MicroRNA-34a通过调控Wnt途径影响慢性淋巴细胞白血病进展的机制探讨

doi:10.11958/20250458

天津医药 ›› 2025, Vol. 53 ›› Issue (8): 785-790.doi: 10.11958/20250458

• 细胞与分子生物学 • 下一篇

MicroRNA-34a通过调控Wnt途径影响慢性淋巴细胞白血病进展的机制探讨

刘虹(), 张玥玥, 王一琳, 王彩丽, 王晓敏, 毛敏, 李燕^△()

新疆维吾尔自治区人民医院血液病科（邮编830001）

收稿日期:2025-02-10 修回日期:2025-05-14 出版日期:2025-08-15 发布日期:2025-08-12
通讯作者: ^△E-mail：liyan232917@139.com
作者简介:刘虹（1976），女，副主任医师，主要从事血液肿瘤细胞遗传学和分子生物学方面的研究。E-mail：54240724@qq.com
基金资助:
新疆维吾尔自治区自然科学基金项目(2021D01C192)

The research on the mechanism of microRNA-34a influencing the progression of chronic lymphocytic leukemia by regulating the Wnt pathway

LIU Hong(), ZHANG Yueyue, WANG Yilin, WANG Caili, WANG Xiaomin, MAO Min, LI Yan^△()

Department of Hematology, People’s Hospital of Xinjiang Uygur Autonomous Region, Urumqi 830001, China

Received:2025-02-10 Revised:2025-05-14 Published:2025-08-15 Online:2025-08-12
Contact: ^△E-mail：liyan232917@139.com

刘虹, 张玥玥, 王一琳, 王彩丽, 王晓敏, 毛敏, 李燕. MicroRNA-34a通过调控Wnt途径影响慢性淋巴细胞白血病进展的机制探讨[J]. 天津医药, 2025, 53(8): 785-790.

LIU Hong, ZHANG Yueyue, WANG Yilin, WANG Caili, WANG Xiaomin, MAO Min, LI Yan. The research on the mechanism of microRNA-34a influencing the progression of chronic lymphocytic leukemia by regulating the Wnt pathway[J]. Tianjin Medical Journal, 2025, 53(8): 785-790.

摘要/Abstract

摘要：

目的探讨微小RNA（miRNA）-34a通过Wnt途径影响慢性淋巴细胞白血病（CLL）疾病进展的作用机制。方法选用人慢性B细胞白血病细胞MEC-1。实验一分组：p53激动剂组和Control组；实验二分组：Control组、miR-34a-5p mimic组及对照组、miR-34a-5p inhibitor组及对照组、miR-34a-5p inhibitor+Wnt抑制剂XAV-939组。采用实时荧光定量PCR（qPCR）检测各组细胞中miR-34a-5p表达水平；CCK8检测细胞增殖能力；Transwell迁移实验检测细胞迁移能力；双萤光素酶报告实验检测p53与miR-34a-5p及miR-34a-5p与Wnt1的靶向关系；Western blot检测Wnt/β-catenin通路相关蛋白β-连环蛋白（β-catenin）、细胞周期蛋白D1（Cyclin D1）的表达。结果在MEC-1细胞中：① p53激动剂组中miR-34a表达升高，增殖受抑制（P<0.05）；双萤光素酶报告实验证实miR-34a-5p与p53之间呈现负调控相关性。② miR-34a-5p mimic组miR-34a-5p表达升高，增殖受抑制，迁移能力降低，β-catenin、Cyclin D1蛋白表达下调（P<0.05）；miR-34a-5p inhibitor组miR-34a-5p表达降低，增殖升高，迁移能力增强，β-catenin、Cyclin D1蛋白表达上调（P<0.05）。③ miR-34a-5p和Wnt1呈现负调控相关性。④ 与miR-34a-5p inhibitor组相比，XAV-939组的miR-34a-5p表达升高，迁移细胞数减少，β-catenin和Cyclin D1蛋白表达量显著降低（P<0.05）。结论 miR-34a在CLL中扮演着抑癌基因的角色，过表达miR-34a可抑制Wnt/β-catenin信号通路，降低细胞的增殖活性和迁移能力，促进细胞凋亡。

关键词: 白血病, B细胞, Wnt信号通路, 肿瘤抑制蛋白质p53, 细胞增殖, microRNA-34a

Abstract:

Objective To investigate the effects of microRNA-34a (miR-34a-5p) on the progression of chronic lymphocytic leukemia (CLL) through the Wnt/β-catenin signaling pathway. Methods Human chronic B-cell leukemia MEC-1 cells were selected for experimentation. MEC cells were divided into two groups, group one: the p53 agonist group and the control group; group two: the control group, the miR-34a-5p mimic group and its corresponding negative control, the miR-34a-5p inhibitor group and its corresponding negative control, as well as the miR-34a-5p inhibitor + Wnt inhibitor XAV-939 group. The expression levels of miR-34a-5p in each group were measured using real-time fluorescence quantitative PCR (qPCR). Cell proliferation was assessed by CCK-8 assay, while cell migration ability was evaluated using Transwell migration assay. Dual-luciferase reporter assay was employed to validate the targeting relationships between p53 and miR-34a-5p, as well as between miR-34a-5p and Wnt1. Western blot analysis was used to detect the protein expressions of β-catenin and Cyclin D1, which were key components of the Wnt/β-catenin signaling pathway. Results In MEC-1 cells: ① compared with the control group, there was a increased miR-34a-5p expression and inhibited cell proliferation in the p53 agonist group (P<0.05). Dual-luciferase reporter assay confirmed a negative regulatory correlation between miR-34a-5p and p53. ② the miR-34a-5p mimic group showed significantly upregulated miR-34a-5p expression compared to the control group, leading to suppressed cell proliferation, reduced migration capability and decreased protein expressions of β-catenin and Cyclin D1 (P<0.05). Conversely, the miR-34a-5p inhibitor group demonstrated significantly downregulated miR-34a-5p expression, resulting in enhanced cell proliferation, increased migratory capacity and upregulated protein levels of β-catenin and Cyclin D1 compared to those of the control group (P<0.05). ③ A targeting relationship was observed between miR-34a-5p and Wnt1. ④ Compared with the miR-34a-5p inhibitor group, the XAV-939 group exhibited significantly upregulated miR-34a-5p expression, markedly decreased numbers of migrated cells and substantially reduced protein expression levels of β-catenin and Cyclin D1 (P<0.05). Conclusion miR-34a plays the role of a tumor suppressor gene in CLL. Overexpression of miR-34a can inhibit the Wnt/β-catenin signaling pathway, reduce the proliferation activity and migration ability of cells, and promote cell apoptosis.

Key words: leukemia, B-cell, Wnt signaling pathway, tumor suppressor protein p53, cell proliferation, microRNA-34a

中图分类号:

R733.72

图/表 11

表1 引物序列

Tab.1 The sequence of primer

基因名称	引物序列（5′→3′）	产物大小/bp
miR-34a-5p	上游：TGGCAGTGTCTTAGCTGGTTG	58
miR-34a-5p	下游：CTCAACTGGTGTCGTGGAGTC	58
U6	上游：CTCGCTTCGGCAGCACAT	94
U6	下游：AACGCTTCACGAATTTGCGT	94
p53	上游：CCGTGTAAAGATCCGGTACC CATTCTCCACTTCTTGTTCC	540
p53	下游：TCCTCGAGGATATCGGATCC GGTCAAGTTCTAGACCCCAT	540
Wnt1	上游：CCGTGTAAAGATCCGGTACCA CAGACTCGCTAGCACTCAA	540
Wnt1	下游：TCCTCGAGGATATCGGATCCTC ATTTCCACATCATCACAG	540

表2 Control组和p53激动剂组细胞增殖率和miR-34a-5p表达水平比较

Tab.2 Comparison of cell proliferation rate and miR-34a-5p expression levels between the control group and the p53 agonist group （n=3，$\bar{x}±s$）

组别	细胞增殖率/%	miR-34a-5p
Control组	102.31±7.75	1.02±0.02
p53激动剂组	83.16±3.89	2.06±0.10
t	3.824^＊	16.930^＊＊

图1 miR-34a-5p与p53 3′-UTR结合位点

Fig.1 The binding site of miR-34a-5p on the 3'-UTR of p53

表3 miR-34a-5p与p53的靶向关系验证结果

Tab.3 Validation results of the targeting relationship between miR-34a-5p and p53 （n=3，$\bar{x}±s$）

组别	p53 WT	p53 MUT
miR-34a-5p mimics NC组	1.11±0.04	1.12±0.10
miR-34a-5p mimics组	0.85±0.06	1.11±0.03
t	6.462^＊＊	0.169

表4 过表达和抑制miR-34a-5p对MEC-1细胞增殖、迁移能力的影响

Tab.4 The effects of overexpression and inhibition of miR-34a-5p on the proliferation and migration abilities of MEC-1 cells （n=3，$\bar{x}±s$）

组别	miR-34a-5p	细胞增殖率/%	迁移细胞数/（个/视野）
Control组	1.03±0.08	104.91±4.62	238.80±17.94
miR-34a-5p mimics NC组	1.06±0.08	103.86±7.33	234.20±16.72
miR-34a-5p mimics组	1.81±0.09^ab	64.66±6.44^ab	69.40±5.64^ab
miR-34a-5p inhibitor NC组	1.06±0.04	106.83±5.63	242.40±11.74
miR-34a-5p inhibitor组	0.46±0.01^ac	122.64±8.25^ac	299.20±8.23^ac
F	147.682^＊＊	32.006^＊＊	223.252^＊＊

图2 Transwell实验观察各组MEC-1细胞迁移情况

Fig.2 The migration of MEC-1 cells in each group observed by Transwell assay

图3 miR-34a-5p与Wnt1 3’UTR结合位点

Fig.3 The binding site of miR-34a-5p on the 3'UTR of Wnt1

表5 miR-34a-5p与Wnt1靶向关系验证结果

Tab.5 Validation results of the targeting relationship between miR-34a-5p and Wnt1 （n=3，$\bar{x}±s$）

组别	Wnt1 WT	Wnt1 MUT
miR-34a-5p mimics NC组	1.08±0.03	1.12±0.02
miR-34a-5p mimics组	0.62±0.06	1.14±0.06
t	12.796^＊＊	0.371

表6 XAV-939调控miR-34a-5p表达及其对MEC-1细胞增殖、迁移能力的影响

Tab.6 Regulation of the expression of miR-34a-5p by XAV-939 and its effects on the proliferation and migration of MEC-1 cells （n=3，$\bar{x}±s$）

组别	miR-34a-5p	细胞增殖率/%	迁移细胞数/（个/视野）
Control组	1.03±0.08	104.91±4.62	238.80±17.94
miR-34a-5p inhibitor NC组	1.06±0.04	106.83±5.63	242.40±11.74
miR-34a-5p inhibitor组	0.46±0.01^ab	122.64±8.25^ab	299.20±8.23^ab
miR-34a-5p inhibitor+XAV-939组	0.81±0.09^ac	111.78±3.61	184.60±3.21^ac
F	59.106^＊＊	5.649^＊	81.547^＊＊

图4 Western blot检测各组MEC-1细胞β-catenin、Cyclin D1蛋白表达变化 A：Control组；B：miR-34a-5p mimics组；C：miR-34a-5p mimics NC组；D：miR-34a-5p inhibitor组；E：miR-34a-5p inhibitor NC组；F：miR-34a-5p inhibitor+XAV-939组。

Fig.4 The protein expressions levels of β-catenin and Cyclin D1 in MEC-1 cells of each group detected by Western blot assay

表7 各组细胞β-catenin、Cyclin D1的蛋白表达比较

Tab.7 Comparison of the protein expression levels of β-catenin and Cyclin D1 between the six groups （n=3，$\bar{x}±s$）

组别	β-catenin	Cyclin D1
Control组	0.50±0.01	0.52±0.01
miR-34a-5p mimics NC组	0.47±0.02	0.50±0.03
miR-34a-5p mimics组	0.15±0.02^ab	0.12±0.01^ab
miR-34a-5p inhibitor NC组	0.50±0.01	0.53±0.03
miR-34a-5p inhibitor组	0.86±0.01^ac	0.94±0.02^ac
miR-34a-5p inhibitor+XAV-939组	0.68±0.01^d	0.66±0.04^d
F	911.200^＊＊	366.803^＊＊

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MicroRNA-34a通过调控Wnt途径影响慢性淋巴细胞白血病进展的机制探讨

The research on the mechanism of microRNA-34a influencing the progression of chronic lymphocytic leukemia by regulating the Wnt pathway

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