Article Title: Circular RNA CpG island hypermethylation-associated silencing in human cancer
Figure Lengend Snippet: circRNA effects on linear transcripts, miRNAs and tumor growth ( A ) Upon efficient transduction of TUSC3 circ104557 in HCT-116 cells (harboring a methylated CpG island), the TUSC3 linear RNA levels did not change: it was not detected in any of the conditions tested. ( B ) TUSC3 circ104557 transduction in DKO cells (harboring an unmethylated CpG island) did not affect the levels of TUSC3 linear RNA. RNA levels were determined using circular or linear specific qRT-PCR primers. The lentiviral transduction of the empty vector (Mock condition) was used as a control. Experiments were performed in technical triplicates. ( C ) Expression of candidate miRNAs putatively targeted by ATRNL1 circ100686 (miR-378a-3p), SAMD4A circ101356 (miR-660-5p and miR-330-3p) and TUSC3 circ104557 (miR-330-3p) was significantly downregulated in DKO cells, evaluated in three biological replicates by real-time quantitative PCR using TaqMan Advanced MicroRNA Assays. Expression levels were normalized using hsa-miR-345-5p, hsa-miR-191-5p and hsa-miR-423-3p advanced Control miRNA Assays. ( D ) Using the same strategy, expression of miR-330-3p, putatively targeted by TUSC3 circ104557, was also assessed in the gain-of-function cellular model. A significant downregulation of miR-330-3p was detected upon TUSC3 circ104557 transduction in HCT116 cells. ( E ) HCT116-Mock and HCT116-TUSC3 circular cells were injected in the left or right flank of 10 mice, respectively. Tumor volume measured over time (left panel) and tumor weight upon sacrifice (right panel) are shown. Tumor growth was significantly reduced upon TUSC3 circular ectopic expression. ns, nonsignificant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, using Student’s t -test. Error bars show means ± s.d.
Article Snippet: In brief, TaqMan™ Advanced miRNA cDNA Synthesis Kit (ThermoFisher, Cat. No. ) was used for retrotranscription.
Techniques: Transduction, Methylation, Quantitative RT-PCR, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Injection, Mouse Assay