Research Paper Volume 13, Issue 4 pp 5383—5402

CircMRE11A_013 binds to UBXN1 and integrates ATM activation enhancing lens epithelial cells senescence in age-related cataract

Identification of circMRE11A, a circular RNA implicated in LECs of ARC and SRA01/04 cell lines, circMRE11A was up-regulated in ARCC compared with controls. (A). Schematic illustration showed that the genomic location of circMRE11A generated from its host gene, validating by Sanger sequencing. Sanger sequencing analysis showed head-to-tail splicing junction of circMRE11A. (B). RT-PCR or PCR assay indicated the detection of circMRE11A using divergent or convergent primers from cDNA or genomic DNA (gDNA) of LECs with and without RNase R treatment (RNase R– / +). (C). Northern blot using a junction-specific the Bioth-labeled probe indicated the endogenous existence of circMRE11A treated with and without RNase R (2 U/μg) in the cell lines at 10 min or 30 min. 18S was used as a specific probe for the internal control. (D). RNA-FISH assay showed the cytoplasmic and nuclear localization of circMRE11A in SRA04/01 cells using a junction specific probe (red), with the nuclei staining with DAPI (blue). Scale bar: 20 μm. (E). qRT-PCR indicated the distribution of circMRE11A, GAPDH, β-actin and U6 in the cytoplasmic and nuclear fractions of SRA04/01 cells. U6 was treated as a nuclear control while GAPDH or β-actin was a cytoplasmic control, ***p F). qTR-PCR assays determined circMRE11A levels in LECs of ARCs (controls (n=10), ARC-C(n=10), ARC-N (n=10), ARC-P(n=10)). GAPDH was an internal control. *p

Figure 1. Identification of circMRE11A, a circular RNA implicated in LECs of ARC and SRA01/04 cell lines, circMRE11A was up-regulated in ARCC compared with controls. (A). Schematic illustration showed that the genomic location of circMRE11A generated from its host gene, validating by Sanger sequencing. Sanger sequencing analysis showed head-to-tail splicing junction of circMRE11A. (B). RT-PCR or PCR assay indicated the detection of circMRE11A using divergent or convergent primers from cDNA or genomic DNA (gDNA) of LECs with and without RNase R treatment (RNase R– / +). (C). Northern blot using a junction-specific the Bioth-labeled probe indicated the endogenous existence of circMRE11A treated with and without RNase R (2 U/μg) in the cell lines at 10 min or 30 min. 18S was used as a specific probe for the internal control. (D). RNA-FISH assay showed the cytoplasmic and nuclear localization of circMRE11A in SRA04/01 cells using a junction specific probe (red), with the nuclei staining with DAPI (blue). Scale bar: 20 μm. (E). qRT-PCR indicated the distribution of circMRE11A, GAPDH, β-actin and U6 in the cytoplasmic and nuclear fractions of SRA04/01 cells. U6 was treated as a nuclear control while GAPDH or β-actin was a cytoplasmic control, ***p < 0.001. (F). qTR-PCR assays determined circMRE11A levels in LECs of ARCs (controls (n=10), ARC-C(n=10), ARC-N (n=10), ARC-P(n=10)). GAPDH was an internal control. *p < 0.05.