miR-193a-3p通过靶向TRIM14对乳腺癌干细胞迁移和侵袭的影响

doi:10.19401/j.cnki.1007-3639.2025.11.002

摘要/Abstract

摘要：

背景和目的：乳腺癌干细胞在癌症的发生、发展中发挥重要作用，乳腺癌患者死亡率较高与癌症的复发及转移密切相关，然而乳腺癌干细胞的自我更新和分化能力能够引发化疗耐药性，进而影响肿瘤的复发和转移。本研究旨在探究miR-193a-3p通过靶向三结构域蛋白14（tripartite motif-containing protein 14，TRIM14）对乳腺癌干细胞迁移和侵袭的影响。 方法：选择人乳腺癌细胞T47D作为研究对象，随机分组分为Control组、NC mimics组（转染NC mimics）、miR-193a-3p mimics组（转染miR-193a-3p mimics）、miR-193a-3p mimics＋pcDNA-NC组（转染miR-193a-3p mimics+pcDNA-NC）和miR-193a-3p mimics＋pcDNA-TRIM14组（转染miR-193a-3p mimics+pcDNA-TRIM14）。采用流式细胞术分离干细胞并检测细胞成球能力；通过细胞计数试剂盒-8（cell counting kit-8，CCK-8）实验检测细胞增殖；使用transwell实验检测细胞迁移和侵袭；通过流式细胞术检测细胞凋亡率；采用蛋白质印迹法（Western blot）检测细胞中细胞周期蛋白D1（cyclin D1）、基质金属蛋白酶-2（matrix metalloproteinase-2，MMP-2）、Bcl-2相关X蛋白（Bcl-2-associated X protein，Bax）及TRIM14蛋白的表达；采用双萤光素酶报告基因实验检测miR-193a-3p与TRIM14的相互作用。 结果：T47D干细胞具有成球能力，并随着时间推移增强，T47D干细胞球体积逐渐增大。与Control组、NC mimics组相比，miR-193a-3p mimics组细胞miR-193a-3p表达、凋亡率、Bax蛋白表达升高（P<0.05），TRIM14 mRNA和蛋白表达、存活率、克隆数、迁移数、侵袭数、cyclin D1、MMP-2降低（P <0.05）。与miR-193a-3p mimics组、miR-193a-3p mimics＋pcDNA-NC组相比，miR-193a-3p mimics＋pcDNA-TRIM14组细胞凋亡率、Bax蛋白表达降低（P <0.05），TRIM14 mRNA和蛋白表达、存活率、克隆数、迁移数、侵袭数、cyclin D1、MMP-2升高（P<0.05）。miR-193a-3p与TRIM14之间存在多个结合位点。与miR-NC＋TRIM14-WT组相比，miR-193a-3p mimics＋TRIM14-WT组双萤光素酶活性明显降低（P<0.05）。 结论：miR-193a-3p可能通过抑制TRIM14表达，发挥抑制乳腺癌干细胞迁移和侵袭的作用。

关键词: 乳腺癌, 干细胞, miR-193a-3p, 三结构域蛋白14, 迁移, 侵袭

Abstract:

Background and purpose: Breast cancer stem cells play an important role in the occurrence and development of cancer. The high mortality of breast cancer patients is closely related to the recurrence and metastasis of cancer. However, the self-renewal and differentiation ability of breast cancer stem cells can lead to chemotherapy resistance, thus affecting the recurrence and metastasis of cancer. This study aimed to explore the impact of miR-193a-3p on the migration and invasion of breast cancer stem cells by targeting the tripartite motif-containing protein 14 (TRIM14). Methods: Human breast cancer cell T47D was randomly assigned into control group, NC mimics group (transfected with NC mimics), miR-193a-3p mimics group (transfected with miR-193a-3p mimics), miR-193a-3p mimics+pcDNA-NC group (transfected with miR-193a-3p mimics+pcDNA-NC) and miR-193a-3p mimics+pcDNA-TRIM14 group (transfected with miR-193a-3p mimics+pcDNA-TRIM14). Separation of stem cells using flow cytometry and detection of cell spheroidization ability were carried out. Cell counting kit-8 (CCK-8) experiment was used to detect cell proliferation. Transwell experiment was used to measure cell migration and invasion. Flow cytometry was used to detect cell apoptotic rate. Western blot was used to detect the expressions of cyclin D1, matrix metalloproteinase-2 (MMP-2), Bcl-2-associated X protein (Bax), and TRIM14 protein in cells. Dual luciferase assay was used to detect the interaction between miR-193a-3p and TRIM14. Results: T47D stem cells had the ability to form spheroids, and with increasing time, the spheroid volume of T47D stem cells gradually increased. Compared with the Control group and NC mimics group, the miR-193a-3p mimics group showed increased miR-193a-3p expression, apoptotic rate, and Bax protein expression (P<0.05), and decreased TRIM14 mRNA and protein expression, survival rate, clone number, migration number, invasion number, cyclin D1 and MMP-2 (P<0.05). Compared with the miR-193a-3p mimics group and the miR-193a-3p mimics+pcDNA NC group, the miR-193a-3p mimics+pcDNA-TRIM14 group showed decreased cell apoptosis rate and Bax protein (P<0.05), and increased TRIM14 mRNA and protein expression, survival rate, clone number, migration number, invasion number, cyclin D1 and MMP-2 (P<0.05). There were multiple binding sites between miR-193a-3p and TRIM14. Compared with the miR-NC+TRIM14-WT group, the miR-193a-3p mimics+TRIM14-WT group showed a prominent decrease in dual luciferase activity (P<0.05). Conclusion: MiR-193a-3p may inhibit the migration and invasion of breast cancer stem cells through inhibiting TRIM14.

Key words: Breast cancer, Stem cells, miR-193a-3p, Tripartite motif-containing protein 14, Migration, Invasion

中图分类号:

R737.9

王欣荣, 王佩显, 任海青, 王欢. miR-193a-3p通过靶向TRIM14对乳腺癌干细胞迁移和侵袭的影响[J]. 中国癌症杂志, 2025, 35(11): 1001-1009.

WANG Xinrong, WANG Peixian, REN Haiqing, WANG Huan. Impact of miR-193a-3p on migration and invasion of breast cancer stem cells through targeting TRIM14[J]. China Oncology, 2025, 35(11): 1001-1009.

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Gene	Forward Primer (5'-3' )	Reverse Primer (5'-3' )
miR-193a-3p	TTTGAGGGATATTTAGAGTTT	AACCTAAAAAACAACCTAACC
TRIM14	GGATTTGTGTCTCCGTTCTG	TCTGTCTGCCTGGTATTCTG
U6	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT
GAPDH	CACCCACTCCTCCACCTTTG	CCACCACCCTGTTGCTGTAG

Group	miR-193a-3p	TRIM14
Control group	1.00±0.12	1.00±0.11
NC mimics group	0.98±0.10	0.99±0.10
miR-193a-3p mimics group	1.75±0.22^ab	0.37±0.05^ab
miR-193a-3p mimics＋pcDNA-NC group	1.72±0.19	0.43±0.06
miR-193a-3p mimics＋pcDNA-TRIM14 group	1.73±0.21	0.86±0.09^cd
F value	43.437	77.893
P value	0.000	0.000

Group	survival rate(%)	Number of clones (pieces)
Control group	92.38±0.57	128.67±13.43
NC mimics group	87.52±9.25	127.49±13.06
miR-193a-3p mimics group	52.84±5.69^ab	67.42±7.38^ab
miR-193a-3p mimics＋pcDNA-NC group	51.04±5.22	68.94±7.44
miR-193a-3p mimics＋pcDNA-TRIM14 group	85.22±9.03^cd	115.82±12.34^cd
F value	53.552	46.983
P value	0.000	0.000

Group	Number of migrations (pieces)
Control group	135.62±14.92
NC mimics group	136.28±14.76
miR-193a-3p mimics group	62.54±7.38^ab
miR-193a-3p mimics＋pcDNA-NC group	64.28±7.14
miR-193a-3p mimics＋pcDNA-TRIM14 group	129.63±13.57^cd
F value	61.448
P value	0.000

Group	Number of invasions (pieces)
Control group	109.63±18.52
NC mimics group	105.62±11.27
miR-193a-3p mimics group	57.86±7.34^ab
miR-193a-3p mimics＋pcDNA-NC group	60.24±7.59
miR-193a-3p mimics＋pcDNA-TRIM14 group	95.38±10.05^cd
F value	27.324
P value	0.000