胞株Huh-7和HCCL-M3中表达增高。si-Yap1-3转染细胞蛋白水平沉默效率达87.004%。MTT结果显示丹参酮ⅡA能够有效抑制Huh-7细胞增殖，其半数抑制浓度为8.683 μmol/L。si-Yap1-3与丹参酮ⅡA联合处理Huh-7肝癌细胞，下游增殖迁移标志物E-钙粘蛋白表达增加，波形蛋白表达降低。Transwell实验结果示，相较于si-NC组，丹参酮ⅡA联合si-Yap1-3处理组细胞迁移、侵袭能力明显降低(43.19±2.88 vs 132.20±10.03，t=8.527，P=0.001；53.95±4.20 vs 179.10±11.11，t=4.484，P=0.011)。si-Yap1-3与丹参酮ⅡA联合处理组细胞凋亡相关标志物Bax表达增加，Bcl-2蛋白表达降低，且联合处理组细胞早期凋亡率为25.98%，高于si-NC组的9.21%(χ²=4.078，P<0.05)。结论沉默促癌基因Yap1联合丹参酮ⅡA能够促进肝癌细胞Huh-7凋亡，并抑制其迁移侵袭，为临床用药提供一定的指导意义。
- 癌, 肝细胞 /
- 基因沉默 /
- 丹参酮
Abstract: Objective To investigate the effect of the Yap1 gene and tanshinone ⅡA on the proliferation, migration, and invasion abilities of Huh-7 hepatoma cells. Methods A total of 10 pairs of human hepatocellular carcinoma (HCC) samples and adjacent tissue samples were collected in Zhongnan Hospital of Wuhan University from June 1 to December 1, 2019. Quantitative real-time PCR and Western blotting were used to measure the expression of the Yap1 gene and phenotype-related molecules. MTT cell proliferation detection reagent was used to measure the inhibition rate of cell proliferation after the treatment with different concentrations of tanshinone ⅡA. Western blotting was used to measure the changes in the expression of apoptosis-and migration-related markers after different interventions. Flow cytometry and Transwell assay were used to measure apoptosis and cell migration and invasion abilities. The data of 375 cases of liver cancer and 50 cases of relatively normal liver tissue samples were downloaded from The Cancer Genome Atlas, including clinicopathological information. The t-test was used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between two groups. Results In 8 of the 10 pairs of HCC samples and adjacent tissue samples, HCC samples had significantly higher expression of Yap1 than the adjacent tissue samples. Compared with the normal human liver epithelial cells L02, the Huh-7 and HCCL-M3 hepatoma cells had a significant increase in the expression of Yap1. The silencing efficiency of si-Yap1-3 transfection reached 87.004% at the protein level. MTT results showed that tanshinone ⅡA effectively inhibited the proliferation of Huh-7 cells, with a half inhibitory concentration of 8.683 μmol/L. After the cells were treated with si-Yap1-3 and tanshinone ⅡA, there was an increase in the expression of the downstream marker for proliferation and migration E-cadherin and a reduction in the expression of vimentin, and the results of Transwell assay showed that compared with the si-NC group, the tanshinone ⅡA+si-Yap1-3 group had significant reductions in the migration and invasion abilities of Huh-7 cells (migration: 43.19±2.88 vs 132.20±10.03, t=8.527, P=0.001; invasion: 53.95±4.20 vs 179.10±11.11, t=4.484, P=0.011). The group treated with si-Yap1-3 and tanshinone ⅡA had an increase in the expression of the apoptosis-related marker Bax and a reduction in the expression of Bcl-2, as well as a significantly higher early apoptosis rate than the si-NC group (25.98% vs 9.21%, χ²=4.078, P < 0.05). Conclusion Oncogene Yap1 silencing combined with tanshinone ⅡA can promote the apoptosis of Huh-7 hepatoma cells and inhibit their migration and invasion, which can provide certain guiding significance for clinical medication.
- Carcinoma, Hepatocellular /
- Gene Silencing /
- TANSHINONE

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图 1 10对人源癌旁和对应肝癌标本中Yap1的表达

注：B，癌旁标本；T，肝癌标本。

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图 2 基因Yap1在不同细胞系中的蛋白表达水平

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图 3 si-Yap1瞬时转染Huh-7细胞Yap1的表达水平

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图 4 不同药物浓度作用于Huh-7细胞的细胞增殖抑制率

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图 5 不同药物浓度作用于Huh-7细胞下游表型相关分子表达变化

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图 6 si-Yap1-3与药物共处理Huh-7细胞的细胞增殖抑制率

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图 7 si-Yap1-3和丹参酮ⅡA联合处理下游表型相关分子变化

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图 8 不同处理下Huh-7的细胞凋亡变化情况

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图 9 对Huh-7进行不同干预后细胞迁移情况

注：a，×100；b，×200。

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图 10 对Huh-7进行不同干预后细胞侵袭情况

注：a，×100；b，×200。

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表 1 si-Yap1瞬时转染Huh-7细胞灰度值分析

指标	si-NC	si-Yap1-1	si-Yap1-2	si-Yap1-3
Yap1	3745.0±131.6	2641.0±74.5	2253.0±74.7	4939.0±6.8
GAPDH	8235.0±129.6	7461.0±129.4	6716.0±108.3	8613.0±61.2
t值		15.33	21.68	25.69
P值		0.004	0.002	0.002

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表 2 si-Yap转染Huh-7后mRNA水平抑制Yap1的表达

指标	si-NC	si-Yap1-1	si-Yap1-2	si-Yap1-3
Yap1(Ct值)	26.62±0.33	26.72±0.11	26.79±0.06	27.72±0.02
GAPDH(Ct值)	16.69±0.25	16.62±0.08	16.26±0.07	16.66±0.22
t值		0.739	10.92	98.97
P值		0.501	<0.001	<0.001

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