miR-216b-5p promotes late apoptosis/necroptosis in trastuzumab-resistant SK-BR-3 cells.

Breast cancer is the most common cancer in women. The human epidermal growth factor receptor 2 (HER2) overexpressing subtype is related to poor prognosis with an aggressive phenotype and is reported as one of the most commonly seen subtypes. Trastuzumab is prevalently used as a treatment method for HER2+ breast cancer however, resistance to the drug frequently occurs following the treatment. MicroRNAs (miRNAs) are 19-23 nucleotide long small RNAs, which regulate gene expression at post-transcriptional level and studies show that there are differentially expressed miRNAs between drug sensitive and resistant groups, indicating that they might have some key roles in drug effectiveness. In this study, the aim is to find out the role of miR-216b-5p in trastuzumab resistance. SK-BR-3 cells developed resistance to trastuzumab after continuous treatment with increasing concentrations of the drug for 6 months. To investigate the effect of miR-216b-5p on cancer cell behavior in resistance state, proliferation, motility, and invasion capacities of these resistant cells were analyzed by xCELLigence real-time cell analyzer. To further understand the molecular mechanisms underlying the regulation of resistant SK-BR-3 cells by miR-216b-5p, microarray analysis was performed. Apoptosis analysis was also performed since the pathway enrichment analysis pointed out cell death related pathways. The proliferation, motility, and invasion capacities of the miR-216b-5p transfected resistant cells were diminished compared to sensitive cells. We identified the necroptosis signaling pathway as the result of microarray and pathway enrichment analyses. STAT1, IRF9, and PKR were validated as the significant elements of the pathway, which are also the putative targets of miR-216b-5p. Our apoptosis analysis showed that a significant amount of trastuzumab resistant SK-BR-3 cells entered to late apoptosis/necrosis stage upon miR-216b-5p overexpression, it could be concluded that reexpression of miR-216b-5p sensitizes trastuzumab resistance through necroptosis in breast cancer.

Experimental MicroRNA Targeting Validation.

microRNAs (miRNAs) have recently been recognized as a new dimension of posttranscriptional regulation. It is well defined that most human protein-coding genes are regulated by one or more miRNAs. Therefore, it is crucial to identify genes targeted by the miRNAs to better understand their functions. Although bioinformatics tools have the ability to identify target candidates it is still essential to identify physiological targets by experimental approaches. Currently, the majority of miRNA-target experimental validation approaches assess the changes in target expression in mRNA or protein level upon miRNA upregulation or downregulation. Additionally, finding out direct physical interactions between miRNAs and their targets is also among the experimental techniques. In this chapter we reviewed the existing experimental techniques for miRNA target identification by considering their advantages and potential drawbacks.

Overexpression of ß-Arrestins inhibits proliferation and motility in triple negative breast cancer cells.

ß-Arrestins (ßArrs) are intracellular signal regulating proteins. Their expression level varies in some cancers and they have a significant impact on cancer cell function. In general, the significance of ßArrs in cancer research comes from studies examining GPCR signalling. Given the diversity of different GPCR signals in cancer cell regulation, contradictory results are inevitable regarding the role of ßArrs. Our approach examines the direct influence of ßArrs on cellular function and gene expression profiles by changing their expression levels in breast cancer cells, MDA-MB-231 and MDA-MB-468. Reducing expression of ßArr1 or ßArr2 tended to increase cell proliferation and invasion whereas increasing their expression levels inhibited them. The overexpression of ßArrs caused cell cycle S-phase arrest and differential expression of cell cycle genes, CDC45, BUB1, CCNB1, CCNB2, CDKN2C and reduced HER3, IGF-1R, and Snail. Regarding to the clinical relevance of our results, low expression levels of ßArr1 were inversely correlated with CDC45, BUB1, CCNB1, and CCNB2 genes compared to normal tissue samples while positively correlated with poorer prognosis in breast tumours. These results indicate that ßArr1 and ßArr2 are significantly involved in cell cycle and anticancer signalling pathways through their influence on cell cycle genes and HER3, IGF-1R, and Snail in TNBC cells.

A ranking-based meta-analysis reveals let-7 family as a meta-signature for grade classification in breast cancer.

Breast cancer is one of the most important causes of cancer-related deaths worldwide in women. In addition to gene expression studies, the progressing work in the miRNA area including miRNA microarray studies, brings new aspects to the research on the cancer development and progression. Microarray technology has been widely used to find new biomarkers in research and many transcriptomic microarray studies are available in public databases. In this study, the breast cancer miRNA and mRNA microarray studies were collected according to the availability of their data and clinical information, and combined by a newly developed ranking-based meta-analysis approach to find out candidate miRNA biomarkers (meta-miRNAs) that classify breast cancers according to their grades and explain the relation between miRNAs and mRNAs. This approach provided meta-miRNAs specific to breast cancer grades, pointing out let-7 family members as grade classifiers. The qRT-PCR studies performed with independent breast tumors confirmed the potential biomarker role of let-7 family members (meta-miRNAs). The concordance between the meta-mRNAs and miRNA target genes specific to tumor grade (common genes) supported the idea of mRNAs as miRNA targets. The pathway analysis results showed that most of the let-7 family miRNA targets, and also common genes, were significantly taking part in cancer-related pathways. The qRT-PCR studies, together with bioinformatic analyses, confirmed the results of meta-analysis approach, which is dynamic and allows combining datasets from different platforms.

High-throughput approaches for microRNA expression analysis.

Profiling microRNA (miRNA) expression is of widespread interest due to their critical roles in diverse biological processes, including development, cell proliferation, differentiation, and apoptosis. Profiling can be achieved via three major methods: amplification-based (real-time quantitative PCR, qRT-PCR), hybridization-based (microarrays), and sequencing-based (next-generation sequencing (NGS)) technologies. The gold standard is qRT-PCR and serves as a platform for single reverse PCR amplification experiments and for a large number of miRNAs in parallel, both by multiplexing and plate based arrays. Currently, qRT-PCR is used for the validation of miRNA profiling results from other platforms. Hybridization based miRNA profiling by microarrays has become a widely used method especially for biomarker and therapeutic target identification. The data obtained from microarrays also enables functional prediction of miRNAs by correlating miRNA expression patterns to corresponding mRNA and protein profiles. Additionally, miRNA profiling strategies based on deep sequencing allow both the identification of novel miRNAs and relative quantification of miRNAs. Each miRNA profiling strategy has specific strengths and challenges that have to be considered depending on the nature of the research context.In this chapter the high-throughput approaches that can be applied to microRNA profiling are discussed starting from small-scale qRT-PCR technology to a wider one, NGS.