Fig. 4: tsRNA-0032 regulates HLECs function by targeting PKM2.

A Fluorescence in situ hybridization (FISH) assays were conducted to detect the intracellular distribution of tsRNA-0032. Scale bar: 10 μm. B Nucleoplasmic separation assays were conducted to quantify the intracellular distribution of tsRNA-0032, with β-Actin and U6 serving as the cytoplasmic and nuclear controls, respectively. C Cellular fractions were isolated from HLECs and immunoprecipitated using Ago2 or IgG antibodies. The amount of tsRNA-0032 in the immunoprecipitates was determined by qRT-PCRs (n = 4, *P < 0.05, Student’s t-test). Western blots were conducted to detect the specific interaction between tsRNA-0032 and Ago2 (n = 4). D qRT-PCR assays were conducted to detect mRNA expression levels of PKM2 and FASN in HLECs transfected with negative control (NC) mimics, tsRNA-0032 mimics, NC inhibitors, tsRNA-0032 inhibitors, or left untreated (Ctrl) (n = 4, *P < 0.05, one-way ANOVA followed by Bonferroni test). E, F Western blots were conducted to detect the levels of PKM2 and FASN protein in HLECs transfected with NC mimics, tsRNA-0032 mimics, NC inhibitors, tsRNA-0032 inhibitors, or left untreated (Ctrl) (n = 4, one-way ANOVA followed by Bonferroni’s post hoc test, *P < 0.05 versus Ctrl group). G Nucleobase complementary alignment between tsRNA-0032 and PKM2 is shown. H The luciferase activity of WT-Luc-PKM2 or mutant Luc-PKM2 was determined after transfection with tsRNA-0032 mimics or NC mimics in HLECs (n = 4, Student’s t-test, *P < 0.05 versus Ctrl group). I The 3ʹ-end biotinylated tsRNA-0032 or biotinylated miR-484 was transfected into HLECs. After streptavidin capture, the levels of PKM2 and GAPDH in the input and bound fractions were detected by qRT-PCRs (n = 4, Student’s t-test, *P < 0.05 versus Ctrl group).