Effects of aconitine regulating miR-181d-5p/DDX3 axis on proliferation and apoptosis of HeLa cells of cervical cancer

doi:10.11958/20221825

Abstract

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

CLC Number:

R737.33

Figures/Tables 9

References 21

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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^＊