Identification of senescence-associated circular RNAs (SAC-RNAs) reveals senescence suppressor CircPVT1

Abstract

Using RNA sequencing (RNA-Seq), we compared the expression patterns of circular RNAs in proliferating (early-passage) and senescent (late-passage) human diploid WI-38 fibroblasts. Among the differentially expressed senescence-associated circRNAs (which we termed ‘SAC-RNAs’), we identified CircPVT1, generated by circularization of an exon of the PVT1 gene, as a circular RNA showing markedly reduced levels in senescent fibroblasts. Reducing CircPVT1 levels in proliferating fibroblasts triggered senescence, as determined by a rise in senescence-associated β-galactosidase activity, higher abundance of CDKN1A/P21 and TP53, and reduced cell proliferation. Although several microRNAs were predicted to bind CircPVT1, only let-7 was found enriched after pulldown of endogenous CircPVT1, suggesting that CircPVT1 might selectively modulate let-7 activity and hence expression of let-7-regulated mRNAs. Reporter analysis revealed that CircPVT1 decreased the cellular pool of available let-7, and antagonizing endogenous let-7 triggered cell proliferation. Importantly, silencing CircPVT1 promoted cell senescence and reversed the proliferative phenotype observed after let-7 function was impaired. Consequently, the levels of several proliferative proteins that prevent senescence, such as IGF2BP1, KRAS and HMGA2, encoded by let-7 target mRNAs, were reduced by silencing CircPVT1. Our findings indicate that the SAC-RNA CircPVT1, elevated in dividing cells and reduced in senescent cells, sequesters let-7 to enable a proliferative phenotype.

Detection and Analysis of Circular RNAs by RT-PCR

“…As shown in Figure 3, following RNase R treatment, the linear X mRNA and Y mRNA are depleted to a level lower than 10%, while Y circRNA was not degraded (Table 1). The fact that Y circRNA level did not show depletion while the counterpart linear Y mRNA depleted to a minimal level with RNase R treatment supports the notion that RNase R degrades linear RNAs specifically leading to enrichment of circRNA population (Figure 3) (Panda et al ., 2017c). …”

“…The PCR product sequence should match exactly the expected circRNA junction sequence as predicted from the RNA-seq (Panda et al ., 2017a and 2017c). However, this analysis does not inform on whether the backsplice junction sequence is coming from a scrambled exon linear transcript or a real backsplice junction.…”

“…The vast family of ncRNAs comprises diverse regulatory RNAs, such as microRNAs and long noncoding (lnc)RNAs, but also the poorly explored class of circular (circ)RNAs. Although first discovered more than three decades ago by electron microscopy, only the advent of high-throughput RNA-sequencing (RNA-seq) and the development of innovative bioinformatic pipelines have begun to allow the systematic identification of circRNAs (Szabo and Salzman, 2016; Panda et al ., 2017b; Panda et al ., 2017c). However, the validation of true circRNAs identified by RNA sequencing requires other molecular biology techniques including reverse transcription (RT) followed by conventional or quantitative (q) polymerase chain reaction (PCR), and Northern blot analysis (Jeck and Sharpless, 2014).…”

Circular RNA circPVT1 Promotes Proliferation and Invasion Through Sponging miR-125b and Activating E2F2 Signaling in Non-Small Cell Lung Cancer

“…Verduci et al also revealed an oncogenic role of circPVT1 in head and neck squamous cell carcinoma through the mutant p53/YAP/TEAD transcription-competent complex [16]. In addition, Panda et al found that circPVT1 is a senescence-associated circular RNA, and it is elevated in dividing cells and reduced in senescent cells, sequesters let-7 to enable a proliferative phenotype [18]. The specific role of circPVT1 in lung cancer is still unknown.…”

“…CircPVT1 is a circular RNA derived from one exon of the PVT1 gene and flanks two long introns (35269 bp and 41466 bp), which harbor many Alu repeats and may facilitate circPVT1 formation. CircPVT1 was first identified as circ6 by Memczak et al [15] and then named circPVT1 after its host gene PVT1 in subsequent work [16][17][18]. They demonstrated that circPVT1 plays an oncogenic role in gastric cancer and head and neck squamous cell carcinoma, however, the specific role of this circular RNA is largely unknown, including NSCLC.…”