miR-186-5p靶向MTDH抑制HER2阳性乳腺癌移植瘤生长的实验研究

miR-186-5p suppresses the growth of HER2-positive breast cancer xenografts by targeting MTDH

摘要:

背景与目的 人表皮生长因子受体2（HER2）阳性乳腺癌侵袭性强、预后较差，微小RNA（miRNA）在其发生发展中发挥重要调控作用。miR-186-5p作为新近报道的肿瘤相关miRNA，其在HER2阳性乳腺癌中的作用机制尚未明确。本研究旨在探讨miR-186-5p对异黏蛋白（MTDH）的靶向调控关系及其对HER2阳性乳腺癌移植瘤生长的影响。方法 采用TargetScan预测miR-186-5p与MTDH的靶向结合位点，并通过双荧光素酶报告实验验证。构建miR-186-5p过表达、MTDH过表达及联合转染的BT-474细胞系，检测各组miR-186-5p与MTDH的mRNA和蛋白表达。将转染后的细胞接种于裸鼠皮下建立HER2阳性乳腺癌移植瘤模型，动态观察肿瘤生长情况，并于第28天测定肿瘤体积与质量，同时检测移植瘤组织中miR-186-5p和MTDH的表达水平。结果 双荧光素酶报告实验表明，miR-186-5p可直接靶向结合MTDH mRNA的3'-UTR。与阴性对照组比较，miR-186-5p过表达可明显下调MTDH mRNA及蛋白表达，而MTDH过表达可上调其表达，并可部分逆转miR-186-5p对MTDH的抑制作用（均P<0.05）。体内实验结果显示，miR-186-5p过表达组移植瘤的生长速度、肿瘤体积及质量均明显低于阴性对照组，MTDH过表达组则明显升高（均P<0.05），而miR-186-5p与MTDH联合转染组与阴性对照组差异无统计学意义（均P>0.05）。体内实验进一步证实，在移植瘤组织中，miR-186-5p过表达组miR-186-5p水平明显升高，MTDH的mRNA及蛋白表达明显下调；MTDH过表达组MTDH表达显著升高；联合转染组MTDH表达与阴性对照组差异无统计学意义（均P>0.05）。免疫组化结果与分子检测结果一致。结论 miR-186-5p可通过靶向抑制MTDH的表达，显著抑制HER2阳性乳腺癌小鼠移植瘤的生长，二者可能构成HER2阳性乳腺癌的重要调控通路，为其分子靶向治疗提供了新的实验依据。

Abstract:

Background and Aims Human epidermal growth factor receptor 2 (HER2)-positive breast cancer is characterized by high aggressiveness and poor prognosis. MicroRNAs (miRNAs) play critical roles in tumor progression. miR-186-5p has recently been identified as a tumor-related miRNA, but its function in HER2-positive breast cancer remains unclear. This study aimed to investigate the regulatory relationship between miR-186-5p and metadherin (MTDH) and its effect on tumor growth in a HER2-positive breast cancer xenograft model.Methods The potential binding site between miR-186-5p and MTDH was predicted by TargetScan and verified by dual-luciferase reporter assay. BT-474 cells were transfected with miR-186-5p mimic, MTDH overexpression plasmid, or both. The mRNA and protein levels of miR-186-5p and MTDH were detected. Xenograft models were established by subcutaneous inoculation of transfected cells into nude mice. Tumor volume and weight were measured on day 28, and the expression of miR-186-5p and MTDH in tumor tissues was analyzed.Results Dual-luciferase reporter assay demonstrated that miR-186-5p directly targets the 3'-UTR of MTDH mRNA. Compared with the negative control group, overexpression of miR-186-5p significantly downregulated the mRNA and protein expression of MTDH, whereas MTDH overexpression upregulated its expression and partially reversed the inhibitory effect of miR-186-5p on MTDH (all P<0.05). In vivo, tumor growth rate, volume, and weight were significantly reduced in the miR-186-5p overexpression group but significantly increased in the MTDH overexpression group compared with the negative control group (all P<0.05), while no significant differences were observed in the miR-186-5p + MTDH co-transfection group (all P>0.05). Further in vivo analysis revealed that miR-186-5p expression was markedly increased, whereas MTDH mRNA and protein expression were significantly decreased in xenograft tumors of the miR-186-5p overexpression group; MTDH expression was significantly elevated in the MTDH overexpression group; and no significant difference in MTDH expression was observed between the co-transfection group and the negative control group (all P>0.05). Immunohistochemical findings were consistent with the molecular results.Conclusion miR-186-5p suppresses the growth of HER2-positive breast cancer xenografts by targeting and downregulating MTDH, suggesting that the miR-186-5p/MTDH axis may serve as a potential therapeutic target for HER2-positive breast cancer.