The biogenesis, biology and characterization of circular RNAs

Pan

et al. 2020

Thoracic Cancer

Background Circular RNAs (circRNAs) participate in the development of human cancers by regulating multiple cell processes. CircRNA antisense to the cerebellar degeneration‐related protein 1 transcript (circCDR1as) expression is dysregulated in many cancers, including non‐small‐cell lung cancer (NSCLC). However, the mechanism by which circCDR1as mediates the development of NSCLC remains unknown. Methods A total of 30 paired cancer and normal tissues were collected from patients with NSCLC. The expression levels of circCDR1as, microRNA (miR)‐219a‐5p and Sex determining region Y‐box protein 5 (SOX5) were measured in tissues or cells by quantitative real‐time polymerase chain reaction or western blot. Cell viability, apoptosis, migration and invasion were detected by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide, colony formation, flow cytometry and transwell assays, respectively. The target relationship between miR‐219a‐5p and circCDR1as or SOX5 was validated by dual‐luciferase reporter assay. Results CircCDR1as expression was elevated in NSCLC tissues and cells in comparison to the matched controls. Interference of circCDR1as led to obvious inhibition of cell viability, migration and invasion and increase of apoptosis in NSCLC cells. MiR‐219a‐5p acted as a target of circCDR1as and miR‐219a‐5p downregulation attenuated the regulatory effect of circCDR1as silencing on NSCLC progression. Moreover, miR‐219a‐5p targeted SOX5 to repress the progression of NSCLC in vitro. Besides, circCDR1as knockdown reduced the expression of SOX5 by increasing miR‐219a‐5p level. Conclusion Knockdown of circCDR1as inhibited the progression of NSCLC by decreasing cell viability, migration and invasion and increasing apoptosis by upregulating miR‐219a‐5p and downregulating SOX5.

Section: Introductionmentioning

confidence: 99%

“…5 Among these types, circular RNA (circRNA) consists of a continuous closed loop. 6 Several circRNAs have been reported to be aberrantly expressed and associated with the tumorigenesis of lung cancer. 7 Furthermore, emerging evidence suggests that several circRNAs might contribute to the progression of NSCLC.…”

Section: Introductionmentioning

confidence: 99%

CircRNA CDR1as knockdown inhibits progression of non‐small‐cell lung cancer by regulating miR‐219a‐5p/SOX5 axis

Pan

et al. 2020

Thoracic Cancer

“…Circular RNAs (circRNAs) are a newly discovered type of endogenous noncoding RNAs (ncRNAs) that are stably expressed in the eukaryotic transcriptome . Unlike other linear RNAs, circRNAs have a covalently closed, continuous loop structure .…”

Section: Introductionmentioning

confidence: 99%

Circular RNA hsa_circ_0000654 promotes esophageal squamous cell carcinoma progression by regulating the miR‐149‐5p/IL‐6/STAT3 pathway

Xu¹,

Xiaojing²,

Li³

et al. 2019

IUBMB Life

Circular RNAs (circRNAs) are novel noncoding RNAs (ncRNAs) with covalently closed‐loop structures that play an essential regulatory role in diverse malignancies, including esophageal squamous cell cancer (ESCC). Circ_0000654 expression in ESCC and its mechanism of action remains unclear. Real‐time PCR (RT‐PCR) was employed to detect circ_0000654 and miR‐149‐5p expression in ESCC tissues. Circ_0000654 and miR‐149‐5p expression in ESCC cells was selectively regulated. Furthermore, interleukin 6 (IL‐6) and signal transducer and activator of transcription 3 (STAT3) expression in cells was detected by RT‐PCR and western blot analysis, while CCK8, BrdU, flow cytometry, and transwell assays were used to monitor cell proliferation, apoptosis, migration and invasion, respectively. The dual‐luciferase reporter assay and RIP assay were used to verify the targeting relationship between circ_0000654 and miR‐149‐5p, miR‐149‐5p and IL‐6. The function of circ_0000654 on ESCC cell proliferation and metastasis in vivo was examined using a subcutaneous xenograft model and a tail intravenous injection model in nude mice. Circ_0000654 was significantly upregulated in ESCC tissues and cell lines, and its high expression was remarkably associated with an increased T stage and local lymph node metastasis in ESCC patients. Circ_0000654 overexpression and knockdown experiments revealed that circ_0000654 regulated ESCC cell proliferation, migration, invasion, and apoptosis in vitro. Circ_0000654 was identified as a sponge of miR‐149‐5p and facilitated ESCC progression by indirectly activating the IL‐6/STAT3 signaling pathway. Additionally, knocking down circ_0000654 strikingly repressed ESCC growth and metastasis in vivo. In summary, circ_0000654 functions as an oncogenic circRNA in ESCC and accelerates ESCC progression via adsorbing miR‐149‐5p and activating the IL‐6/STAT3 signaling pathway.

“…Thousands of circRNAs have been discovered across species with cell-type-and tissue-specific expression patterns 3-6 . However, the functional repertoire of circRNAs remains mostly uncharacterized to date, which is mainly due to the unique properties of circRNAs and limitations of current approaches in circRNA studies 7,8 . The rapid development of CRISPR-Cas9 based genomics screens has dramatically enhanced speed and precision of functional characterization of both coding genes and linear non-coning RNAs 9 .…”

mentioning

confidence: 99%

Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying essential circRNAs

Nguyen

et al. 2020

Preprint

Circular RNAs (circRNAs) are widely expressed, but their functions remain largely unknown.To study circRNAs in a high-throughput manner, short hairpin RNA (shRNA) screens 1 have recently been used to deplete circRNAs by targeting their unique back-splicing junction (BSJ) sites. Here, we report frequent discrepancies between shRNA-mediated circRNA knockdown efficiency and the corresponding biological effect, raising pressing concerns about the robustness of shRNA screening for functional circRNAs. To address this issue, we leveraged the CRISPR/Cas13d system 2 for circRNAs functional screenings. We optimized a strategy for designing single guide RNAs to deplete circRNAs. We then performed shRNA and CRISPR/Cas13d parallel screenings and demonstrated that shRNA-mediated circRNAs screening yielded a high rate of false positives phenotypes, while optimized CRISPR/Cas13d led to the identification of bona-fide functional circRNAs. Collectively, we developed a specific and reliable approach to functionalize circRNAs in a high-throughput manner. MainCircular RNAs (circRNAs) are covalently closed, single stranded transcripts, which are produced by back-splicing of precursor mRNAs (pre-mRNAs). Thousands of circRNAs have been discovered across species with cell-type-and tissue-specific expression patterns 3-6 . However, the functional repertoire of circRNAs remains mostly uncharacterized to date, which is mainly due to the unique properties of circRNAs and limitations of current approaches in circRNA studies 7,8 . The rapid development of CRISPR-Cas9 based genomics screens has dramatically enhanced speed and precision of functional characterization of both coding genes and linear non-coning RNAs 9 . Nevertheless, the majority of circRNAs are generated from protein-coding genes 10 , and hence the sequences of circRNAs are completely overlapping with their cognate linear RNAs