Tumor Suppressive Function of mir-205 in Breast Cancer Is Linked to HMGB3 Regulation

Identifying targets of dysregulated microRNAs (miRNAs) will enhance our understanding of how altered miRNA expression contributes to the malignant phenotype of breast cancer. The expression of miR-205 was reduced in four breast cancer cell lines compared to the normal-like epithelial cell line MCF10A and in tumor and metastatic tissues compared to adjacent benign breast tissue. Two predicted binding sites for miR-205 were identified in the 3’ untranslated region of the high mobility group box 3 gene, HMGB3. Both dual-luciferase reporter assay and Western blotting confirmed that miR-205 binds to and regulates HMGB3. To further explore miR-205 targeting of HMGB3, WST-1 proliferation and in vitro invasion assays were performed in MDA-MB-231 and BT549 cells transiently transfected with precursor miR-205 oligonucleotide or HMGB3 small interfering RNA (siRNA). Both treatments reduced the proliferation and invasion of the cancer cells. The mRNA and protein levels of HMGB3 were higher in the tumor compared to adjacent benign specimens and there was an indirect correlation between the expression of HMGB3 mRNA and patient survival. Treatment of breast cancer cells with 5-Aza/TSA derepressed miR-205 and reduced HMGB3 mRNA while knockdown of the transcriptional repressor NRSF/REST, reduced miR-205 and increased HMGB3. In conclusion, regulation of HMGB3 by miR-205 reduced both proliferation and invasion of breast cancer cells. Our findings suggest that modulating miR-205 and/or targeting HMGB3 are potential therapies for advanced breast cancer.鉴定失调的微小RNA（microRNAs，miRNAs）的靶标，将加深我们对miRNA表达异常如何促成乳腺癌恶性表型的理解。相较于正常样上皮细胞系MCF10A，miR-205在四种乳腺癌细胞系中的表达水平显著下调；相较于癌旁良性乳腺组织，miR-205在肿瘤组织及转移性组织中的表达亦显著降低。研究人员在高迁移率族盒蛋白3（HMGB3）基因的3'非翻译区中，鉴定到两个miR-205的预测结合位点。双荧光素酶报告基因实验与蛋白质免疫印迹（Western blotting）实验均证实，miR-205可结合并调控HMGB3。为进一步探究miR-205对HMGB3的靶向调控作用，研究人员分别将miR-205前体寡核苷酸或HMGB3小干扰RNA（siRNA）瞬时转染至MDA-MB-231与BT549乳腺癌细胞中，随后开展WST-1增殖实验与体外侵袭实验。两种干预手段均能显著抑制乳腺癌细胞的增殖与侵袭能力。相较于癌旁良性标本，肿瘤组织中HMGB3的mRNA与蛋白表达水平均显著升高；且HMGB3 mRNA的表达水平与患者生存率呈间接相关关系。用5-氮杂胞苷/曲古抑菌素A（5-Aza/TSA）处理乳腺癌细胞后，miR-205的表达被去抑制，而HMGB3的mRNA水平则有所降低；而敲低转录阻遏因子NRSF/REST后，miR-205的表达被下调，HMGB3的表达则出现升高。综上，miR-205对HMGB3的调控可抑制乳腺癌细胞的增殖与侵袭能力。本研究结果提示，调控miR-205的表达或靶向HMGB3，有望成为晚期乳腺癌的潜在治疗策略。