CircBACH1调节miR-140-5p/MDM2轴对多发性骨髓瘤细胞增殖、凋亡和化疗敏感性的影响

doi:10.11958/20230126

天津医药 ›› 2023, Vol. 51 ›› Issue (11): 1170-1175.doi: 10.11958/20230126

CircBACH1调节miR-140-5p/MDM2轴对多发性骨髓瘤细胞增殖、凋亡和化疗敏感性的影响

刘荟敏(), 许旋旋, 王远丽, 熊涛, 唐元艳^△()

荆州市中心医院血液内科（邮编434020）

收稿日期:2023-02-03 修回日期:2023-04-07 出版日期:2023-11-15 发布日期:2023-11-07
通讯作者: ^△E-mail：louzte05236@163.com
作者简介:刘荟敏（1984），女，主治医师，主要从事白血病和骨髓造血干细胞方面研究。E-mail：bbp3ldn@163.com
基金资助:
湖北省卫生计生委科研项目(WJ2019F125)

Impacts of CircBACH1 on proliferation, apoptosis and chemosensitivity of multiple myeloma cells by regulating miR-140-5p /MDM2 axis

LIU Huimin(), XU Xuanxuan, WANG Yuanli, XIONG Tao, TANG Yuanyan^△()

Department of Hematology, Jingzhou Central Hospital, Jingzhou 434020, China

Received:2023-02-03 Revised:2023-04-07 Published:2023-11-15 Online:2023-11-07
Contact: ^△E-mail：louzte05236@163.com

刘荟敏, 许旋旋, 王远丽, 熊涛, 唐元艳. CircBACH1调节miR-140-5p/MDM2轴对多发性骨髓瘤细胞增殖、凋亡和化疗敏感性的影响[J]. 天津医药, 2023, 51(11): 1170-1175.

LIU Huimin, XU Xuanxuan, WANG Yuanli, XIONG Tao, TANG Yuanyan. Impacts of CircBACH1 on proliferation, apoptosis and chemosensitivity of multiple myeloma cells by regulating miR-140-5p /MDM2 axis[J]. Tianjin Medical Journal, 2023, 51(11): 1170-1175.

摘要/Abstract

摘要：

目的探讨CircBACH1对多发性骨髓瘤（MM）细胞增殖、凋亡和化疗敏感性的影响以及在此过程中对miR-140-5p/MDM2轴的调节机制。方法收集MM组和正常对照组骨髓浆细胞，将人MM细胞系U266细胞分为未转染（Control）组、si-NC组、si1-CircBACH1组、si2-CircBACH1组、si-CircBACH1+anti-miR-NC组、si-CircBACH1+anti-miR-140-5p组。实时荧光定量PCR检测细胞中CircBACH1、miR-140-5p、鼠双微体2（MDM2）mRNA的表达；CCK-8法检测细胞增殖活性；流式细胞术检测细胞凋亡及对硼替佐米的敏感性；Western blot检测细胞中B细胞淋巴因子2（Bcl-2）、Bcl-2相关X蛋白（Bax）、裂解的胱天蛋白酶（cleaved caspase）-3蛋白的表达；双萤光素酶报告基因实验验证CircBACH1、miR-140-5p、MDM2三者的靶向关系。结果与正常对照组相比，MM组的骨髓浆细胞及U266细胞中CircBACH1、MDM2 mRNA表达升高，miR-140-5p表达降低（P<0.05）；与Control组和si-NC组相比，si-CircBACH1组细胞中CircBACH1、MDM2的mRNA表达、细胞增殖活性、Bcl-2蛋白表达均降低，miR-140-5p的表达、细胞凋亡率、化疗敏感性及cleaved caspase-3、Bax表达均升高（P<0.05）；而回补实验中，si-CircBACH1对MM细胞的影响被anti-miR-140-5p逆转，且MDM2的表达也升高（P<0.05）。双萤光素酶基因报告实验验证了CircBACH1、MDM2均与miR-140-5p存在靶向关系。结论敲低CircBACH1能够上调miR-140-5p表达，下调MDM2的表达，抑制MM细胞的增殖，促进MM细胞凋亡，提高化疗敏感性。

关键词: 多发性骨髓瘤, 细胞增殖, 细胞凋亡, CircBACH1, miR-140-5p, MDM2轴, 化疗敏感性

Abstract:

Objective To investigate the effect of CircBACH1 on proliferation, apoptosis and chemotherapy sensitivity of multiple myeloma (MM) cells and the regulatory mechanism on miR-140-5p/MDM2 axis during this process. Methods Bone marrow plasma cells were collected from the MM patient group and normal control group. Human MM cell line U266 cells were divided into the untransfected group, the si-NC group, the si1-CircBACH1 group, the si2-CircBACH1 group, the si-CircBACH1+anti-miR-NC group and the si-CircBACH1+anti-miR-140-5p group. The mRNA expressions of CircBACH1, miR-140-5p and mouse double microsoma 2 (MDM2) were detected by real-time quantitative PCR. Cell proliferation activity was detected by CCK-8 assay. Flow cytometry was used to detect apoptosis and sensitivity to bortezomib. The expression levels of B-cell lymphofactor 2 (Bcl-2), Bcl-2 associated X protein (Bax) and cleaved caspase -3 were detected by Western blot assay. Dual luciferase reporter gene assay was used to verify the targeting relationship of CircBACH1, miR-140-5p and MDM2. Results Compared with the normal control group, the mRNA expression levels of CircBACH1 and MDM2 were increased in bone marrow plasma cells and U266 cells in the MM group, while the expression of miR-140-5p was decreased (P<0.05). Compared with the control group and the si-NC group, the mRNA expression of CircBACH1 and MDM2, cell proliferation activity and Bcl-2 protein expression were decreased in the transfected si-CircBACH1 group. The expression of miR-140-5p, cell apoptosis rate, chemotherapy sensitivity, cleaved caspase-3 and Bax expressions were increased (P<0.05). In the anti-miR-140-5p supplementation experiment, the effect of si-CircBACH1 on MM cells was reversed, and the expression of MDM2 was increased (P<0.05). Dual luciferase gene reporting experiment verified the targeting relationship between CircBACH1 and MDM2 and miR-140-5p. Conclusion Knock down of CircBACH1 can up-regulate the expression of miR-140-5p, down-regulate the expression of MDM2, inhibit the proliferation of MM cells, promote the apoptosis of MM cells and improve the chemosensitivity.

Key words: multiple myeloma, cell proliferation, apoptosis, CircBACH1, miR-140-5p, mouse double minute 2, chemosensitivity

中图分类号:

R733.3

图/表 8

表1 qRT-PCR引物

Tab.1 qRT-PCR primers

基因名称	引物序列（5′→3′）	产物大小/bp
CircBACH1	上游：GCTGTCGCAAGAGAAAACTTGA	198
CircBACH1	下游：AAACTCCACACATTTGCACACT	198
MDM2	上游：CAGTAGCAGTGAATCTACAGGGA	139
MDM2	下游：CTGATCCAACCAATCACCTGAAT	139
GAPDH	上游：CAGAACATCATCCCTGCCTCTAC	216
GAPDH	下游：TTGAAGTCAGAGGAGACCACCTG	216
miR-140-5p	上游：CAGTGGTTTTACCCTA	142
miR-140-5p	下游：GTGCAGGGTCCGAGGT	142
U6	上游：CTCGCTTCGGCAGCACA	89
U6	下游：AACGCTTCACGAATTTGCG	89

表2 各组CircBACH1、miR-140-5p、MDM2 mRNA表达水平比较（n=18，$\bar{x}±s$）

Tab.2 Comparison of mRNA expression levels of CircBACH1, miR-140-5p and MDM2 between three groups

组别	CircBACH1	miR-140-5p	MDM2
正常对照组	1.02±0.07	0.98±0.06	0.96±0.05
MM组	1.97±0.15^a	0.37±0.03^a	1.61±0.11^a
U266细胞	2.01±0.12^a	0.34±0.03^a	1.68±0.10^a
F	405.689^＊＊	1304.333^＊＊	346.024^＊＊

表3 各转染组细胞中CircBACH1、miR-140-5p、MDM2 mRNA表达水平比较（n=3，$\bar{x}±s$）

Tab.3 Comparison of mRNA expression levels of CircBACH1, miR-140-5p and MDM2 between five groups of cells

组别	CircBACH1	miR-140-5p	MDM2
Control组	1.00±0.00	1.00±0.00	1.00±0.00
si-NC组	0.98±0.05	1.02±0.05	1.01±0.06
si-CircBACH1组	0.46±0.03^ab	1.93±0.13^ab	0.39±0.02^ab
si-CircBACH1+anti- miR-NC组	0.44±0.02	1.91±0.14	0.41±0.03
si-CircBACH1+anti- miR-140-5p组	0.46±0.03	1.32±0.08^c	0.77±0.05^c
F	276.128^＊＊	69.823^＊＊	187.459^＊＊

图1 流式细胞术检测各组细胞的凋亡率

Fig.1 Apoptosis of cells in each group detected by flow cytometry

图2 流式细胞术检测加入硼替佐米后各组细胞的凋亡率

Fig.2 The apoptosis of cells in each group after adding bortezomib detected by flow cytometry

图3 各组细胞中Bcl-2、Bax、cleaved caspase-3蛋白表达情况 A：Control组；B：si-NC组；C：si-CircBACH1组；D：si-CircBACH1+anti-miR-NC组；E：si-CircBACH1+anti-miR-140-5p组。

Fig.3 Bcl-2, Bax, cleaved caspase-3 protein expression in each group

表4 各组细胞中Bcl-2、Bax、cleaved caspase-3蛋白表达水平比较（n=3，$\bar{x}±s$）

Tab.4 Comparison of expression levels of Bcl-2, Bax, and cleaved caspase-3 between five groups of cells

组别	Bcl-2	Bax	cleaved caspase-3
Control组	1.02±0.05	0.97±0.04	0.92±0.04
si-NC组	1.01±0.05	0.95±0.04	0.93±0.05
si-CircBACH1组	0.41±0.02^ab	1.52±0.06^ab	1.44±0.06^ab
si-CircBACH1+anti- miR-NC组	0.42±0.02	1.51±0.06	1.45±0.07
si-CircBACH1+anti- miR-140-5p组	0.72±0.04^c	1.21±0.05^c	1.17±0.05^c
F	182.493	89.791	67.222

图4 miR-140-5p与CircBACH1、MDM2之间的靶向结合位点

Fig.4 Targeted binding sites between miR-140-5p and CircBACH1 and MDM2

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CircBACH1调节miR-140-5p/MDM2轴对多发性骨髓瘤细胞增殖、凋亡和化疗敏感性的影响

Impacts of CircBACH1 on proliferation, apoptosis and chemosensitivity of multiple myeloma cells by regulating miR-140-5p /MDM2 axis

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