CircFISH: A Novel Method for the Simultaneous Imaging of Linear and Circular RNAs

Circular RNAs (circRNAs) are regulatory RNAs which have recently been shown to have clinical significance in several diseases, including, but not limited to, various cancers, neurological diseases and cardiovascular diseases. The function of such regulatory RNAs is largely dependent on their subcellular localization. Several circRNAs have been shown to conduct antagonistic roles compared to the products of the linear isoforms, and thus need to be characterized distinctly from the linear RNAs. However, conventional fluorescent in situ hybridization (FISH) techniques cannot be employed directly to distinguish the signals from linear and circular isoforms because most circRNAs share the same sequence with the linear RNAs. In order to address this unmet need, we adapted the well-established method of single-molecule FISH by designing two sets of probes to differentiate the linear and circular RNA isoforms by virtue of signal colocalization. We call this method ‘circular fluorescent in situ hybridization’ (circFISH). Linear and circular RNAs were successfully visualized and quantified at a single-molecule resolution in fixed cells. RNase R treatment during the circFISH reduced the levels of linear RNAs while the circRNA levels remain unaltered. Furthermore, cells with shRNAs specific to circRNA showed the loss of circRNA levels, whereas the linear RNA levels were unaffected. The optimization of the in-situ RNase R treatment allowed the multiplexing of circFISH to combine it with organelle staining. CircFISH was found to be compatible with multiple sample types, including cultured cells and fresh-frozen and formalin-fixed tissue sections. Thus, we present circFISH as a versatile method for the simultaneous visualization and quantification of the distribution and localization of linear and circular RNA in fixed cells and tissue samples.

Comprehensive Assessment of Serum hsa_circ_0070354 as a Novel Diagnostic and Predictive Biomarker in Non-small Cell Lung Cancer

Background: More and more studies have shown that circular RNAs (circRNAs) play an essential role in the occurrence and development of tumors. Hence, they can be used as biomarkers to assist in diagnosing tumors. This study focuses on exploring the role of circular RNA (hsa_circ_0070354) in the diagnosis and prognosis of non-small cell lung cancer (NSCLC).Materials and Methods: First of all, high-throughput sequencing was used to find the difference in the expression of circular RNA between NSCLC and adjacent tissues. The circRNAs with higher differences in expression were selected to verify their expressions in tissues, cells, and serum using qRT-PCR. Secondly, the hsa_circ_0070354 with a significant difference was chosen as the research goal, and the molecular properties were verified by agarose gel electrophoresis and Sanger sequencing, etc. Then, actinomycin D and repeated freeze-thaw were used to explore the stability and repeatability of hsa_circ_0070354. Finally, the expression of hsa_circ_0070354 in serum of 133 patients with NSCLC and 97 normal donors was detected, and its sensitivity, specificity, and prognosis as tumor markers were statistically analyzed.Results: Hsa_circ_0070354 was highly expressed in tissues, cells, and serum of NSCLC, and it has the characteristics of sensitivity, stability, and repeatability. The ROC curve indicates that hsa_circ_0070354 is superior to conventional tumor markers in detecting NSCLC, and the combined diagnosis is of more significance in the diagnosis. The high expression of hsa_circ_0070354 is closely related to the late-stage, poor differentiation of the tumor and the short survival time of the patients, which is an independent indicator of poor prognosis.Conclusion: Hsa_circ_0070354 is not only a novel sensitive index for the diagnosis of NSCLC but also a crucial marker for bad biological behavior.

The circular RNA circGlis3 protects against islet β-cell dysfunction and apoptosis during obesity

The molecular link between obesity and β-cell decompensation that causes diabetes remains incompletely understood. Here we found that circGlis3, a circular RNA derived from Glis3, plays a critical role in islet β-cell compensation. circGlis3 was increased in islets of obese mouse models and moderately diabetic individuals with compensated β-cell function by Quaking (QKI)-mediated splicing. Overexpression of circGlis3 functions to restrain islet β-cell dysfunction and maintain β-cell mass in high-fat diet (HFD) fed mice and Leprdb/db mice. The cellular levels of circGlis3 modulate both insulin synthesis and secretion and lipotoxicity-induced apoptosis, resulting in profound changes in β-cell compensation. In an obesity model, circGlis3 promotes the synthesis and secretion of insulin by upregulating NeuroD1 and Creb1 through sponging miR-124-3p. In addition, we identified SCOTIN and fused in sarcoma (FUS) as interacting proteins using quantitative mass spectrometry. We demonstrated that the binding of SCOTIN to circGlis3 regulated the apoptosis of β-cell. And more, FUS binding to circGlis3 could decrease free circGlis3 in cytoplasm and block mechanism of circGlis3 via abnormal stable formation of stress granules (SGs) in hyperactive response to chronic stresses in obesity that is thought to contribute to the β-cell decompensation. These findings highlight a physiological role for circRNAs in compensation and indicate that modulation of circGlis3 expression may represent a potential strategy to protect against islet β-cell dysfunction and apoptosis during obesity.