miR-221 stimulates breast cancer cells and cancer-associated fibroblasts (CAFs) through selective interference with the A20/c-Rel/CTGF signaling.
miR-221 stimulates breast cancer cells and cancer-associated fibroblasts (CAFs) through selective interference with the A20/c-Rel/CTGF signaling.
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DOI：
10.1186/s13046-018-0767-6
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发表时间：
2018-05-02
期刊：
影响因子：
--
通讯作者：
Vivacqua A
中科院分区：
文献类型：
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作者：
Santolla MF;Lappano R;Cirillo F;Rigiracciolo DC;Sebastiani A;Abonante S;Tassone P;Tagliaferri P;Di Martino MT;Maggiolini M;Vivacqua A
MicroRNA (miRNA) 是非编码小 RNA 分子，通过抑制靶 mRNA 的翻译来调节基因表达。在人类癌症中几种失调的 miRNA 中，miR-221 的上调与多种血液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。 TaqMan microRNA 检测用于检测正常细胞、MDA-MB 231 和 SkBr3 乳腺癌细胞以及肿瘤微环境的主要参与者（即癌症相关成纤维细胞 (CAF)）中的 miR-221 水平。 miR-221模拟序列和锁核酸(LNA)-i-miR-221构建体分别用于诱导或抑制所用细胞中的miR-221表达。进行定量 PCR 和蛋白质印迹分析以评估 miR-221 靶基因 A20 (TNFAIP3) 以及 NF-kB 复合体成员即 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) 测定以确定 c-Rel 向 CTFG 启动子的募集。最后，通过细胞计数、集落形成和博伊登室测定来评估在 LNA-i-miR-221 存在或通过特定短发夹沉默 c-Rel 和 CTGF 的情况下的细胞生长和迁移。通过方差分析进行统计分析。我们首先在我们的实验模型中证明 LNA-i-miR-221 抑制 miR-221 的内源性和异位表达。接下来，我们发现使用 LNA-i-miR-221，miR-221 诱导的 A20 下调以及 c-Rel 上调不再明显。此外，我们确定使用 LNA-i-miR-221 可以阻止 miR-221 依赖的 c-Rel 募集到位于 CTGF 启动子区域内的 NF-kB 结合位点。此外，我们确定使用 LNA-i-miR221 和沉默 c-Rel，miR-221 对 CTGF mRNA 和蛋白质水平的上调不再明显。最后，我们评估了 LNAi-miR-221 和沉默 c-Rel 或 CTGF 消除了 MDA-MB 231 和 SkBr3 乳腺癌细胞以及 CAF 中 miR-221 诱导的细胞生长和迁移。总体而言，这些数据为 miR-221 在乳腺癌细胞和 CAF 中的刺激作用提供了新的见解，表明其抑制可被考虑用于乳腺癌患者的靶向治疗方法。
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.
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影响因子：
--
作者：
Gilmore, Thomas D;Gerondakis, Steve
通讯作者：
Gerondakis, Steve
影响因子：
--
作者：
Ben-Hamo R;Efroni S
通讯作者：
Efroni S
干性的维持与肝细胞癌自分泌的 LRP6 和肝素结合蛋白 CCN2 的相互作用有关
DOI：
10.1186/s13046-017-0576-3
发表时间：
2017-09-04
期刊：
Journal of experimental & clinical cancer research : CR
影响因子：
--
作者：
Jia Q;Bu Y;Wang Z;Chen B;Zhang Q;Yu S;Liu Q
通讯作者：
Liu Q
影响因子：
11.2
作者：
Iorio, MV;Ferracin, M;Croce, CM
通讯作者：
Croce, CM
DOI：
10.1016/j.jpba.2017.12.027
发表时间：
2018-02-20
影响因子：
3.4
作者：
Franzoni, S.;Vezzelli, A.;Breda, M.
通讯作者：
Breda, M.