Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad 9177948974, Iran.
Department of Computer Engineering, Sharif University of Technology, Tehran 11155-11365, Iran.
Int J Mol Sci. 2021 Jun 23;22(13):6768. doi: 10.3390/ijms22136768.
miR-29b2 and miR-29c play a suppressive role in breast cancer progression. (also named ) is the host gene for generating both microRNAs. However, the region also expresses longer transcripts with unknown functions. We employed bioinformatics and experimental approaches to decipher expression and function in breast cancer tissues. We also used the CRISPR/Cas9 technique to excise a predicted distal promoter and followed the behavior of the edited cells by real-time PCR, flow cytometry, migration assay, and RNA-seq techniques. We observed that long transcript is significantly downregulated in triple-negative breast cancer. We also identified a promoter for the longer transcripts of whose functionality was demonstrated by transfecting MCF7 cells with a promoter-GFP construct. Knocking-out the promoter by means of CRISPR/Cas9 revealed no alterations in the expression of the neighboring genes and , while the expression of miR-29c was reduced by half. Furthermore, the promoter knockout elevated the migration ability of the edited cells. RNA sequencing revealed many up- and downregulated genes involved in various cellular pathways, including epithelial to mesenchymal transition and mammary gland development pathways. Altogether, we are reporting here the existence of an additional/distal promoter with an enhancer effect on miR-29 generation and an inhibitory effect on cell migration.
miR-29b2 和 miR-29c 在乳腺癌的进展中发挥抑制作用。（也称为）是产生这两个 microRNA 的宿主基因。然而，该区域还表达具有未知功能的更长转录本。我们采用生物信息学和实验方法来破译乳腺癌组织中 的表达和功能。我们还使用了 CRISPR/Cas9 技术来切除预测的远端启动子，并通过实时 PCR、流式细胞术、迁移实验和 RNA-seq 技术来跟踪编辑细胞的行为。我们观察到，长转录本在三阴性乳腺癌中显著下调。我们还鉴定了一个更长转录本的启动子，其功能通过转染 MCF7 细胞的一个启动子-GFP 构建体得到证明。通过 CRISPR/Cas9 敲除启动子，邻近基因 和 的表达没有改变，而 miR-29c 的表达减少了一半。此外，启动子敲除提高了编辑细胞的迁移能力。RNA 测序揭示了许多涉及各种细胞途径的上调和下调基因，包括上皮间质转化和乳腺发育途径。总的来说，我们在这里报告了一个额外/远端启动子的存在，该启动子对 miR-29 的生成具有增强作用，并对细胞迁移具有抑制作用。
