Department of Biotechnology and Molecular Medicine, Faculty of Medicine, Arak University of Medical Sciences, Arak, Iran.
Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Mol Carcinog. 2023 Dec;62(12):1960-1973. doi: 10.1002/mc.23628. Epub 2023 Oct 3.
An anticancer drug known as Rapamycin acts by inhibiting the mammalian target of the Rapamycin pathway. This agent has recently been investigated for its potential therapeutic benefits in sensitizing drug-resistant breast cancer (BC) treatment. The molecular mechanism underlying these effects, however, is still a mystery. Using a systems biology method and in vitro experiment, this study sought to discover essential genes and microRNAs (miRNAs) targeted by Rapamycin in triple-negative BC (TNBC) cells to aid prospective new medications with less adverse effects in BC treatment. We developed the transcription factor-miRNA-gene and protein-protein interaction networks using the freely accessible microarray data sets. FANMOD and MCODE were utilized to identify critical regulatory motifs, clusters, and seeds. Then, functional enrichment analyses were conducted. Using topological analysis and motif detection, the most important genes and miRNAs were discovered. We used quantitative real-time polymerase chain reaction (qRT-PCR) to examine the effect of Rapamycin on the expression of the selected genes and miRNAs to verify our findings. We performed flow cytometry to investigate Rapamycin's impact on cell cycle and apoptosis. Furthermore, wound healing and migration assays were done. Three downregulated (PTGS2, EGFR, VEGFA) and three upregulated (c-MYC, MAPK1, PIK3R1) genes were chosen as candidates for additional experimental verification. There were also three upregulated miRNAs (miR-92a, miR-16, miR-20a) and three downregulated miRNAs (miR-146a, miR-145, miR-27a) among the six selected miRNAs. The qRT-PCR findings in MDA-MB-231 cells indicated that c-MYC, MAPK1, PIK3R1, miR-92a, miR-16, and miR-20a expression levels were considerably elevated following Rapamycin treatment, whereas PTGS2, EGFR, VEGFA, miR-146a, and miR-145 expression levels were dramatically lowered (p < 0.05). These genes are engaged in cancer pathways, transcriptional dysregulation in cancer, and cell cycle, according to the top pathway enrichment findings. Migration and wound healing abilities of the cells declined after Rapamycin treatment, and the number of apoptotic cells increased. We demonstrated that Rapamycin suppresses cell migration and metastasis in the TNBC cell line. In addition, our data indicated that Rapamycin induces apoptosis in this cell line. The discovered vital genes and miRNAs affected by Rapamycin are anticipated to have crucial roles in the pathogenesis of TNBC and its therapeutic resistance.
一种名为雷帕霉素的抗癌药物通过抑制雷帕霉素靶蛋白途径来发挥作用。最近，人们研究了这种药物在增强耐药性乳腺癌（BC）治疗中的潜在治疗益处。然而，这些影响的分子机制仍然是一个谜。本研究采用系统生物学方法和体外实验，旨在发现雷帕霉素在三阴性 BC（TNBC）细胞中作用的关键靶基因和 microRNA（miRNA），以帮助开发具有较少不良反应的 BC 治疗新药物。我们使用免费的 microarray 数据集开发了转录因子-miRNA-基因和蛋白质-蛋白质相互作用网络。使用 FANMOD 和 MCODE 来识别关键的调控基序、簇和种子。然后，进行功能富集分析。通过拓扑分析和基序检测，发现了最重要的基因和 miRNA。我们使用定量实时聚合酶链反应（qRT-PCR）检测雷帕霉素对选定基因和 miRNA 表达的影响，以验证我们的发现。我们进行了流式细胞术来研究雷帕霉素对细胞周期和凋亡的影响。此外，还进行了划痕愈合和迁移实验。选择三个下调（PTGS2、EGFR、VEGFA）和三个上调（c-MYC、MAPK1、PIK3R1）的基因作为进一步实验验证的候选基因。在六个选定的 miRNA 中，还有三个上调的 miRNA（miR-92a、miR-16、miR-20a）和三个下调的 miRNA（miR-146a、miR-145、miR-27a）。qRT-PCR 结果显示，在 MDA-MB-231 细胞中，c-MYC、MAPK1、PIK3R1、miR-92a、miR-16 和 miR-20a 的表达水平在雷帕霉素处理后显著升高，而 PTGS2、EGFR、VEGFA、miR-146a 和 miR-145 的表达水平则显著降低（p < 0.05）。根据通路富集分析的结果，这些基因参与癌症通路、癌症转录失调和细胞周期。雷帕霉素处理后，细胞的迁移和伤口愈合能力下降，凋亡细胞数量增加。我们证明了雷帕霉素抑制了 TNBC 细胞系的迁移和转移。此外，我们的数据表明雷帕霉素诱导了该细胞系的细胞凋亡。雷帕霉素作用下受影响的关键基因和 miRNA 预计在 TNBC 的发病机制及其治疗耐药性中发挥关键作用。
