Fig. 2

TDP43 inhibits the biogenesis of circPSD3. (A) Putative TDP43 binding sites in PSD3 pre-mRNA were predicted using the online CircInteractome database. (B) Schematic showing the positions of qRT-PCR primers (A–E) on PSD3 pre-mRNA. A (UG)₁₁ repeat was identified at position E. (C) The direct binding of TDP43 to the PSD3 pre-mRNA was confirmed using a RIP assay. (D) The relative mRNA levels of TDP43 in 48 paired HCC and non-cancerous tissues were measured by qRT-PCR. (E) The protein levels of TDP43 in six paired HCC and non-cancerous tissues were measured using western blotting. (F) Immunohistochemical staining revealing the protein levels of TDP43 in a patient with HCC and PVTT. Scale bar, upper = 50 μm; lower = 20 μm. (G) Pearson correlation analysis showing a negative correlation between TDP43 and circPSD3 in 48 HCC tissues. (H) Western blotting verification of TDP43 knockdown in HCC cell lines transfected with the indicated siRNAs. (I and J) Relative levels of circPSD3 in HCC-LM9 and SK-Hep-1 cells transfected with TDP43 siRNAs. Data are shown as the mean ± SD. Statistical analyses were performed using unpaired Student’s t-tests (NS, no statistical significance; ***p < 0.001; ****p < 0.0001). circPSD3, circRNA pleckstrin and Sect. 7 domain containing 3; HCC, hepatocellular carcinoma; mRNA, messenger RNA; PVTT, portal vein tumor thrombosis; qRT-PCR, quantitative real-time PCR; RIP, RNA immunoprecipitation; siRNA, small interfering RNA; TDP43, TAR DNA-binding protein 43;N, non-tumorous tissues; T, tumor tissues