白皮杉醇调控miR-106b-5p/RUNX3轴对宫颈癌细胞迁移及侵袭的影响

doi:10.11958/20221529

天津医药 ›› 2023, Vol. 51 ›› Issue (8): 814-819.doi: 10.11958/20221529

白皮杉醇调控miR-106b-5p/RUNX3轴对宫颈癌细胞迁移及侵袭的影响

王玉宁¹(), 宋聚星², 田志刚³, 郝国荣¹, 申浩¹

1 石家庄市第四医院妇科（邮编050035）
2 保定市第二中心医院病理科
3 保定市第二中心医院检验科

收稿日期:2022-09-22 修回日期:2023-03-03 出版日期:2023-08-15 发布日期:2023-08-10
作者简介:王玉宁（1983），男，主治医师，主要从事妇科肿瘤方面研究。E-mail：wang_yn1983w@163.com
基金资助:
石家庄市科学技术研究与发展计划项目(191201233)

The effect of piceatannol on the migration and invasion of cervical cancer cells by regulating miR-106b-5p/RUNX3 axis

WANG Yuning¹(), SONG Juxing², TIAN Zhigang³, HAO Guorong¹, SHEN Hao¹

1 Department of Gynecology, Shijiazhuang No.4 Hospital, Shijiazhuang 050035, China
2 Department of Pathology, Baoding Second Central Hospital
3 Department of Clinical Laboratory, Baoding Second Central Hospital

Received:2022-09-22 Revised:2023-03-03 Published:2023-08-15 Online:2023-08-10

王玉宁, 宋聚星, 田志刚, 郝国荣, 申浩. 白皮杉醇调控miR-106b-5p/RUNX3轴对宫颈癌细胞迁移及侵袭的影响[J]. 天津医药, 2023, 51(8): 814-819.

WANG Yuning, SONG Juxing, TIAN Zhigang, HAO Guorong, SHEN Hao. The effect of piceatannol on the migration and invasion of cervical cancer cells by regulating miR-106b-5p/RUNX3 axis[J]. Tianjin Medical Journal, 2023, 51(8): 814-819.

摘要/Abstract

摘要：

目的探究白皮杉醇（PIC）调控微小RNA-106b-5p（miR-106b-5p）/RUNT相关转录因子3（RUNX3）轴对宫颈癌（CC）细胞迁移及侵袭的影响。方法使用不同浓度PIC（0、20、40、80和160 μmol/L）培养液处理人CC Hela细胞，通过CCK-8法检测PIC对细胞增殖活力的影响，以确定PIC最佳使用浓度。将Hela细胞分为Control组、PIC组、PIC+NC mimics组、PIC+miR-106b-5p mimics组、NC inhibitor组、miR-106b-5p inhibitor组、miR-106b-5p inhibitor+si-RNA组及miR-106b-5p inhibitor+si-RUNX3组。实时荧光定量PCR检测各组Hela细胞miR-106b-5p表达水平；Transwell法检测各组Hela细胞迁移及侵袭能力；Western blot法检测各组Hela细胞中RUNX3、基质金属蛋白酶（MMP）2和MMP9蛋白表达水平；双萤光素酶报告基因实验检测miR-106b-5p与RUNX3靶向关系。结果 Hela细胞增殖活力随着PIC处理浓度的增高而呈现降低趋势（P<0.05），其中80 μmol/L PIC对Hela细胞的抑制作用接近半数抑制浓度（IC₅₀），故选择80 μmol/L为后续研究的PIC浓度。与Control组相比，PIC组miR-106b-5p、MMP2和MMP9表达水平均降低，迁移和侵袭细胞数量减少，RUNX3表达水平增加（P<0.05）；与PIC+NC mimics组相比，PIC+miR-106b-5p mimics组miR-106b-5p、MMP2和MMP9表达水平增加，迁移和侵袭细胞数量增多，RUNX3表达水平降低（P<0.05）。双萤光素酶报告基因实验证实RUNX3为miR-106b-5p的靶基因。与NC inhibitor组相比，miR-106b-5p inhibitor组RUNX3表达水平增加，miR-106b-5p、MMP2与MMP9表达水平降低，迁移和侵袭细胞数量减少（P<0.05）；与miR-106b-5p inhibitor+si-RNA组相比，miR-106b-5p inhibitor+si-RUNX3组RUNX3表达水平降低，miR-106b-5p、MMP2和MMP9表达水平增加，迁移和侵袭细胞数量增多（P<0.05）。结论 PIC通过抑制miR-106b-5p表达、促进RUNX3表达来抑制CC细胞迁移及侵袭。

关键词: 微RNAs, 核心结合因子α3亚基, 宫颈肿瘤, 细胞运动, 肿瘤浸润, 白皮杉醇, miR-106b-5p, RUNT相关转录因子3

Abstract:

Objective To explore the effect of piceatannol (PIC) on migration and invasion of cervical cancer (CC) cells by regulating microRNA-106b-5p (miR-106b-5p)/RUNT-related transcription factor 3 (RUNX3) axis. Methods Firstly, human CC cells Hela were treated with culture media of different concentrations of PIC (0, 20, 40, 80 and 160 μmol/L), and the effect of PIC on cell proliferation was detected by CCK-8 assay to determine the optimal concentration of PIC. Hela cells were divided into the control group, the PIC group, the PIC+NC mimics group, the PIC+ miR-106b-5p mimics group, the NC inhibitor group, the miR-106b-5p inhibitor group, the miR-106b-5p inhibitor+si-RNA group and the miR-106b-5p inhibitor+si-RUNX3 group. qRT-PCR was used to detect the expression level of miR-106b-5p in Hela cells in each group. Transwell method was used to detect the migration and invasion abilities of Hela cells in each group. Western blot assay was used to detect the protein levels of RUNX3, MMP2 and MMP9 in Hela cells of each group. Dual luciferase reporter gene experiment was used to detect the targeting relationship between miR-106b-5p and RUNX3. Results The proliferation activity of Hela cells decreased with the increase of PIC treatment concentration (P<0.05). The inhibitory effect of 80 μmol/L PIC on Hela cells was nearly to half inhibitory concentration (IC₅₀), so 80 μmol/L was selected as the PIC concentration for subsequent study. Compared with the control group, the expression levels of miR-106b-5p, MMP2 and MMP9 decreased in the PIC group, the numbers of migrating and invasive cells decreased, and the expression level of RUNX3 increased (P<0.05). Compared with the PIC+NC mimics group, the expression levels of miR-106b-5p, MMP2 and MMP9 increased in the PIC+miR-106b-5p mimics group, numbers of migrating and invasive cells increased, and the expression level of RUNX3 decreased (P<0.05). Double luciferase reporter gene assay confirmed RUNX3 as the target gene of miR-106b-5p. Compared with the NC inhibitor group, the expression level of RUNX3 increased in the miR-106b-5p inhibitor group, and the expression levels of miR-106b-5p, MMP2 and MMP9 decreased, and numbers of migrating and invasive cells decreased (P<0.05). Compared with the miR-106b-5p inhibitor+si-RNA group, the miR-106b-5p inhibitor+si-RUNX3 group showed lower RUNX3 expression level, increased miR-106b-5p, MMP2 and MMP9 expression levels, and increased numbers of migrating and invading cells (P<0.05). Conclusion PIC inhibits the miR-106b-5p expression and promotes RUNX3 expression to inhibit CC cell migration and invasion.

Key words: microRNAs, core binding factor alpha 3 subunit, uterine cervical neoplasms, cell movement, neoplasm invasiveness, piceatannol, miRNA-106b-5p, RUNT-related transcription factor 3

中图分类号:

R285，R349.5

图/表 9

图1 不同浓度PIC对Hela细胞增殖活力的影响 a与0 μmol/L比较，b与20 μmol/L比较，c与40 μmol/L比较，d与80 μmol/L比较，P<0.05。

Fig.1 Effects of different concentrations of PIC on the proliferative activity of Hela cells

表1 各组Hela细胞中miR-106b-5p、RUNX3表达水平及迁移和侵袭能力比较（n=6，$\bar{x}±s$）

Tab.1 Comparison of miR-106b-5p and RUNX3 expression levels, migration and invasion ability between the four groups of Hela cells

组别	miR-106b-5p	RUNX3	迁移数量/（个/视野）	侵袭数量/（个/视野）	MMP2	MMP9
Control组	1.00±0.03	0.31±0.02	296.31±33.24	221.27±20.16	1.24±0.10	1.35±0.13
PIC组	0.24±0.04^a	1.12±0.10^a	94.20±4.67^a	64.38±8.17^a	0.37±0.04^a	0.42±0.04^a
PIC+NC mimics组	0.28±0.05^a	1.15±0.12^a	98.86±4.97^a	69.31±6.19^a	0.41±0.05^a	0.51±0.06^a
PIC+miR-106b-5p mimics组	0.63±0.07^c	0.71±0.08^c	183.31±15.83^c	137.29±11.54^c	0.79±0.08^c	0.92±0.09^c
F	304.869^＊＊	120.789^＊＊	153.635^＊＊	200.789^＊＊	192.263^＊＊	144.477^＊＊

图2 各组Hela细胞迁移和侵袭情况（结晶紫染色，×200）

Fig.2 Migration and invasion of Hela cells in each group (crystal violet staining, ×200)

图3 各组Hela细胞中RUNX3、MMP2和MMP9蛋白印迹图 A：Control组；B：PIC组；C：PIC+NC mimics组；D：PIC+miR-106b-5p mimics组。

Fig.3 Western blot results of RUNX3, MMP2 and MMP9 in Hela cells of each group

图4 miRTarBase网站预测miR-106b-5p与RUNX3靶向结合位点图

Fig.4 Targeted binding site map of miR-106b-5p and RUNX3 predicted by miRTarBase website

表2 2组RUNX3-WT、RUNX3-MUT荧光素酶活性比较（n=6，$\bar{x}±s$）

Tab.2 Comparison of luciferase activities of RUNX3-WT and RUNX3-MUT between the two groups

组别	RUNX3-WT	RUNX3-MUT
miR-NC组	1.00±0.01	1.01±0.02
miR-106b-5p mimics组	0.29±0.05	0.99±0.03
t	34.107^＊＊	1.359

表3 各组Hela细胞中RUNX3表达水平及迁移和侵袭能力比较（n=6，$\bar{x}±s$）

Tab.3 Comparison of RUNX3 expression levels and migration and invasion ability between the four groups of Hela cells

组别	miR-106b-5p	RUNX3	迁移数量/（个/视野）	侵袭数量/（个/视野）	MMP2	MMP9
NC inhibitor组	1.02±0.05	0.25±0.03	284.29±30.74	211.28±20.93	1.30±0.11	1.42±0.14
miR-106b-5p inhibitor组	0.32±0.04^a	0.99±0.08^a	86.14±3.20^a	51.27±7.38^a	0.31±0.04^a	0.37±0.03^a
miR-106b-5p inhibitor+si-RNA组	0.28±0.04^a	1.07±0.12^a	93.75±4.18^a	58.16±3.15^a	0.27±0.03^a	0.30±0.05^a
miR-106b-5p inhibitor+si-RUNX3组	0.57±0.06^c	0.63±0.05^c	171.23±13.16^c	143.14±11.65^c	0.85±0.07^c	1.05±0.10^c
F	298.559^＊＊	140.000^＊＊	177.429^＊＊	217.499^＊＊	294.677^＊＊	213.552^＊＊

图5 各组Hela细胞迁移和侵袭情况（结晶紫染色，×200） A：NC inhibitor组；B：miR-106b-5p inhibitor组；C：miR-106b-5p inhibitor+si-RNA组；D：miR-106b-5p inhibitor+si-RUNX3组。

Fig.5 Migration and invasion of Hela cells in each group (crystal violet staining, ×200)

图6 各组Hela细胞中RUNX3、MMP2和MMP9蛋白印迹图 A：NC inhibitor组；B：miR-106b-5p inhibitor组；C：miR-106b-5p inhibitor+si-RNA组；D：miR-106b-5p inhibitor+si-RUNX3组。

Fig.6 Western blot assay of RUNX3, MMP2 and MMP9 in Hela cells of each group

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白皮杉醇调控miR-106b-5p/RUNX3轴对宫颈癌细胞迁移及侵袭的影响

The effect of piceatannol on the migration and invasion of cervical cancer cells by regulating miR-106b-5p/RUNX3 axis

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