Fig 2: TIMP4 was a target of miR-92b-5p, and TIMP4 inhibition reversed the effects of miR-92b-5p knockdown on proliferation, apoptosis, and ECM degradation in LPS-induced OA chondrocytes. A The target genes predicted by TargetScan for miR-92b-5p were intersected with the significantly down-regulated mRNAs in OA cartilage from GSE113825. B The mRNAs expression of these target genes was tested by qRT-PCR in chondrocyte with miR-92b-5p overexpression or knockdown. C RIP-Ago2 analysis indicated binding of miR-92b-5p to TIMP4. D Interaction between miR-92b-5p and TIMP4 was affirmed by dual luciferase reporter assay. E Chondrocyte transfected with miR-92b-5p inhibitor or NC inhibitor was treated with 40 ng/mL LPS for 48 h. RT-qPCR was used to detect the mRNA expression of miR-92b-5p and TIMP4. F Chondrocyte was transfected with miR-92b-5p inhibitor alone or in combination with shTIMP4, and then treated with 40 ng/mL LPS for 48 h. CCK-8 was detected the viability of chondrocyte. G Chondrocyte was transfected with miR-92b-5p inhibitor alone or in combination with shTIMP4, and then treated with 40 ng/mL LPS for 48 h. The apoptosis of chondrocyte was tested by flow cytometry. H Chondrocyte was transfected with miR-92b-5p inhibitor alone or in combination with shTIMP4, and then treated with 40 ng/mL LPS for 48 h. Western blotting was applied to assess the protein expression of MMP13, ADAMTS5, Aggrecan, and Collagen II. LPS, lipopolysaccharide; NC mim., negative control corresponding to miR-92b-5p mimics; miR mim., miR-92b-5p mimics; NC inh., negative control corresponding to miR-92b-5p inhibitor; miR inh., miR-92b-5p inhibitor; shTIMP4, TIMP4 knockdown adenovirus. N = 3. n.s.P > 0.05, *P < 0.05, **P < 0.01, and.***P < 0.001

Fig 3: Overall schematic of epigenetic silencing of TIMP4 in heart failure.

Fig 4: WTAP knockdown inhibited LPS-induced OA chondrocyte damage by upregulating TIMP4. Chondrocyte was infected with WTAP knockdown adenovirus alone or in combination with TIMP4 knockdown adenovirus, and then treated with 40 ng/mL LPS for 48 h. A CCK-8 was detected the viability of chondrocyte. B EdU staining was applied to detect the proliferation of chondrocyte. C and E The apoptosis of chondrocyte was assessed by flow cytometry. D The caspase-3 activity was measured by the caspase-3 activity kit. F Western blotting was applied to assess the protein expression levels of MMP13, ADAMTS5, Aggrecan, and Collagen II. LPS, lipopolysaccharide; shTIMP4, TIMP4 knockdown adenovirus; shWTAP, WTAP knockdown adenovirus. N = 3. *P < 0.05, **P < 0.01, and.***P < 0.001

Fig 5: Altered tissue inhibitors of metalloprotease (TIMP) levels in nonfailing control (NFC) and dilated cardiomyopathy (DCM) hearts from pediatric and adult populations. Representative Western blots (A) and averaged protein quantification (B) for TIMP1, TIMP2, TIMP3, and TIMP4. n=12 to 16 per group. Data represent median±interquartile range. (C) A collective array showing a summary of protein expression levels for the molecules analyzed in this study in pediatric and adult NFC and DCM hearts. The color scale indicates the relative expression level. *P<0.05 compared with the corresponding control, † P<0.05 compared with the corresponding pediatric group. ADAMS indicates A disintegrin and metalloproteinases; ADAMTSs, a disintegrin and metalloproteinase with thrombospondin motifs; AU, arbitrary units; MMPs, matrix metalloproteases; sGAGs, sulfated glycosaminoglycans; SPARC, secreted protein acidic and cysteine‐rich; THBS1, thrombosondin‐1.