嵌合RNA的形成机制及生物学意义

传统观念认为,嵌合RNA只是由染色体重排导致的基因融合,且融合基因及其产物(RNA和蛋白质)曾被认为是癌症的独有特征。然而,随着测序技术的进步和生物信息学软件工具的开发,通过对RNA-Seq数据库分析,越来越多的嵌合RNA被分离和鉴定出来。近年的研究表明,嵌合RNA并不是癌症所特有的现象,它广泛存在于人类多种正常组织和细胞中。除了染色体重排之外,嵌合RNA还有多种不同的分子形成机制,包括相邻基因的顺式剪接和反式剪接等。未发生染色体改变的嵌合RNA在转录水平上受到调控,从而呈现出独特的调控模式,其失调可能影响细胞分化并诱导肿瘤的发生。此外,嵌合RNA还发挥特定的生理功能,包括影响正常细胞生长和迁移能力,调控细胞周期及凋亡。通过影响染色体重排从而诱导基因组畸变,亦可作为潜在的竞争性内源RNA,以及影响干细胞的分化等。了解嵌合RNA在组织和细胞发育不同阶段的特异性表达,将有助于发掘潜在的临床诊疗生物标志物。深入且准确地对嵌合RNA的组织学图谱进行研究,可能实现从崭新的视角对特定细胞类型进行嵌合RNA治疗。越来越多的实验数据表明,嵌合RNA广泛存在于癌症和正常组织中,且具有重要的生理功能,其表达水平和模式也是高等动物拓展基因组功能的方式之一。     

RNA-driven JAZF1-SUZ12 gene fusion in human endometrial stromal cells

Oncogenic fusion genes as the result of chromosomal rearrangements are important for understanding genome instability in cancer cells and developing useful cancer therapies. To date, the mechanisms that create such oncogenic fusion genes are poorly understood. Previously we reported an unappreciated RNA-driven mechanism in human prostate cells in which the expression of chimeric RNA induces specified gene fusions in a sequence-dependent manner. One fundamental question yet to be addressed is whether such RNA-driven gene fusion mechanism is generalizable, or rather, a special case restricted to prostate cells. In this report, we demonstrated that the expression of designed chimeric RNAs in human endometrial stromal cells leads to the formation of JAZF1-SUZ12, a cancer fusion gene commonly found in low-grade endometrial stromal sarcomas. The process is specified by the sequence of chimeric RNA involved and inhibited by estrogen or progesterone. Furthermore, it is the antisense rather than sense chimeric RNAs that effectively drive JAZF1-SUZ12 gene fusion. The induced fusion gene is validated both at the RNA and the genomic DNA level. The ability of designed chimeric RNAs to drive and recapitulate the formation of JAZF1-SUZ12 gene fusion in endometrial cells represents another independent case of RNA-driven gene fusion, suggesting that RNA-driven genomic recombination is a permissible mechanism in mammalian cells. The results could have fundamental implications in the role of RNA in genome stability, and provide important insight in early disease mechanisms related to the formation of cancer fusion genes.     

Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras

Technologies that mediate targeted delivery of small interfering RNAs (siRNAs) are needed to improve their therapeutic efficacy and safety. Therefore, we have developed aptamer-siRNA chimeric RNAs capable of cell type-specific binding and delivery of functional siRNAs into cells. The aptamer portion of the chimeras mediates binding to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium, whereas the siRNA portion targets the expression of survival genes. When applied to cells expressing PSMA, these RNAs are internalized and processed by Dicer, resulting in depletion of the siRNA target proteins and cell death. In contrast, the chimeras do not bind to or function in cells that do not express PSMA. These reagents also specifically inhibit tumor growth and mediate tumor regression in a xenograft model of prostate cancer. These studies demonstrate an approach for targeted delivery of siRNAs with numerous potential applications, including cancer therapeutics.     

嵌合RNA检测和验证技术

嵌合RNA(chimeric RNA)是由来自不同基因的外显子片段组成的融合转录本。传统的嵌合RNA检测方法有染色体核型分析、荧光原位杂交（FISH）等,但这些技术的特异性、灵敏性和准确性较差。随着测序技术的发展,二代测序技术展现出强大的数据处理能力,可以通过高通量序列分析来检测嵌合RNA,目前基于高通量测序的检测方法有FusionCatcher、SOAPfuse、EricScript等。目前较为常用的对检测到的嵌合RNA的验证方法有聚合酶链反应（PCR）、核糖核酸酶保护实验（RPA）、琼脂糖凝胶电泳、Sanger测序等。多种检测技术的开发使得越来越多的嵌合RNA被发现,但现有的检测技术各有优劣,主要体现于检测成本、假阳性率、检测时间等方面的差异。本文对嵌合RNA的检测方法、验证方法及各方法的优劣性进行阐述。     

A general approach to high-yield biosynthesis of chimeric RNAs bearing various types of functional small RNAs for broad applications

RNA research and therapy relies primarily on synthetic RNAs. We employed recombinant RNA technology toward large-scale production of pre-miRNA agents in bacteria, but found the majority of target RNAs were not or negligibly expressed. We thus developed a novel strategy to achieve consistent high-yield biosynthesis of chimeric RNAs carrying various small RNAs (e.g. miRNAs, siRNAs and RNA aptamers), which was based upon an optimal noncoding RNA scaffold (OnRS) derived from tRNA fusion pre-miR-34a (tRNA/mir-34a). Multi-milligrams of chimeric RNAs (e.g. OnRS/miR-124, OnRS/GFP-siRNA, OnRS/Neg (scrambled RNA) and OnRS/MGA (malachite green aptamer)) were readily obtained from 1 l bacterial culture. Deep sequencing analyses revealed that mature miR-124 and target GFP-siRNA were selectively released from chimeric RNAs in human cells. Consequently, OnRS/miR-124 was active in suppressing miR-124 target gene expression and controlling cellular processes, and OnRS/GFP-siRNA was effective in knocking down GFP mRNA levels and fluorescent intensity in ES-2/GFP cells and GFP-transgenic mice. Furthermore, the OnRS/MGA sensor offered a specific strong fluorescence upon binding MG, which was utilized as label-free substrate to accurately determine serum RNase activities in pancreatic cancer patients. These results demonstrate that OnRS-based bioengineering is a common, robust and versatile strategy to assemble various types of small RNAs for broad applications.     

Crosstalk between lncRNAs in the apoptotic pathway and therapeutic targets in cancer

The assertion that a significant portion of the mammalian genome has not been translated and that non-coding RNA accounts for over half of polyadenylate RNA have received much attention. In recent years, increasing evidence proposes non-coding RNAs (ncRNAs) as new regulators of various cellular processes, including cancer progression and nerve damage. Apoptosis is a type of programmed cell death critical for homeostasis and tissue development. Cancer cells often have inhibited apoptotic pathways. It has recently been demonstrated that up/down-regulation of various lncRNAs in certain types of tumors shapes cancer cells' response to apoptotic stimuli. This review discusses the most recent studies on lncRNAs and apoptosis in healthy and cancer cells. In addition, the role of lncRNAs as novel targets for cancer therapy is reviewed here. Finally, since it has been shown that lncRNA expression is associated with specific types of cancer, the potential for using lncRNAs as biomarkers is also discussed.     

Fusion Peptides from Oncogenic Chimeric Proteins as Putative Specific Biomarkers of Cancer

Chromosomal translocations encoding chimeric fusion proteins constitute one of the most common mechanisms underlying oncogenic transformation in human cancer. Fusion peptides resulting from such oncogenic chimeric fusions, though unique to specific cancer subtypes, are unexplored as cancer biomarkers. Here we show, using an approach termed fusion peptide multiple reaction monitoring mass spectrometry, the direct identification of different cancer-specific fusion peptides arising from protein chimeras that are generated from the juxtaposition of heterologous genes fused by recurrent chromosomal translocations. Using fusion peptide multiple reaction monitoring mass spectrometry in a clinically relevant scenario, we demonstrate the specific, sensitive, and unambiguous detection of a specific diagnostic fusion peptide in clinical samples of anaplastic large cell lymphoma, but not in a diverse array of benign lymph nodes or other forms of primary malignant lymphomas and cancer-derived cell lines. Our studies highlight the utility of fusion peptides as cancer biomarkers and carry broad implications for the use of protein biomarkers in cancer detection and monitoring.A cancer biomarker is generally an analyte that indicates the presence or extent of a specific form of cancer. A useful cancer biomarker should reliably distinguish between benign and malignant states and, ideally, distinguish one form of cancer from other, related differential diagnoses. Many human cancers contain recurrent chromosomal translocations and chimeric gene fusions that could be exploited as cancer-specific biomarkers (1, 2). Indeed, several structural aberrations are specific and pathognomonic for distinct types of cancer (3). Moreover, as new molecular therapies increasingly target oncogenic fusion proteins, the detection and quantitation of these proteins may also provide important, direct therapeutic guidance (4–6). Although genomic techniques targeting fusion partner genes are routinely used for diagnosing cancers, fusion peptides resulting from oncogenic chimeric fusions are unexplored as biomarker candidates for cancer detection. The specificity and qualitative/binary nature (i.e. present or absent) of fusion proteins in specific tumor types make these analytes attractive candidates for cancer detection.Advances in mass spectrometry permit the direct and unbiased interrogation of proteins and peptides in complex mixtures with unambiguous identification of specific proteins (7, 8). Multiple reaction monitoring (MRM)¹ via mass spectrometry is a powerful approach for the targeted detection of biomarker candidates in a complex background (9). MRM involves the focused interrogation of specific m/z windows for the precursor analyte, as well as selected fragment ions, following MS/MS analysis. By focusing only on specific m/z windows, one increases the sensitivity of detection dramatically, and within the context of a complex mixture there is the potential for a reproducible dynamic range spanning ≥4 orders of magnitude (10, 11).Despite their enormous potential as biomarkers, fusion peptides resulting from oncogenic chimeric fusions have not been exploited for the specific and sensitive detection of cancer. Here we demonstrate the detection of unique fusion peptides that are specific for various forms of cancer. To demonstrate applicability in a clinically relevant scenario, we show the utility of our MRM-based MS approach combined with an innovative double stable isotope strategy for the identification of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion peptide arising from the corresponding chimeric fusion protein for the identification of NPM-ALK-positive anaplastic large cell lymphoma (ALCL). We show the exquisite specificity and sensitivity of this fusion peptide (FP) MRM approach and the extraordinary accuracy of its application with clinical biopsy material.