乌头碱调控miR-181d-5p/DDX3轴对宫颈癌HeLa细胞增殖、凋亡的影响

doi:10.11958/20221825

摘要/Abstract

摘要：

目的探讨乌头碱通过调控微小RNA-181d-5p（miR-181d-5p）/DEAD-box RNA解旋酶3（DDX3）轴对宫颈癌HeLa细胞增殖、凋亡的影响。方法使用不同剂量乌头碱处理宫颈癌HeLa细胞，四甲基偶氮唑盐法检测细胞增殖确定给药剂量。将HeLa细胞分为对照组（正常培养，不进行处理），乌头碱低剂量组（4 mg/L）、中剂量组（8 mg/L）、高剂量组（16 mg/L），顺铂组（5 mg/L），乌头碱高剂量+miR-NC组[16 mg/L乌头碱+转染miR-181d-5p siRNA阴性对照（miR-NC）质粒]及乌头碱高剂量+miR-181d-5p低表达组[16 mg/L乌头碱+转染miR-181d-5p小干扰RNA（siRNA）质粒]。平板克隆形成实验及流式细胞仪分别检测各组HeLa细胞克隆形成数与凋亡情况；实时荧光定量PCR检测HeLa细胞中miR-181d-5p和DDX3 mRNA表达水平；蛋白免疫印迹法检测HeLa细胞中DDX3、细胞周期蛋白D1（Cyclin D1）、增殖细胞核抗原（PCNA）、B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、胱天蛋白酶-3（Caspase-3）蛋白表达水平；双萤光素酶报告基因实验验证miR-181d-5p与DDX3的靶向关系。结果以0.5~64 mg/L的乌头碱分别处理HeLa细胞24 h、48 h、72 h后，对HeLa细胞均有不同程度的抑制作用，选择剂量为4 mg/L、8 mg/L、16 mg/L的乌头碱用于后续实验。与对照组比较，乌头碱低、中、高剂量组及顺铂组HeLa细胞克隆形成数、DDX3 mRNA和蛋白、Cyclin D1、PCNA、Bcl-2蛋白表达水平依次降低（P<0.05），凋亡率、miR-181d-5p、Bax、Caspase-3蛋白表达水平依次升高（P<0.05）；与乌头碱高剂量组和乌头碱高剂量+miR-NC组比较，乌头碱高剂量+miR-181d-5p低表达组HeLa细胞克隆形成数、DDX3 mRNA和蛋白、Cyclin D1、PCNA、Bcl-2蛋白表达水平升高（P<0.05），凋亡率、miR-181d-5p、Bax、Caspase-3蛋白表达水平降低（P<0.05）；双萤光素酶报告基因检测证实miR-181d-5p与DDX3存在靶向关系。结论乌头碱可调控miR-181d-5p/DDX3轴，促进miR-181d-5p表达，抑制DDX3表达，进而抑制宫颈癌HeLa细胞增殖，促进细胞凋亡。

关键词: 乌头碱, 微小RNA-181d-5p, DEAD-box RNA解旋酶3, 宫颈癌HeLa细胞

Abstract:

Objective To investigate effects of aconitine on proliferation and apoptosis of cervical cancer HeLa cells by regulating the microRNA-181d-5p (miR-181d-5p)/DEAD-box RNA helicase 3 (DDX3) axis. Methods Cervical cancer HeLa cells were treated with different doses of aconitine, and cell proliferation was detected by tetramethylazolium salt method to determine the dose. HeLa cells were divided into the control group (normal culture, no treatment), the aconitine low dose group (4 mg/L), the aconitine medium dose group (8 mg/L), the aconitine high dose group (16 mg/L), the cisplatin group (5 mg/L), the aconitine high dose + miR-NC group [16 mg/L aconitine + transfected with miR-181d-5p siRNA negative control (miR-NC) plasmid] and the aconitine high dose + miR-181d-5p low expression group [16 mg/L aconitine + transfected miR-181d-5p small interfering RNA (siRNA) plasmid]. Plate clone formation experiment and flow cytometry were used to detect the clonal formation number and apoptosis of HeLa cells in each group. Expression levels of miR-181d-5p and DDX3 messenger RNA (mRNA) in HeLa cells were detected by real-time fluorescent quantitative PCR. Western blot assay was used to detect expression levels of DDX3, Cyclin D1, proliferating cell nuclear antigen (PCNA), B cell lymphocytoma-2 (Bcl-2), Bcl-2 related X protein (Bax) and Caspase-3 protein in HeLa cells. Dual luciferase reporter gene experiment was used to verify the targeting relationship between miR-181d-5p and DDX3. Results HeLa cells were treated with 0.5-64 mg/L aconitine for 24 h, 48 h and 72 h, respectively, and HeLa cells were inhibited to varying degrees. Aconitine doses of 4 mg/L, 8 mg/L and 16 mg/L were selected for follow-up experiments. Compared with the control group, HeLa cell clonal formation number, DDX3 mRNA and protein, Cyclin D1, PCNA and Bcl-2 protein expression levels were decreased successively in the aconitine low, medium and high dose groups and the cisplatin groups (P<0.05), and the apoptosis rate, expression levels of miR-181d-5p, Bax and Caspase-3 protein were increased successively (P<0.05). Compared with the aconitine high dose group and the aconitine high dose + miR-NC group, HeLa cell clonal formation number, DDX3 mRNA and protein, Cyclin D1, PCNA and Bcl-2 protein expression levels were increased in the aconitine high dose + miR-181d-5p low expression group (P<0.05), and the apoptosis rate, expression levels of miR-181d-5p, Bax and Caspase-3 protein were decreased (P<0.05). Dual luciferase reporter gene detection confirmed the targeting relationship between miR-181d-5p and DDX3. Conclusion Aconitine can regulate the miR-181d-5p/DDX3 axis, promote the expression of miR-181d-5p, inhibit the expression of DDX3, and then inhibit the proliferation of cervical cancer HeLa cells and promote cell apoptosis.

Key words: aconitine, microRNA-181d-5p, DEAD-box RNA helicase 3, cervical cancer HeLa cells

中图分类号:

R737.33

图/表 9

参考文献 21

[1]	彭巧华, 吕卫国. 2022年第1版《NCCN子宫颈癌临床实践指南》解读[J]. 实用肿瘤杂志, 2022, 37(3):205-214.
	PENG Q H, LYU W G. Interpretation of NCCN guidelines for cervical cancer,version 1. 2022[J]. Journal of Practical Oncology, 2022, 37(3):205-214. doi:10.13267/j.cnki.syzlzz.2022.034.
[2]	ZHAO M, GU R Y, DING S R, et al. Risk factors of cervical cancer among ethnic minorities in Yunnan Province,China:a case-control study[J]. Eur J Cancer Prev, 2022, 31(3):287-292. doi:10.1097/CEJ.0000000000000704.
[3]	LI L, ZHANG L, LIAO T, et al. Advances on pharmacology and toxicology of aconitine[J]. Fundam Clin Pharmacol, 2022, 36(4):601-611. doi:10.1111/fcp.12761.
[4]	SONG N, MA J, ZHANG X, et al. Lappaconitine hydrochloride induces apoptosis and S phase cell cycle arrest through MAPK signaling pathway in human liver cancer HepG2 cells[J]. Pharmacogn Mag, 2021, 17(74):334-341.doi:10.4103/pm.pm_251_20.
[5]	熊慧生, 蒋参, 高瑞, 等. 乌头碱对肝癌MHCC97细胞生长、侵袭和迁移的调控作用及机制研究[J]. 中国免疫学杂志, 2018, 34(5):688-691.
	XIONG H S, JIANG C, GAO R, et al. Regulatory effects and mechanism of aconitine on proliferation,invasion and migration of hepatoma carcinoma cell MHCC97[J]. Chinese Journal of Immunology, 2018, 34(5):688-691. doi:10.3969/j.issn.1000-484X.2018.05.009.
[6]	DING M, ZHAO X, CHEN X, et al. Cancer-associated fibroblasts promote the stemness and progression of renal cell carcinoma via exosomal miR-181d-5p[J]. Cell Death Discov, 2022, 8(1):439. doi:10.1038/s41420-022-01219-7.
[7]	夏炳辉, 张振山, 姚天宇, 等. miR-181-5p下调人非小细胞肺癌细胞KLF6表达并抑制其增殖、迁移和侵袭[J]. 生物技术, 2022, 32(5):587-591.
	XIA B H, ZHANG Z S, YAO T Y, et al. miR-181-5p down-regulates the expression of human non-small cell lung cancer cell KLF6 and inhibits its proliferation,migration and invasion[J]. Biotechnology, 2022, 32(5):587-591. doi:10.16519/j.cnki.1004-311x.2022.05.0093.
[8]	郭芳芳, 赵瑞皎, 李杜娟, 等. DDX3表达上调在人宫颈癌细胞增殖中的作用分析[J]. 中华病理学杂志, 2021, 50(2):119-124.
	GUO F F, ZHAO R J, LI D J, et al. Role of up-regulated DDX3 in the proliferation of human cervical cancer cells[J]. Chin J Pathol, 2021, 50(2):119-124. doi:10.3760/cma.j.cn112151-20200519-00394.
[9]	李萍, 姬白嫣, 魏娟, 等. 藏红花素对人宫颈癌Hela细胞顺铂耐药性的影响[J]. 新乡医学院学报, 2021, 38(5):401-405.
	LI P, JI B Y, WEI J, et al. Effect of crocin on cisplatin resistance of human cervical cancer Hela cells[J]. Journal of Xinxiang Medical University, 2021, 38(5):401-405. doi:10.7683/xxyxyxb.2021.05.001.
[10]	GAO Y, FAN H, NIE A, et al. Aconitine:A review of its pharmacokinetics,pharmacology,toxicology and detoxification[J]. J Ethnopharmacol, 2022, 293:115270. doi:10.1016/j.jep.2022.115270.
[11]	LUAN S, GAO Y, LIANG X, et al. Aconitine linoleate,a natural lipo-diterpenoid alkaloid,stimulates anti-proliferative activity reversing doxorubicin resistance in MCF-7/ADR breast cancer cells as a selective topoisomerase Ⅱα inhibitor[J]. Naunyn Schmiedebergs Arch Pharmacol, 2022, 395(1):65-76. doi:10.1007/s00210-021-02172-5.
[12]	周小剑, 李玉华, 唐湘, 等. 乌头碱抑制食管癌EC-1细胞增殖与侵袭并诱导其凋亡作用研究[J]. 中国医药生物技术, 2019, 14(4):335-340.
	ZHOU X J, LI Y H, TANG X, et al. Aconitine inhibits the proliferation and invasion while induces the apoptosis of esophageal cancer EC-1 cells[J]. Chinese Medicinal Biotechnology, 2019, 14(4):335-340. doi:10.3969/j.issn.1673-713X.2019.04.008.
[13]	WANG X, LIN Y, ZHENG Y. Antitumor effects of aconitine in A2780 cells via estrogen receptor β-mediated apoptosis,DNA damage and migration[J]. Mol Med Rep, 2020, 22(3):2318-2328. doi:10.3892/mmr.2020.11322.
[14]	JI B L, XIA L P, ZHOU F X, et al. Aconitine induces cell apoptosis in human pancreatic cancer via NF-κB signaling pathway[J]. Eur Rev Med Pharmacol Sci, 2016, 20(23):4955-4964.
[15]	柯东平, 朱金祥, 毛俊倩, 等. miR-181d-5p对人结肠癌细胞凋亡的影响及机制[J]. 贵州医科大学学报, 2019, 44(7):814-820.
	KE D P, ZHU J X, MAO J Q, et al. Effect and mechanism of microRNA-181d-5p on apoptosis of human colon cancer cells[J]. Journal of Guizhou Medical University, 2019, 44 (7):814-820. doi:10.19367/j.cnki.1000-2707.2019.07.014.
[16]	DONG X, LIU Y, DENG X, et al. C1GALT1,Negatively regulated by miR-181d-5p,promotes tumor progression via upregulating RAC1 in lung adenocarcinoma[J]. Front Cell Dev Biol, 2021, 9:707970. doi:10.3389/fcell.2021.707970.
[17]	KHALIL S, FABBRI E, SANTANGELO A, et al. miRNA array screening reveals cooperative MGMT-regulation between miR-181d-5p and miR-409-3p in glioblastoma[J]. Oncotarget, 2016, 7(19):28195-28206. doi:10.18632/oncotarget.8618.
[18]	李祥双, 于海霞, 叶燕珍, 等. miR-181d-5p对宫颈癌细胞放射敏感性的影响及机制[J]. 山东医药, 2022, 62(22):42-46.
	LI X S, YU H X, YE Y Z, et al. Influence and mechanism of miR-181d-5p on radiosensitivity of cervical cancer cells[J]. Shandong Medical Journal, 2022, 62(22):42-46. doi:10.3969/j.issn.1002-266X.2022.22.010.
[19]	NI J, CHEN L, LING L, et al. MicroRNA-196a promotes cell proliferation and inhibits apoptosis in human ovarian cancer by directly targeting DDX3 and regulating the PTEN/PI3K/AKT signaling pathway[J]. Mol Med Rep, 2020, 22(2):1277-1284. doi:10.3892/mmr.2020.11236.
[20]	VAN DER POL C C, MOELANS C B, MANSON Q F, et al. Cytoplasmic DDX3 as prognosticator in male breast cancer[J]. Virchows Arch, 2021, 479(4):647-655. doi:10.1007/s00428-021-03107-4.
[21]	HUA Q, LIU Y, LI M, et al. Tobacco-related exposure upregulates Circ_0035266 to exacerbate inflammatory responses in human bronchial epithelial cells[J]. Toxicol Sci, 2021, 179(1):70-83. doi:10.1093/toxsci/kfaa163.

乌头碱剂量/ （mg/L）	增殖抑制率
乌头碱剂量/ （mg/L）	24 h	48 h	72 h
0.5	1.03±0.11	1.34±0.14	1.56±0.19
1	3.16±0.24^a	4.63±0.60^a	5.71±0.62^a
2	7.54±0.69^ab	8.86±0.95^ab	10.27±1.93^ab
4	15.57±2.51^abc	18.73±2.90^abc	22.81±3.34^abc
8	25.62±3.83^abcd	28.04±4.66^abcd	32.80±4.63^abcd
16	36.04±4.85^abcde	40.52±5.04^abcde	46.72±5.37^abcde
32	42.09±5.33^abcde	51.05±5.89^abcdef	56.92±6.74^abcdef
64	57.11±6.60^abcdefg	62.84±6.99^abcdefg	70.55±8.04^abcdefg
F	168.383^＊	176.023^＊	175.180^＊

乌头碱剂量/ （mg/L）	增殖抑制率
乌头碱剂量/ （mg/L）	24 h	48 h	72 h
0.5	1.03±0.11	1.34±0.14	1.56±0.19
1	3.16±0.24^a	4.63±0.60^a	5.71±0.62^a
2	7.54±0.69^ab	8.86±0.95^ab	10.27±1.93^ab
4	15.57±2.51^abc	18.73±2.90^abc	22.81±3.34^abc
8	25.62±3.83^abcd	28.04±4.66^abcd	32.80±4.63^abcd
16	36.04±4.85^abcde	40.52±5.04^abcde	46.72±5.37^abcde
32	42.09±5.33^abcde	51.05±5.89^abcdef	56.92±6.74^abcdef
64	57.11±6.60^abcdefg	62.84±6.99^abcdefg	70.55±8.04^abcdefg
F	168.383^＊	176.023^＊	175.180^＊

组别	细胞克隆形成数/ （个/视野）	凋亡率/%
对照组	154.63±4.57	2.39±0.45
乌头碱低剂量组	128.54±4.37^a	11.62±1.57^a
乌头碱中剂量组	105.46±3.78^ab	19.54±2.41^ab
乌头碱高剂量组	85.42±4.56^abc	28.03±3.25^abc
顺铂组	72.43±2.95^abcd	35.53±4.31^abcd
乌头碱高剂量+miR-NC组	87.28±4.41	27.62±3.57
乌头碱高剂量+miR-181d-5p 低表达组	134.16±5.65^de	14.85±2.34^de
F	183.402^＊	96.632^＊

组别	细胞克隆形成数/ （个/视野）	凋亡率/%
对照组	154.63±4.57	2.39±0.45
乌头碱低剂量组	128.54±4.37^a	11.62±1.57^a
乌头碱中剂量组	105.46±3.78^ab	19.54±2.41^ab
乌头碱高剂量组	85.42±4.56^abc	28.03±3.25^abc
顺铂组	72.43±2.95^abcd	35.53±4.31^abcd
乌头碱高剂量+miR-NC组	87.28±4.41	27.62±3.57
乌头碱高剂量+miR-181d-5p 低表达组	134.16±5.65^de	14.85±2.34^de
F	183.402^＊	96.632^＊

组别	miR-181d-5p	DDX3 mRNA
对照组	1.01±0.03	1.00±0.01
乌头碱低剂量组	1.73±0.15^a	0.71±0.09^a
乌头碱中剂量组	2.31±0.22^ab	0.49±0.06^ab
乌头碱高剂量组	2.85±0.27^abc	0.30±0.04^abc
顺铂组	3.42±0.31^abcd	0.20±0.02^abcd
乌头碱高剂量+miR-NC组	2.89±0.33^abc	0.32±0.05^abc
乌头碱高剂量+miR-181d-5p 低表达组	1.68±0.19^de	0.75±0.10^de
F	77.515^＊	135.392^＊