Restoring expression of tumour suppressor PTEN by engineered circular RNA‐enhanced Osimertinib sensitivity in non‐small cell lung cancer

Abstract

Dear Editor,This study provides a new strategy to construct circular RNA (circRNA) in vitro named NeoAna, with splicing sites concealed in CVB3_IRES. Re-storing phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression by engineered circRNA enhances sensitivity to Osimertinib in non-small lung cancer (NSCLC).Previous Anabaena permuted intron-exon system could permit the circularisation of sequences up to 5 kb in length, significantly longer than previously reported; however, it is important to acknowledge the presence of 'scar sequences' in the final products (Figure 1A). [1][2][3] We designed the NeoAna systems to synthesise circRNAs (Figure 1B) without scar sequences (Figure S1). As shown in Figure 1C, the circRNA (enhanced green fluorescent protein [EGFP], as an example) is clearly observed and resistance to RNase R treatment. Indeed, the formation of circRNA was further confirmed by PCR and the exact splicing site was determined by Sanger sequencing (Figure 1D,E). Successful protein translation was confirmed in cells (Figure 1F,H). Then, in vitro transcription (IVT) products of NeoAna system were subjected to high-performance liquid chromatography and each fraction was transfected into 293T cells, and the main peak showed strongest protein expression (Figure 1G,I-K). Then, we synthesised pseudo-uridinemodified linear EGFP (m1ψ-EGFP), cEGFP_Ana and cEGFP_NeoAna (Figure S2A) and transfected three RNAs into 293T and H1299 cells and green fluorescence and protein expression were observed in cells (Figure S2B,C). Compared with cEGFP_Ana, cEGFP_NeoAna induced weaker innate immunity response in 293T cells (Figure 1L). Besides, we found that the stability of cEGFP_NeoAna is comparable to that of cEGFP_Ana (Figure S3A).