miR-373-3p调控小窝蛋白1逆转膀胱癌细胞顺铂耐药的机制研究

金月伟; 毛祖杰; 胡林义; 泮永志; 王俊杰; 杨盼; 郑敏超; 郑永强; 朱龙

doi:10.16766/j.cnki.issn.1674-4152.002762

miR-373-3p调控小窝蛋白1逆转膀胱癌细胞顺铂耐药的机制研究

doi: 10.16766/j.cnki.issn.1674-4152.002762

1.
浙江省人民医院浙东南院区泌尿外科，浙江台州 317300
2.
浙江省人民医院泌尿外科，浙江杭州 310000

基金项目:

浙江省医药卫生科技计划项目 2019RC102

台州市科技计划项目 20ywb178

详细信息

通讯作者:
毛祖杰, E-mail: jiejie981@126.com

中图分类号: R73-36 R730.53 R737.14
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出版历程
- 收稿日期: 2022-11-02
- 网络出版日期: 2023-02-07

Mechanism of miR-373-3p in reversing cisplatin resistance in bladder cancer cells by regulating caveolin 1

1.
Department of Urology Surgery, East and South Zhejiang Hospital of Zhejiang Provincial

摘要

摘要: 目的探讨微小RNA-373-3p(miR-373-3p)靶向小窝蛋白1(Cav1)对膀胱癌细胞顺铂(DDP)耐药性的影响。方法建立膀胱癌DDP耐药细胞株T24/DDP，RT-qPCR检测T24细胞与T24/DDP细胞中miR-373-3p表达，在T24/DDP细胞中过表达miR-373-3p或抑制Cav1表达，每组实验设置6个复孔，MTT法检测DDP对T24/DDP细胞的半数抑制浓度(IC₅₀)及细胞增殖活性；流式细胞术检测细胞凋亡；免疫印迹法检测细胞中Cav1、多药耐药相关蛋白(MRP)、P-糖蛋白(P-gp)蛋白表达；双荧光素酶报告基因实验验证miR-373-3p与Cav1的靶向关系。结果与T24细胞比较，T24/DDP细胞中miR-373-3p表达水平降低(0.34±0.06 vs. 1.00±0.18，t=8.521，P＜0.001)，Cav1蛋白表达水平升高(0.95±0.10 vs. 0.41±0.06，t=11.342，P＜0.001)；双荧光素酶报告基因实验证明miR-373-3p可靶向负调控Cav1表达；T24/DDP细胞中过表达miR-373-3p或抑制Cav1表达，细胞凋亡率显著升高(均P＜0.05)，细胞增殖活性、DDP对细胞的IC₅₀值及MRP、P-gp蛋白表达水平显著降低(均P＜0.05)；过表达Cav1可逆转过表达miR-373-3p对T24/DDP细胞耐药性的抑制作用。结论 miR-373-3p可靶向下调Cav1表达逆转T24/DDP细胞对DDP的耐药性。
- 微小RNA-373-3p /
- 小窝蛋白1 /
- 膀胱癌细胞 /
- 顺铂 /
- 耐药性
Abstract: Objective To investigate the effect of microRNA-373-3p (miR-373-3p) targeting caveolin 1 (Cav1) on the cisplatin (DDP) resistance of bladder cancer cell. Methods The DDP-resistant bladder cancer cell line T24/DDP were established. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-373-3p in T24 and T24/DDP cells. miR-373-3p was overexpressed, or the expression of Cav1 was inhibited in T24/DDP cells. Each group of experiments was set up with six replicate wells. MTT method was used to detect the half inhibitory concentration (IC₅₀) of DDP on T24/DDP cells and cell proliferation activity. Flow cytometry was used to detect cell apoptosis, and Western blotting was used to determine the expressions of Cav1, multidrug resistance associated protein (MRP) and P-glycoprotein (P-gp) proteins in cells. Dual luciferase reporter gene experiment was used to verify the targeting relationship between miR-373-3p and Cav1. Results Compared with T24 cells, the expression level of miR-373-3p in T24/DDP cells decreased (0.34±0.06 vs. 1.00±0.18, t=8.521, P < 0.001), and that of Cav1 protein increased (0.95±0.10 vs. 0.41±0.06, t=11.342, P < 0.001). Dual luciferase reporter experiment proved that miR-373-3p could target and negatively regulate Cav1 expression. After the overexpression of miR-373-3p or inhibition of Cav1 expression in T24/DDP cells, the apoptosis rate significantly increased (all P < 0.05), and the cell proliferation activity, IC₅₀ of DDP to the cell and protein expression levels of MRP and P-gp significantly decreased (all P < 0.05). The overexpressed Cav1 could reverse the inhibitory effect of the overexpression of miR-373-3p on the drug resistance of T24/DDP cells. Conclusion MiR-373-3p can target and down regulate Cav1 expression and reverse the DDP resistance of T24/DDP cells.
- MicroRNA-373-3p /
- Caveolin 1 /
- Bladder cancer cell /
- Cisplatin /
- Drug resistance

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图 1 T24、T24/DDP细胞中Cav1蛋白表达情况

Figure 1. Expression of Cav1 protein in T24 and T24/DDP cells

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图 2 过表达miR-373-3p对T24/DDP细胞增殖的影响

注：与miR-NC组比较，^aP＜0.05。

Figure 2. The effect of overexpression of miR-373-3p on the proliferation of T24/DDP cells

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图 3 抑制Cav1表达对T24/DDP细胞增殖的影响

注：与si-NC组比较，^aP＜0.05。

Figure 3. The effect of inhibiting Cav1 expression on the proliferation of T24/DDP cells

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图 4 各组T24/DDP细胞增殖曲线

注：与miR-373-3p mimic+pcDNA组比较，^aP＜0.05。

Figure 4. Proliferation curve of T24/DDP cells in each group

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表 1 miR-373-3p、Cav1在T24、T24/DDP细胞中的表达水平比较(x±s)

Table 1. Expression of miR-373-3p and Cav1 in T24 and T24/DDP cells (x±s)

细胞	n	miR-373-3p	Cav1
T24	6	1.00±0.18	0.41±0.06
T24/DDP	6	0.34±0.06	0.95±0.10
t值		8.521	11.342
P值		＜0.001	＜0.001

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表 2 过表达miR-373-3p对T24/DDP细胞DDP耐药性的影响(x±s)

Table 2. Overexpression of miR-373-3p reduces the resistance of T24/DDP cells to DDP (x±s)

组别	n	miR-373-3p	细胞凋亡率(%)	IC₅₀(μg/mL)	MRP	P-gp
对照组	6	1.00±0.00	5.45±0.87	17.76±2.34	1.15±0.13	1.01±0.11
miR-NC组	6	0.97±0.10	4.97±0.95	16.17±2.56	1.10±0.12	1.03±0.10
miR-373-3p mimic组	6	1.53±0.42^a	23.45±2.51^a	8.54±1.24^a	0.51±0.08^a	0.36±0.05^a
F值		9.583	250.923	32.230	60.493	106.317
P值		0.002	＜0.001	＜0.001	＜0.001	＜0.001
注：与miR-NC组比较，^aP＜0.05。

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表 3 抑制Cav1表达对T24/DDP细胞DDP耐药性的影响(x±s)

Table 3. Inhibition of Cav1 expression reduces the resistance of T24/DDP cells to DDP(x±s)

组别	n	细胞凋亡率(%)	IC₅₀(μg/mL)	Cav1	MRP	P-gp
对照组	6	4.95±0.82	16.78±2.31	0.85±0.07	1.10±0.13	0.98±0.10
si-NC组	6	5.13±0.96	15.46±2.52	0.87±0.08	1.07±0.12	1.01±0.13
si-Cav1组	6	20.62±2.24^a	7.68±1.52^a	0.23±0.04^a	0.47±0.08^a	0.33±0.05^a
F值		220.304	31.096	184.744	60.302	90.388
P值		＜0.001	＜0.001	＜0.001	＜0.001	＜0.001
注：与si-NC组比较，^aP＜0.05。

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表 4 过表达Cav1与miR-373-3p对T24/DDP细胞耐药性的影响(x±s)

Table 4. Effects of overexpression of Cav1 and miR-373-3p on drug resistance of T24/DDP cells(x±s)

组别	n	细胞凋亡率(%)	IC₅₀(μg/mL)	Cav1	MRP	P-gp
miR-373-3p mimic	6	22.48±2.92	7.45±1.37	0.25±0.04	0.48±0.07	0.35±0.05
miR-373-3p mimic+pcDNA组	6	23.56±2.87	8.23±1.41	0.23±0.03	0.52±0.08	0.33±0.04
miR-373-3p mimic+Cav1组	6	11.63±1.58^a	12.57±1.86^a	0.78±0.06^a	1.07±0.12^a	0.93±0.10^a
F值		40.688	18.701	287.115	76.132	148.256
P值		＜0.001	＜0.001	＜0.001	＜0.001	＜0.001
注：与miR-373-3p mimic+pcDNA组比较，^aP＜0.05。

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