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

doi:10.11958/20250458

Abstract

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

CLC Number:

R733.72

Figures/Tables 11

References 25

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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

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

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^＊＊

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^＊＊

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