Fig 2: let-7b is upregulated in LPS-pre EVs, and knockdown of let-7b inhibits LPS-pre EVs-mediated effects on protein expression in vitro. a SMSCs were transfected with siRNA-Ago2, and transfection efficiency was evaluated using real-time PCR (n = 6, Student’s t test). b Protein expression of Ago-2 analyzed by western blot (n = 3, one-way ANOVA). c Protein expression of aggrecan, COL2A1, and ADAMTS5 in chondrocytes treated with NCKD-LPS-pre EVs or Ago2KD-LPS-pre EVs. d Heat map of miRNA levels between EVs and LPS-pre EVs groups (n = 3, Student’s t test). e Comparisons of the five most significantly upregulated miRNAs between EVs and LPS-pre EVs. f Venn diagram showing the five most significant targets for ADAMTS5 identified by three independent miRNA target prediction programs, and the target sequence of let-7b estimated within 3′-UTR of ADAMTS5. g Inhibitor-let-7b was transfected into chondrocytes, and RT-PCR was performed to evaluate transfection efficiency (n = 6, Student’s t test). h–i Aggrecan, COL2A1, and ADAMTS5 protein expression levels within chondrocytes after let-7b knockdown (n = 3, Student’s t test). j Luciferase reporter assay showing that ADAMTS5 is the target gene for let-7b (n = 3, Student’s t test). a, g, i, j Values are presented as mean ± SD. NS, no significance; *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test and one-way ANOVA

Fig 3: NEAT1 promotes HSCs activation via negatively regulating miR-342.A Schematic diagram of the miR-342 binding sites on NEAT1. B The dual-luciferase activity of the NEAT1 wild-type sequence (NEAT1-WT) and NEAT1 mutant sequence (NEAT1-MUT) in LX-2 cells transfected with miR-342 mimics or miR-342 inhibitor. C Relative NEAT1 expression level in Ago2 immunoprecipitates compared to IgG immunoprecipitates. D NEAT1 expression level was overexpressed or knocked down in LX-2 cells by qRT-PCR assay. E The impact of NEAT1 knockdown or overexpression on miR-342 expression in LX-2 cells was measured by qRT-PCR. F The impact of NEAT1 and miR-342 knockdown on miR-342 expression in LX-2 cells was measured by qRT-PCR. G The LX-2 cell proliferation was examined by the CCK-8 assay. H The cell cycle of the LX-2 was examined by the flow cytometry. I, J The migration of LX-2 was determined by the scratch assay (I) and the transwell assay (J). K The protein levels of collagen I, α-SMA, fibronectin, Sp1, TGF-β1, p-Smad2, Smad2, p-Smad3, and Smad3 were measured by western blot. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 4: TUG1 was upregulated in kidney tissues during renal IRI, and miR‐144‐3p was a direct target of TUG1. A, The TUG1 level was measured in normal and I/R‐injured kidney tissues by RT‐qPCR (n = 6). B, A schematic diagram showing the potential binding site between TUG1 and miR‐144‐3p. C, After luciferase construct and miR‐144‐3p mimic or miR‐NC were co‐transfected into TCMK cells, the relative luciferase activity was measured in TUG1‐Wt group and TUG1‐mut group (n = 3). D, RNA pulldown analysis using TUG1‐Wt showed that TUG1 directly interacted with miR‐144‐3p in TCMK cells, and TUG1‐Mut failed to pull down miR‐144‐3p (n = 3). E, After RIP, RT‐qPCR was used to detect the total amount of TUG1 bound to Ago2 or IgG (n = 3). All data are expressed as the mean ± SD. Differences between two groups were compared using unpaired t test. Data comparisons between multiple groups were performed using one‐way analysis of variance (ANOVA) with Tukey's post hoc test. *p < 0.05, **p < 0.01, NS, no significant difference

Fig 5: FBXL19 was negatively regulated by miR-532-3p. a The expression levels of three predicted mRNAs targeted by miR-532-3p were evaluated via RT-qPCR. b FBXL19 expression was measured by RT-qPCR in cells transfected with NC mimics or miR-532-3p mimics. c, d FBXL19 expression was evaluated by RT-qPCR in cells transfected with sh-SNHG10#1/2 or pcDNA3.1/SNHG10. e Binding sites between FBXL19 and miR-532-3p were shown. f RNA pull down experiments validated that FBXL19 could bind to miR-532-3p. g Luciferase reporter assay certified the influence of miR-532-3p on the luciferase activity of FBXL19-WT/Mut. h RIP validated that SNHG10, miR-532-3p and FBXL19 were co-existed in Ago2-assembled RNA induced silencing complexes (RISCs). *P < 0.05, **P < 0.01, n.s. presented no significance