Identification of a core EMT signature that separates basal-like breast cancers into partial- and post-EMT subtypes.

Epithelial-mesenchymal transition (EMT) is a cellular plasticity program critical for embryonic development and tissue regeneration, and aberrant EMT is associated with disease including cancer. The high degree of plasticity in the mammary epithelium is reflected in extensive heterogeneity among breast cancers. Here, we have analyzed RNA-sequencing data from three different mammary epithelial cell line-derived EMT models and identified a robust mammary EMT gene expression signature that separates breast cancers into distinct subgroups. Most strikingly, the basal-like breast cancers form two subgroups displaying partial-EMT and post-EMT gene expression patterns. We present evidence that key EMT-associated transcription factors play distinct roles at different stages of EMT in mammary epithelial cells.

Distinct Small RNA Signatures in Extracellular Vesicles Derived from Breast Cancer Cell Lines.

Breast cancer is a heterogeneous disease, and different subtypes of breast cancer show distinct cellular morphology, gene expression, metabolism, motility, proliferation, and metastatic potential. Understanding the molecular features responsible for this heterogeneity is important for correct diagnosis and better treatment strategies. Extracellular vesicles (EVs) and their associated molecules have gained much attention as players in intercellular communication, ability to precondition specific organs for metastatic invasion, and for their potential role as circulating cancer biomarkers. EVs are released from the cells and contain proteins, DNA, and long and small RNA species. Here we show by high-throughput small RNA-sequencing that EVs from nine different breast cancer cell lines share common characteristics in terms of small RNA content that are distinct from their originating cells. Most strikingly, a highly abundant small RNA molecule derived from the nuclear 28S rRNA is vastly enriched in EVs. The miRNA profiles in EVs correlate with the cellular miRNA expression pattern, but with a few exceptions that includes miR-21. This cancer-associated miRNA is retained in breast cancer cell lines. Finally, we report that EVs from breast cancer cell lines cluster together based on their small RNA signature when compared to EVs derived from other cancer cell lines. Altogether, our data demonstrate that breast cancer cell lines manifest a specific small RNA signature in their released EVs. This opens up for further evaluation of EVs as breast cancer biomarkers.

Next generation sequencing of microRNAs from isogenic neuroblastoma cell lines isolated before and after treatment.

Neuroblastoma is a pediatric cancer of the developing sympathetic nervous system. High risk neuroblastoma patients typically undergo an initial remission in response to treatment, followed by recurrence of aggressive tumors that have become refractory to further treatment. Recent works have underlined the involvement of microRNAs (miRNAs) in neuroblastoma development and evolution of drug resistance. In this study we have used deep sequencing technology to identify miRNAs differentially expressed in neuroblastoma cell lines isolated from 6 patients at diagnosis and at relapse after intensive treatments. This approach revealed a panel of 42 differentially expressed miRNAs, 8 of which were upregulated and 34 were downregulated. Most strikingly, the 14q32 miRNA clusters encode 22 of the downregulated miRNAs. Reduced expression of 14q32 miRNAs in tumors associated with poor prognosis factors was confirmed in a cohort consisting of 226 primary neuroblastomas. In order to gain insight into the nature of the genes that may be affected by the differentially expressed miRNAs we utilized Ingenuity Pathway Analysis (IPA). This analysis revealed several biological functions and canonical pathways associated with cancer progression and drug resistance. The results of this study contribute to the identification of miRNAs involved in the complex processes of surviving therapeutic treatment and developing drug resistance in neuroblastoma.

Performance comparison of digital microRNA profiling technologies applied on human breast cancer cell lines.

MicroRNA profiling represents an important first-step in deducting individual RNA-based regulatory function in a cell, tissue, or at a specific developmental stage. Currently there are several different platforms to choose from in order to make the initial miRNA profiles. In this study we investigate recently developed digital microRNA high-throughput technologies. Four different platforms were compared including next generation SOLiD ligation sequencing and Illumina HiSeq sequencing, hybridization-based NanoString nCounter, and miRCURY locked nucleic acid RT-qPCR. For all four technologies, full microRNA profiles were generated from human cell lines that represent noninvasive and invasive tumorigenic breast cancer. This study reports the correlation between platforms, as well as a more extensive analysis of the accuracy and sensitivity of data generated when using different platforms and important consideration when verifying results by the use of additional technologies. We found all the platforms to be highly capable for microRNA analysis. Furthermore, the two NGS platforms and RT-qPCR all have equally high sensitivity, and the fold change accuracy is independent of individual miRNA concentration for NGS and RT-qPCR. Based on these findings we propose new guidelines and considerations when performing microRNA profiling.

RNA reprogramming and repair based on trans-splicing group I ribozymes.

While many traditional gene therapy strategies attempt to deliver new copies of wild-type genes back to cells harboring the defective genes, RNA-directed strategies offer a range of novel therapeutic applications. Revision or reprogramming of mRNA is a form of gene therapy that modifies mRNA without directly changing the transcriptional regulation or the genomic gene sequence. Group I ribozymes can be engineered to act in trans by recognizing a separate RNA molecule in a sequence-specific manner, and to covalently link a new RNA sequence to this separate RNA molecule. Group I ribozymes have been shown to repair defective transcripts that cause human genetic or malignant diseases, as well as to replace transcript sequences by foreign RNA resulting in new cellular functions. This review provides an overview of current strategies using trans-splicing group I ribozymes in RNA repair and reprogramming.

RNA reprogramming of alpha-mannosidase mRNA sequences in vitro by myxomycete group IC1 and IE ribozymes.

Trans-splicing group I ribozymes have been introduced in order to mediate RNA reprogramming (including RNA repair) of therapeutically relevant RNA transcripts. Efficient RNA reprogramming depends on the appropriate efficiency of the reaction, and several attempts, including optimization of target recognition and ribozyme catalysis, have been performed. In most studies, the Tetrahymena group IC1 ribozyme has been applied. Here we investigate the potential of group IC1 and group IE intron ribozymes, derived from the myxomycetes Didymium and Fuligo, in addition to the Tetrahymena ribozyme, for RNA reprogramming of a mutated alpha-mannosidase mRNA sequence. Randomized internal guide sequences were introduced for all four ribozymes and used to select accessible sites within isolated mutant alpha-mannosidase mRNA from mammalian COS-7 cells. Two accessible sites common to all the group I ribozymes were identified and further investigated in RNA reprogramming by trans-splicing analyses. All the myxomycete ribozymes performed the trans-splicing reaction with high fidelity, resulting in the conversion of mutated alpha-mannosidase RNA into wild-type sequence. RNA protection analysis revealed that the myxomycete ribozymes perform trans-splicing at approximately similar efficiencies as the Tetrahymena ribozyme. Interestingly, the relative efficiency among the ribozymes tested correlates with structural features of the P4-P6-folding domain, consistent with the fact that efficient folding is essential for group I intron trans-splicing.

Optimization and application of the group I ribozyme trans-splicing reaction.

Group I ribozymes are naturally occurring catalytic RNAs that are able to excise themselves as introns (group I introns) from a precursor RNA, and to ligate the flanking exons. Group I ribozymes can be engineered to act in trans by recognizing a separate RNA molecule in a sequence specific manner, and to covalently link an RNA sequence to this separate RNA molecule. This ribozyme transesterification reaction has potential in molecular biology and in medicine as a new approach to gene therapy. Here we describe detailed optimized protocols where trans-splicing group I ribozymes are applied in mapping accessible sites in target messenger RNA, and in messenger RNA-repair by correcting mutations.

The ability to form full-length intron RNA circles is a general property of nuclear group I introns.

In addition to splicing, group I intron RNA is capable of an alternative two-step processing pathway that results in the formation of full-length intron circular RNA. The circularization pathway is initiated by hydrolytic cleavage at the 3' splice site and followed by a transesterification reaction in which the intron terminal guanosine attacks the 5' splice site presented in a structure analogous to that of the first step of splicing. The products of the reactions are full-length circular intron and unligated exons. For this reason, the circularization reaction is to the benefit of the intron at the expense of the host. The circularization pathway has distinct structural requirements that differ from those of splicing and appears to be specifically suppressed in vivo. The ability to form full-length circles is found in all types of nuclear group I introns, including those from the Tetrahymena ribosomal DNA. The biological function of the full-length circles is not known, but the fact that the circles contain the entire genetic information of the intron suggests a role in intron mobility.