Abstract：
About 70%-80% of breast cancers (BCs) express estrogen receptors (ER-positive). MicroRNAs (miRNAs) are a group of small endogenous noncoding RNAs that play a critical regulatory role in cancer development and progression, including in BC. MiRNA deficiency promotes the development of BCs. MiR-143-5p is one of the most commonly dysregulated miRNAs in BC but its role as a tumor suppressor remains unclear.
MiR-143-3p and -5p expression in breast tissue was analyzed using TCGA and StarBase databases. Expression in BC subclasses and survival analyses were conducted. Clinical samples were collected, cell cultures created, and gene expression assays performed following previous studies. Protein expression, luciferase reporter, wound healing, DAPI staining, cell cycle, colony formation, spheroid, CD44 FACS, and proliferation assays were conducted following various protocols.
Here, we find that both miR-143-3p and miR-143-5p levels are considerably lower in BC tissue compared to normal breast tissue and low miR-143 expression predicts poor prognosis in ER+ BC patients. In-depth analyses identified 3 miR-143-5p binding sites in the 3\' untranslated region (UTR) of the DNA binding protein High Mobility Group AT-Hook 2 (HMGA2). Luciferase reporter assays using wild-type and mutant HMGA2 3\'UTR sequences and Western blot analyses demonstrated that HMGA2 is a direct and bona fide miR-143-5p target in BC cells. In addition, we show that restoration of miR-143-5p expression suppresses metastasis-related features of ER+ BC cells, including reduced tumor cell migration, increased E-cadherin expression, and decreased vimentin and N-cadherin expression. Furthermore, miR-143-5p reduces cell proliferation, cell cycle entry, and stemness, while promoting apoptosis moderately. Finally, patient sample pathway analyses demonstrated that these mechanisms are also active in BC.
Altogether, our findings shed new light on miR-143-5p\'s anticancer biological functions in BC progression by directly targeting HMGA2. This suggests that restoration of miR-143-5p could be a promising new therapeutic approach for the treatment of ER+ BC.
MiR-143-3p and -5p expression in breast tissue was analyzed using TCGA and StarBase databases. Expression in BC subclasses and survival analyses were conducted. Clinical samples were collected, cell cultures created, and gene expression assays performed following previous studies. Protein expression, luciferase reporter, wound healing, DAPI staining, cell cycle, colony formation, spheroid, CD44 FACS, and proliferation assays were conducted following various protocols.
Here, we find that both miR-143-3p and miR-143-5p levels are considerably lower in BC tissue compared to normal breast tissue and low miR-143 expression predicts poor prognosis in ER+ BC patients. In-depth analyses identified 3 miR-143-5p binding sites in the 3\' untranslated region (UTR) of the DNA binding protein High Mobility Group AT-Hook 2 (HMGA2). Luciferase reporter assays using wild-type and mutant HMGA2 3\'UTR sequences and Western blot analyses demonstrated that HMGA2 is a direct and bona fide miR-143-5p target in BC cells. In addition, we show that restoration of miR-143-5p expression suppresses metastasis-related features of ER+ BC cells, including reduced tumor cell migration, increased E-cadherin expression, and decreased vimentin and N-cadherin expression. Furthermore, miR-143-5p reduces cell proliferation, cell cycle entry, and stemness, while promoting apoptosis moderately. Finally, patient sample pathway analyses demonstrated that these mechanisms are also active in BC.
Altogether, our findings shed new light on miR-143-5p\'s anticancer biological functions in BC progression by directly targeting HMGA2. This suggests that restoration of miR-143-5p could be a promising new therapeutic approach for the treatment of ER+ BC.
摘要：
背景：大约70%-80%的乳腺癌（BC）表达雌激素受体（ER阳性）。microRNAs（miRNAs）是一组小的内源性非编码RNAs，在癌症的发生和发展中起着重要的调节作用。包括BC。miRNA缺乏促进BCs的发育。MiR-143-5p是BC中最常见的失调miRNA之一，但其作为肿瘤抑制因子的作用尚不清楚。
方法：使用TCGA和StarBase数据库分析乳腺组织中MiR-143-3p和-5p的表达。进行BC亚类中的表达和存活分析。收集临床样本，细胞培养物被创造出来,和基因表达测定是根据先前的研究进行的。蛋白质表达，荧光素酶报告基因,伤口愈合，DAPI染色,细胞周期,菌落形成,球体,CD44FACS,和增殖试验按照各种方案进行。
结果：这里，我们发现,与正常乳腺组织相比,BC组织中的miR-143-3p和miR-143-5p水平均显著降低,miR-143低表达预示ER+BC患者预后不良.深入分析确定了DNA结合蛋白高迁移率组AT-Hook2（HMGA2）的3'非翻译区（UTR）中的3个miR-143-5p结合位点。使用野生型和突变型HMGA23'UTR序列和蛋白质印迹分析的荧光素酶报告基因测定表明，HMGA2是BC细胞中直接和真正的miR-143-5p靶标。此外,我们显示miR-143-5p表达的恢复抑制ER+BC细胞的转移相关特征，包括减少肿瘤细胞迁移，E-cadherin表达增加，波形蛋白和N-钙粘蛋白表达降低。此外，miR-143-5p减少细胞增殖，细胞周期进入，和干劲，同时适度促进细胞凋亡。最后,患者样本通路分析表明,这些机制在BC中也是活跃的.
结论：总而言之，我们的研究结果为miR-143-5p通过直接靶向HMGA2在BC进展中的抗癌生物学功能提供了新的思路。这表明miR-143-5p的恢复可能是治疗ER+BC的有希望的新治疗方法。
方法：使用TCGA和StarBase数据库分析乳腺组织中MiR-143-3p和-5p的表达。进行BC亚类中的表达和存活分析。收集临床样本，细胞培养物被创造出来,和基因表达测定是根据先前的研究进行的。蛋白质表达，荧光素酶报告基因,伤口愈合，DAPI染色,细胞周期,菌落形成,球体,CD44FACS,和增殖试验按照各种方案进行。
结果：这里，我们发现,与正常乳腺组织相比,BC组织中的miR-143-3p和miR-143-5p水平均显著降低,miR-143低表达预示ER+BC患者预后不良.深入分析确定了DNA结合蛋白高迁移率组AT-Hook2（HMGA2）的3'非翻译区（UTR）中的3个miR-143-5p结合位点。使用野生型和突变型HMGA23'UTR序列和蛋白质印迹分析的荧光素酶报告基因测定表明，HMGA2是BC细胞中直接和真正的miR-143-5p靶标。此外,我们显示miR-143-5p表达的恢复抑制ER+BC细胞的转移相关特征，包括减少肿瘤细胞迁移，E-cadherin表达增加，波形蛋白和N-钙粘蛋白表达降低。此外，miR-143-5p减少细胞增殖，细胞周期进入，和干劲，同时适度促进细胞凋亡。最后,患者样本通路分析表明,这些机制在BC中也是活跃的.
结论：总而言之，我们的研究结果为miR-143-5p通过直接靶向HMGA2在BC进展中的抗癌生物学功能提供了新的思路。这表明miR-143-5p的恢复可能是治疗ER+BC的有希望的新治疗方法。
