miR-21靶向调控PTEN/PI3K/Akt通路对胆管癌细胞恶性生物学行为的影响

施喆; 周丽媛; 赵国栋; 孙树刚; 薛亮

doi:10.3969/j.issn.1001-5256.2022.09.026

miR-21靶向调控PTEN/PI3K/Akt通路对胆管癌细胞恶性生物学行为的影响

DOI: 10.3969/j.issn.1001-5256.2022.09.026

施喆^a,
周丽媛^b, , ,,
赵国栋^a,
孙树刚^a,
薛亮^a

a.
河北工程大学附属医院普外科, 河北邯郸 056000
b.
河北工程大学附属医院妇科, 河北邯郸 056000

基金项目:

河北省中医药管理局科研计划项目 (2020227)

伦理学声明：本研究于2019年9月27日通过河北工程大学附属医院伦理委员会批准，批号为2019[K]-042。

利益冲突声明：本研究不存在研究者、伦理委员会成员以及与公开研究成果有关的利益冲突。

作者贡献声明：施喆负责课题设计，研究实施，数据分析以及论文撰写；周丽媛、赵国栋负责资料收集，数据分析；孙树刚、薛亮负责课题设计，研究指导，论文修改。

详细信息

通信作者:
周丽媛，zhouliyuan0310@126.com

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出版历程
- 收稿日期: 2022-01-14
- 录用日期: 2022-02-18
- 出版日期: 2022-09-20

Effect of micro-ribonucleic acid-21 on the malignant biological behavior of cholangiocarcinoma cells by targeting the PTEN/PI3K/Akt pathway

a.
Department of General Surgery, The Affiliated Hospital of Hebei University of Engineering, Handan, Hebei 056000, China
b.
Department of Gynecology, The Affiliated Hospital of Hebei University of Engineering, Handan, Hebei 056000, China

Research funding:

Scientific Research Plan Project of Hebei Provincial Administration of Traditional Chinese Medicine (2020227)

More Information

Corresponding author: ZHOU Liyuan, zhouliyuan0310@126.com (ORCID: 0000-0002-6540-0369)

摘要

摘要: 目的探讨微小核糖核酸-21(miR-21)靶向蛋白酪氨酸磷酸酶(PTEN)/磷酸肌醇-3-激酶(PI3K)/蛋白激酶B (Akt)通路对人胆管癌细胞株QBC939恶性生物学行为的影响。方法将对数期生长的胆管癌细胞株QBC939分为4组: 空载组、空白对照组、过表达组、沉默组。倒置荧光显微镜检测转染效率; 四甲基偶氮唑蓝法、流式细胞仪、Transwell小室实验、划痕实验检测细胞增殖活性、凋亡率、侵袭活性和迁移活性。实时逆转录聚合酶链反应(RT-qPCR)检测miR-21、PTEN、PI3K、Akt、哺乳动物雷帕霉素靶蛋白(mTOR) mRNA的表达; 免疫印迹法检测PTEN、PI3K、Akt、磷酸化Akt (p-Akt)和mTOR蛋白表达; 双荧光素酶报告基因实验验证miR-21对PTEN的作用。计量资料多组间比较采用单因素方差分析, 进一步两两差异比较采用SNK-q检验。结果过表达组、沉默组、空载组转染效率分别为(90.27±18.03)%、(91.43±18.26)%和(92.16±18.41)%。与空白对照组和空载组比较, 过表达组QBC939细胞增殖活性明显提高(P值均 < 0.05), 凋亡率显著下降(P值均 < 0.01);与空白对照组、空载组和过表达组比较, 沉默组细胞增殖活性显著降低(P值均 < 0.01), 而凋亡率显著上升(P值均 < 0.01)。与空白对照组和空载组比较, 过表达组QBC939细胞迁移率和穿膜数目均显著增高(P值均 < 0.01);与空白对照组、空载组和过表达组比较, 沉默组迁移率和穿膜数目均显著下降(P值均 < 0.01)。与空白对照组和空载组比较, 过表达组miR-21、PI3K、Akt和mTOR mRNA表达均升高, PTEN mRNA表达下降, 差异均有统计学意义(P值均 < 0.05);与空白对照组、空载组和过表达组比较, 沉默组miR-21、PI3K、Akt和mTOR mRNA表达均下降, PTEN mRNA表达升高, 差异均有统计学意义(P值均 < 0.05)。与空白对照组和空载组比较, 过表达组PI3K、Akt、p-Akt和mTOR蛋白表达均显著升高, PTEN蛋白表达显著下降(P值均 < 0.01);与空白对照组、空载组和过表达组比较, 沉默组PI3K、Akt、p-Akt和mTOR蛋白表达均显著下降, PTEN蛋白表达显著升高(P值均 < 0.01)。miR-21可靶向调控PTEN的表达。结论 miR-21沉默可靶向PTEN/PI3K/Akt通路, 上调PTEN表达, 下调PI3K、Akt、mTOR表达及p-Akt水平, 抑制人胆管癌细胞株QBC939的恶性行为。
- 微RNAs /
- 信号传导 /
- 细胞增殖 /
- 细胞凋亡
Abstract: Objective To investigate the effect of micro-ribonucleic acid-21(miR-21) on the malignant biological behavior of human cholangiocarcinoma cell line QBC939 by targeting the protein tyrosine phosphatase (PTEN)/inositol phosphate 3-kinase (PI3K)/protein kinase B (Akt) pathway. Methods The cholangiocarcinoma cell line QBC939 in the logarithmic growth phase was divided into empty vector group, blank control group, overexpression group, and silencing group.An inverted fluorescence microscope was used to observe transfection efficiency; MTT assay, flow cytometry, Transwell assay, and wound healing assay were used to measure cell proliferative activity, apoptosis rate, invasion activity, and migration activity.Quantitative reverse transcription PCR was used to measure the mRNA expression levels of miR-21, PTEN, PI3K, Akt, and mammalian target of rapamycin (mTOR); Western blotting was used to measure the protein expression levels of PTEN, PI3K, Akt, phosphorylated Akt (p-Akt), and mTOR; dual-luciferase reporter assay was used to verify the effect of miR-21 on PTEN.A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the SNK-q test was used for further comparison between two groups. Results Transfection efficiency was 90.27%±18.03% in the overexpression group, 91.43%±18.26% in the silencing group, and 92.16%±18.41% in the empty vector group.Compared with the blank control group and the empty vector group, the overexpression group had a significant increase in the proliferative activity of QBC939 cells (both P < 0.05) and a significant reduction in apoptosis rate (both P < 0.01);compared with the blank control group, the empty vector group, and the overexpression group, the silencing group had a significant reduction in proliferative activity (P < 0.01) and a significant increase in apoptosis rate (P < 0.01).Compared with the blank control group and the empty vector group, the overexpression group had significant increases in the migration rate of QBC939 cells and number of cells penetrating the membrane (all P < 0.01);compared with the blank control group, the empty vector group, and the overexpression group, the silencing group had significant reductions in migration rate and number of cells penetrating the membrane (all P < 0.01).Compared with the blank control group and the empty vector group, the overexpression group had significant increases in the mRNA expression levels of miR-21, PI3K, Akt, and mTOR and a significant reduction in the mRNA expression level of PTEN (all P < 0.05);compared with the blank control group, the empty vector group, and the overexpression group, the silencing group had significant reductions in the mRNA expression levels of miR-21, PI3K, Akt, and mTOR and a significant increase in the mRNA expression level of PTEN (all P < 0.05).Compared with the blank control group and the empty vector group, the overexpression group had significant increases in the protein expression levels of PI3K, Akt, p-Akt, and mTOR and a significant reduction in the protein expression level of PTEN (all P < 0.01);compared with the blank control group, the empty vector group, and the overexpression group, the silencing group had significant reductions in the protein expression levels of PI3K, Akt, p-Akt, and mTOR and a significant increase in the protein expression level of PTEN (all P < 0.01).Furthermore, miR-21 showed targeted regulation of PTEN expression. Conclusion MiR-21 silencing may inhibit the malignant biological behavior of human cholangiocarcinoma cell line QBC939 by targeting the PTEN/PI3K/Akt signaling pathway to upregulate the expression of PTEN and downregulate the expression of PI3K, Akt, mTOR, and p-Akt.
- MicroRNAs /
- Signal Transduction /
- Cell Proliferation /
- Apoptosis

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图 1 转染后QBC939细胞荧光图(×100)

Figure 1. Photofluorograms of QBC939 cells after transfection (×100)

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图 2 各组QBC939细胞增殖活性

Figure 2. The proliferative activities of QBC939 cells in each group

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图 3 流式细胞检测各组QBC939细胞凋亡率

Figure 3. Flow cytometry examined apoptosis rates of QBC939 cells in each group

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图 4 划痕实验结果

Figure 4. The scratch experiments results

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图 5 Transwell小室试验结果(结晶紫染色，×100)

Figure 5. Transwell experiment results(crystal violet staining, ×100)

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图 6 各组细胞miR-21、PI3K、Akt、mTOR和PTEN mRNA表达的倍比关系

注：与空白对照组比较，#P＜0.05；与空载组比较，△P＜0.05；与过表达组比较，P＜0.05。

Figure 6. The multiple ratio relationships of the expressions of miR-21, PI3K, Akt, mTOR, and PTEN mRNA in each group

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图 7 各组细胞PI3K、Akt、p-Akt、mTOR和PTEN蛋白表达量

Figure 7. Expression levels of PI3K, Akt, p-Akt, mTOR, and PTEN proteins in each group

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图 8 数据库预测miR-21和PTEN的互补序列

Figure 8. Database predicts complementary sequences of miR-21-3p and PTEN

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图 9 双荧光素酶检测结果

注：与转染NC mimics细胞比较，P＜0.05。

Figure 9. Results of the double luciferase assay

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表 1 PCR反应引物序列

Table 1. The primer sequences of PCR reaction

基因	引物序列	扩增产物长度(bp)
miR-21	上游引物5′-GTGCAGGGTCCGAGGT-3′	72
	下游引物5′-GCCGCTAGCTTATCAGACTGATGT-3′
PI3K	上游引物5′-CAAAGCCGAGAACCTATTGC-3′	384
	下游引物5′-TTGAGGGAGTCATTGTGCTG-3′
Akt	上游引物5′-TGGACTACTTGCACTCCGAG-3′	175
	下游引物5′-CGCAGAACGTCTTCATGGTG-3′
mTOR	上游引物5′-ATGACGAGACCCAGGCTAAG-3′	225
	下游引物5′-GCCAGTCCATGCCATCAG-3′
PTEN	上游引物5′-TTATTGCTATGGGATTTCCTGC-3′	198
	下游引物5′-GCTGTGGTGGGTTATGGTCTTC-3′
β-actin	上游引物5′-TGACGTGGACATCCGCAAAG-3′	443
	下游引物5′-CTGGAAGGTGGACAGCGAGG-3′

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表 2 各组QBC939细胞凋亡率

Table 2. The apoptosis rates of QBC939 cells in each group

组别	细胞凋亡率(%)
空白对照组	5.74±1.13
空载组	6.08±1.20
过表达组	3.49±0.65¹⁾²⁾
沉默组	29.87±5.89¹⁾²⁾³⁾
F值	81.767
P值	＜0.001
注：与空白对照组比较，1)P＜0.01；与空载组比较，2)P＜0.01；与过表达组比较，3)P＜0.01。

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表 3 各组QBC939细胞体外侵袭实验结果

Table 3. Results of in vitro invasion experiments of QBC939 cells in each group

组别	迁移率(%)	穿膜数目(个)
空白对照组	49.21±9.81	253.53±50.61
空载组	49.03±9.78	251.49±50.25
过表达组	80.17±16.02¹⁾²⁾	413.01±82.56¹⁾²⁾
沉默组	28.64±5.71¹⁾²⁾³⁾	137.96±27.57¹⁾²⁾³⁾
F值	18.784	20.194
P值	＜0.001	＜0.001
注：与空白对照组比较，1)P＜0.01；与空载组比较，2)P＜0.01；与过表达组比较，3)P＜0.01。

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表 4 各组细胞PI3K、Akt、p-Akt、mTOR和PTEN蛋白相对表达量

Table 4. Relative expression levels of PI3K, Akt, p-Akt, mTOR, and PTEN proteins in each group

组别	PI3K	Akt	p-Akt	mTOR	PTEN
空白对照组	1.35±0.26	0.45±0.09	0.51±0.09	0.79±0.15	0.43±0.08
空载组	1.33±0.24	0.44±0.07	0.49±0.08	0.78±0.14	0.42±0.06
过表达组	2.21±0.41¹⁾²⁾	0.84±0.16¹⁾²⁾	0.95±0.18¹⁾²⁾	1.81±0.36¹⁾²⁾	0.23±0.04¹⁾²⁾
沉默组	0.72±0.13¹⁾²⁾³⁾	0.19±0.03¹⁾²⁾³⁾	0.11±0.02¹⁾²⁾³⁾	0.41±0.08¹⁾²⁾³⁾	0.71±0.14¹⁾²⁾³⁾
F值	24.197	36.489	43.880	40.639	25.059
P值	＜0.001	＜0.001	＜0.001	＜0.001	＜0.001
注：与空白对照组比较，1)P＜0.01；与空载组比较，2)P＜0.01；与过表达组比较，3)P＜0.01。

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