Khajah Maitham A, Al-Ateyah Alyaa, Luqmani Yunus A
Faculty of Pharmacy, Kuwait University, Safat, 13110, Kuwait.
Biochem Biophys Rep. 2022 Jul 20;31:101316. doi: 10.1016/j.bbrep.2022.101316. eCollection 2022 Sep.
MicroRNAs (miRs) regulate gene expression through translation inhibition of target mRNAs. One of the most promising approaches for cancer therapy is through mimicking or antagonizing the action of miRs. In this report, we analyzed the miRnome profile of several human breast cancer cell lines to determine the influence of estrogen receptor (ER) silencing previously shown to result in epithelial to mesenchymal transition (EMT) and enhanced tumor invasion.
MicroRNA extracted from MDA-MB-231 ( ER-) and ER-silenced ( ER-) pII and IM-26 or ER-expressing (YS1.2) siRNA transfected derivatives of MCF7 cells was deep sequenced on Illumina NextSeq500. Respective miRnomes were compared with edgeR package in R and Venny2.1 and target prediction performed with miRTarBase. Mimics and inhibitors of selected differentially expressed miRs associated with EMT mediators (miR-200c-3p targeting ZEB1, miR-449a targeting δ-catenin and miR-29a-3p) were transfected into pII cells and mRNA targets, as well as E-cadherin and keratin 19 (epithelial and mesenchymal markers respectively) were measured using taqman PCR.
Each cell line expressed about 20% of the total known human miRnome; There was a high degree of similarity between the 3 tested ER-lines. Out of these expressed miRs, 50-60% were significantly differentially expressed between ER- and ER + lines. Transfection of miR-200c-3p mimic into pII cells down regulated ZEB1 and vimentin, and increased E-cadherin and keratin 19 with accompanying morphological changes, and reduced cell motility, reflecting a reversal back into an epithelial phenotype. On the other hand, transfecting pII with miR-449a inhibitor reduced cell invasion but did not induce EMT. Transfecting pII cell line with the mimic or inhibitor of miR-29a-3p showed no change in EMT markers or cell invasion suggesting that the EMT induced by loss of ER function can be reversed by blocking some but not just any random EMT-associated genes.
These data suggest that differences in miR expression can be exploited not only as mediators (using mimics) and targets (using miR antagonists) for general cancer therapies aimed at regulating either individual or multiple mRNAs, but also to re-sensitize endocrine resistant breast cancers by turning them back into a type that will be susceptible to endocrine agents.
微小RNA（miR）通过抑制靶mRNA的翻译来调节基因表达。癌症治疗最有前景的方法之一是模拟或拮抗miR的作用。在本报告中，我们分析了几种人乳腺癌细胞系的miR组图谱，以确定先前显示导致上皮-间质转化（EMT）和增强肿瘤侵袭的雌激素受体（ER）沉默的影响。
从MDA-MB-231（ER-）和ER沉默（ER-）的pII和IM-26或ER表达（YS1.2）的MCF7细胞的siRNA转染衍生物中提取的微小RNA在Illumina NextSeq500上进行深度测序。使用R中的edgeR软件包和Venny2.1比较各自的miR组，并使用miRTarBase进行靶标预测。将与EMT介质相关的选定差异表达miR的模拟物和抑制剂（靶向ZEB1的miR-200c-3p、靶向δ-连环蛋白的miR-449a和miR-29a-3p）转染到pII细胞中，并使用taqman PCR测量mRNA靶标以及E-钙黏蛋白和角蛋白19（分别为上皮和间质标志物）。
每个细胞系表达约20%的已知人类miR组；在3个测试的ER-细胞系之间存在高度相似性。在这些表达的miR中，50-60%在ER-和ER +细胞系之间存在显著差异表达。将miR-200c-3p模拟物转染到pII细胞中可下调ZEB1和波形蛋白，并增加E-钙黏蛋白和角蛋白19，同时伴有形态学变化，并降低细胞运动性，反映出逆转回上皮表型。另一方面，用miR-449a抑制剂转染pII可降低细胞侵袭，但不诱导EMT。用miR-29a-3p的模拟物或抑制剂转染pII细胞系，EMT标志物或细胞侵袭没有变化，这表明通过阻断一些但不是任何随机的EMT相关基因，可以逆转由ER功能丧失诱导的EMT。
这些数据表明，miR表达的差异不仅可以作为针对调节单个或多个mRNA的一般癌症治疗的介质（使用模拟物）和靶标（使用miR拮抗剂），还可以通过将内分泌抗性乳腺癌转变回对内分泌药物敏感的类型来使其重新敏感。
