南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (10): 1517-1525.doi: 10.12122/j.issn.1673-4254.2022.10.11

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miR-4324与Talin2在乳腺癌中高表达

高 鹏，朱海涛，裴文浩，许培海，丁勇兴   

  1. 蚌埠医学院附属蚌埠市第三人民医院普外科，安徽 蚌埠 233099；蚌埠医学院癌症转化医学安徽省重点实验室，安徽 蚌埠 233030
  • 出版日期:2022-10-20 发布日期:2022-10-31

Expression of miR-4324 and its targeted gene Talin2 in breast cancer

GAO Peng, ZHU Haitao, PEI Wenhao, XU Peihai, DING Yongxing   

  1. Department of General Surgery, Bengbu Third People's Hospital Affiliated to Bengbu Medical College, Bengbu 233099, China; Anhui Provincial Key Laboratory of Translational Cancer Research, Bengbu Medical College, Bengbu 233030, China
  • Online:2022-10-20 Published:2022-10-31

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摘要： 目的 探讨miR-4324对踝蛋白2（Talin2）调控和乳腺癌细胞的影响及临床意义。方法 利用免疫组化法检验Talin2在乳腺癌组织（BCT）和对应癌旁乳腺组织（PCBT）的表达，结合病理资料分析Talin2与乳腺癌预后及病理特征间的关联；qRT-PCR法测定miR-4324在BCT和PCBT的表达；人乳腺癌细胞株（SKBR-3）体外培养，分为Control对照组（正常培养)及相关转染组（转染相关片段）：miR-4324 mimics组；miR-4324 inhibitor组；miR-4324 NC组；si-Talin2组；miR-4324 inhibitor+si-Talin2组。通过细胞增殖、凋亡、迁移和侵袭实验，检测SKBR-3的生物学活性改变；通过qRT-PCR及Western-blot实验测定Talin2的表达；利用荧光素酶活性实验验证miR-4324对Talin2靶向表达；利用Transwell回复实验验证miR-4324调控Talin2影响SKBR-3细胞的迁移能力。结果 Talin2在BCT中的表达高于PCBT，并与淋巴结转移、HER-2的高表达有关（P<0.05），与年龄、临床分期、组织学分级及雌、孕激素受体（ER、PR）的表达间无明显相关性（P>0.05）；miR-4324在BCT中表达较PCBT降低（P<0.01）；与Control组比较，miR-4324 mimics组SKBR-3细胞增殖降低，凋亡增多（P<0.01），迁移和侵袭能力降低（P<0.05）；双荧光素酶报告基因检测证实，相对于Control组，miR-4324 mimics共转染可显著降低Talin2-3'-UTR WT报告质粒的荧光素酶活性（P<0.05）；miR-4324 mimics组中Talin2的Mrna和蛋白表达较Control组降低（P<0.05）；Transwell迁移实验结果显示，相对于Control组，SKBR-3细胞的迁移能力在miR-4324 inhibitor组中上升（P<0.01）；si-Talin2组中下降（P<0.01）；miR-4324 inhibitor+si-Talin2组中居中（P<0.05）。结论 Talin2高表达与乳腺癌患者淋巴结转移及HER-2过表达相关，下调miR-4324能抑制乳腺癌细胞的增殖、侵袭与迁移，并诱导凋亡，其中抑制迁移可能是其靶向抑制Talin2表达实现的。

关键词: 乳腺癌；miR-4324；踝蛋白2

Abstract: Objective To investigate the regulatory effect of miR-4324 on ankyrin 2 (Talin2) expression and biological behaviors of breast cancer cells and the clinical implications of changes in miR-4324 and Talin2 expressions in breast cancer. Methods In breast cancer and adjacent tissues, the expressions of Talin2 and miR-4324 were examined with immunohistochemistry and qRT-PCR, respectively and the association of Talin2 expression levels with the prognosis and clinicopathological features of breast cancer patients was analyzed. The human breast cancer cell line SKBR-3 was transfected with miR-4324 mimic, miR-4324 inhibitor, si-Talin2, or both miR-4324 inhibitor and si-Talin2, and the changes in biological behaviors of the cells were examined; the cellular expression of Talin2at the mRNA and protein levels were detected with qRT-PCR and Western blotting. Dual luciferase reporter gene assay was used to verify the targeting relationship between miR-4324 and Talin2. The effect of miR-4324-mediated regulation of Talin2 on SKBR-3 cell migration was assessed using Transwell assays. Results Talin2 expression was significantly higher in breast cancer tissues than in the adjacent tissues, and its expression level was correlated with lymph node metastasis and high HER-2 expression in breast cancer (P<0.05) but not with the patient's age, clinical stage, histological grade or expressions of estrogen and progesterone receptors (P>0.05). The expression of miR-4324 was significantly reduced in breast cancer tissues as compared with the adjacent tissues (P<0.01). In SKBR-3 cells, transfection with miR-4324 mimics significantly inhibited proliferation, migration and invasion (P<0.05) and promoted apoptosis (P<0.01) of the cells. Dual luciferase reporter gene assay confirmed that cotransfection with miR-4324 mimics significantly reduced luciferase activity of Talin2-3'-UTR WT reporter plasmid (P<0.05). Transfection of the cells with miR-4324 mimics significantly reduced mRNA and protein expressions of Talin2 (P<0.05). Transwell migration assay showed that the migration ability of SKBR-3 cells was significantly enhanced after transfection with miR- 4324 inhibitor (P<0.01), lowered after transfection with si-Talin2 (P<0.01), and maintained at the intermediate level after co-transfection with miR-4324 inhibitor + si-Talin2 group (P<0.05). Conclusions High expression of Talin2 is associated with lymph node metastasis and HER-2 overexpression in breast cancer patients. Down-regulation of miR-4324 inhibits the proliferation, invasion and migration and induces apoptosis of breast cancer cells, and the inhibitory effect of miR-4324 knockdown on breast cancer cell migration is mediated probably by targeted inhibition of Talin2 expression.

Key words: breast cancer; miR-4324; Talin2