Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende, Italy.
Regional Hospital Cosenza, Cosenza, Italy.
J Exp Clin Cancer Res. 2018 May 2;37(1):94. doi: 10.1186/s13046-018-0767-6.
MicroRNA (miRNAs) are non-coding small RNA molecules that regulate gene expression by inhibiting the translation of target mRNAs. Among several dysregulated miRNAs in human cancer, the up-regulation of miR-221 has been associated with development of a variety of hematologic and solid malignancies. In this study, we investigated the involvement of miR-221 in breast cancer.
TaqMan microRNA assay was used to detect the miR-221 levels in normal cells and in MDA-MB 231 and SkBr3 breast cancer cells as well as in main players of the tumor microenvironment, namely cancer-associated fibroblasts (CAFs). miR-221 mimic sequence and locked nucleic acid (LNA)-i-miR-221 construct were used to induce or inhibit, respectively, the miR-221 expression in cells used. Quantitative PCR and western blotting analysis were performed to evaluate the levels of the miR-221 target gene A20 (TNFAIP3), as well as the member of the NF-kB complex namely c-Rel and the connective tissue growth factor (CTGF). Chromatin immunoprecipitation (ChIP) assay was performed to ascertain the recruitment of c-Rel to the CTFG promoter. Finally, the cell growth and migration in the presence of LNA-i-miR-221 or silencing c-Rel and CTGF by specific short hairpin were assessed by cell count, colony formation and boyden chambers assays. Statistical analysis was performed by ANOVA.
We first demonstrated that LNA-i-miR-221 inhibits both endogenous and ectopic expression of miR-221 in our experimental models. Next, we found that the A20 down-regulation, as well as the up-regulation of c-Rel induced by miR-221 were no longer evident using LNA-i-miR-221. Moreover, we established that the miR-221 dependent recruitment of c-Rel to the NF-kB binding site located within the CTGF promoter region is prevented by using LNA-i-miR-221. Furthermore, we determined that the up-regulation of CTGF mRNA and protein levels by miR-221 is no longer evident using LNA-i-miR221 and silencing c-Rel. Finally, we assessed that cell growth and migration induced by miR-221 in MDA-MB 231 and SkBr3 breast cancer cells as well as in CAFs are abolished by LNAi-miR-221 and silencing c-Rel or CTGF.
Overall, these data provide novel insights into the stimulatory action of miR-221 in breast cancer cells and CAFs, suggesting that its inhibition may be considered toward targeted therapeutic approaches in breast cancer patients.
MicroRNA (miRNAs) 是一类非编码的小分子 RNA 分子，通过抑制靶 mRNA 的翻译来调节基因表达。在人类癌症中，多种失调的 miRNAs 中，miR-221 的上调与多种血液系统和实体恶性肿瘤的发展有关。在这项研究中，我们研究了 miR-221 在乳腺癌中的作用。
使用 TaqMan 微 RNA 检测试剂盒检测正常细胞以及 MDA-MB 231 和 SkBr3 乳腺癌细胞中 miR-221 的水平，以及肿瘤微环境中的主要参与者，即癌症相关成纤维细胞（CAFs）中的 miR-221 水平。使用 miR-221 模拟序列和锁定核酸（LNA）-i-miR-221 构建体分别诱导或抑制细胞中 miR-221 的表达。通过定量 PCR 和 Western blot 分析评估 miR-221 靶基因 A20（TNFAIP3）以及 NF-kB 复合物成员 c-Rel 和结缔组织生长因子（CTGF）的水平。进行染色质免疫沉淀（ChIP）实验以确定 c-Rel 募集到 CTGF 启动子上。最后，通过细胞计数、集落形成和 Boyden 室测定，评估 LNA-i-miR-221 或通过特异性短发夹沉默 c-Rel 和 CTGF 对细胞生长和迁移的影响。通过 ANOVA 进行统计分析。
我们首先证明 LNA-i-miR-221 抑制了我们实验模型中内源性和外源性 miR-221 的表达。接下来，我们发现 miR-221 诱导的 A20 下调以及 c-Rel 的上调不再明显，使用 LNA-i-miR-221。此外，我们确定 miR-221 依赖性 c-Rel 募集到位于 CTGF 启动子区域的 NF-kB 结合位点被 LNA-i-miR-221 阻止。此外，我们确定 miR-221 诱导的 CTGF mRNA 和蛋白水平的上调不再明显，使用 LNA-i-miR221 和沉默 c-Rel。最后，我们评估了 miR-221 在 MDA-MB 231 和 SkBr3 乳腺癌细胞以及 CAFs 中诱导的细胞生长和迁移，使用 LNAi-miR-221 和沉默 c-Rel 或 CTGF 被消除。
总的来说，这些数据为 miR-221 在乳腺癌细胞和 CAFs 中的刺激作用提供了新的见解，表明其抑制可能被认为是针对乳腺癌患者的靶向治疗方法。
