Fig 2: ALKBH5 increases HS3ST3B1-IT1 expression through an m6A-YTHDF2-dependent mechanism(A) Dot blot analyses with an anti-m6A antibody using normal and osteoarthritic cartilages. Methylene blue (MB) staining served as a loading control (n = 5).(B) The m6A modification sites in HS3ST3B1-IT1 were predicted with the online tool SRAMP (http://www.cuilab.cn/sramp).(C) The m6A level of HS3ST3B1-IT1 in chondrocytes was determined by MeRIP assay. The fold enrichment of HS3ST3B11-IT1 in m6A pellet was relative to its matching IgG control. Values were shown as mean ± SD, n = 3 biologically independent samples.(D) qRT-PCR was performed to detect the expression levels of ALKBH5 and HS3ST3B1-IT1 in chondrocytes after transfection with ALKBH5 expression plasmid. Values were shown as mean ± SD (n = 3).(E) The expression levels of ALKBH5 in normal and osteoarthritic cartilage were evaluated by qRT-PCR (n = 50). ß-actin served as an internal control. n = 50 normal cartilage tissues from 50 individuals with no prior medical history of OA and undergoing hip arthroplasty surgery, and n = 50 OA cartilages from 50 patients with OA undergoing total knee replacement surgery. Each dot represented one donor as calculated by log2 transform. The horizontal lines indicated the mean ± SD from different donors per group.(F) Associations between ALKBH5 and HS3ST3B1-IT1 in osteoarthritic cartilage (n = 50) were assessed by Pearson correlation analysis.(G) The m6A level of HS3ST3B1-IT1 was evaluated by MeRIP assay in chondrocytes after transfecting with ALKBH5 expression plasmid. Values were shown as mean ± SD, n = 3 biologically independent samples.(H) The chondrocytes were treated with Act D for the indicated times, and qRT-PCR was performed to evaluate the expression level of HS3ST3B1-IT1 upon ALKBH5 overexpression.(I) Left panel: the wild-type (wt) and m6A consensus sequence mutant (mut) HS3ST3B1-IT1 fused with firefly luciferase reporter. Right panel: luciferase reporter assay in HEK 293T cells transfected with luciferase reporter constructs (wt or mut) and ALKBH5 expression plasmid. Values were shown as mean ± SD, n = 3 biologically independent samples.(J) RIP assay was performed in chondrocytes with anti-YTHDF2 antibody, and the coprecipitated RNAs were subjected to detect HS3ST3B1-IT1 using qRT-PCR. The fold enrichment of HS3ST3B1-IT1 in YTHDF2 pellet is relative to its matching IgG control. Values were shown as mean ± SD, n = 3 biologically independent samples.(K) The chondrocytes were transfected with YTHDF2 wild type or mutant expression plasmids and then treated with Act D for the indicated times. qRT-PCR was performed to evaluate the expression level of HS3ST3B1-IT1. Values were shown as mean ± SD (n = 3). Statistical analysis was performed using an unpaired Student’s two-tailed t test (C–E, G, I–J). OA, osteoarthritis; MB, methylene blue; wt, wild-type; mut, mutant.

Fig 3: HS3ST3B1 increases chondrocyte viability, inhibits chondrocyte apoptosis, and regulates ECM metabolism(A and B) qRT-PCR (A) and western blotting (B) were performed to verify the overexpression and knockdown efficiencies of HS3ST3B1 in chondrocytes transfected with pCMV3-HS3ST3B1 plasmid or HS3ST3B1 siRNAs. Values were shown as mean ± SD (n = 3).(C) CCK-8 assay was used to identify the viability of chondrocytes with HS3ST3B1 overexpression or knockdown. Values were shown as mean ± SD (n = 3).(D) The apoptosis rates were evaluated in chondrocytes with HS3ST3B1 overexpression or knockdown by flow cytometry. Values were shown as mean ± SD (n = 3).(E) The expression levels of apoptosis-associated proteins were detected by western blotting in chondrocytes with HS3ST3B1 overexpression or knockdown. Representative blots from three independent experiments were shown (left panel). Densitometric analyses of the blots were presented as mean ± SD (n = 3, right panel).(F) The expression levels of ECM proteins (COL2A1 and Aggrecan) and cartilage-degrading enzymes (MMP13 and ADAMTS-5) were analyzed by western blotting in chondrocytes with HS3ST3B1 overexpression or knockdown. Representative blots from three independent experiments were shown (left panel). Densitometric analyses of the blots were presented as mean ± SD (n = 3, right panel). Statistical analysis was performed using an unpaired Student’s two-tailed t test (A, D–F) or a two-way ANOVA followed by Sidak’s multiple comparison test (C).

Fig 4: HS3ST3B1-IT1 attenuates OA progression in vivo(A) Schematic diagram illustrating the experimental design. Mice were randomly assigned into three groups: Ctrl+AAV-NC group (n = 7), MIA+AAV-NC group (n = 7), and MIA+ AAV-IT1 group (n = 7). For the induction of OA, mice were given an intra-articular injection of MIA in the knee. One week later, AAVs were administered once per week for 3 consecutive weeks. Six weeks later, mice were euthanized, and the knee joint tissues were separated for the subsequent experiments.(B) Histological sections of cartilage harvested from each group were stained with safranin-O/fast green and toluidine blue staining. Scale bar, 100 µm (n = 4 for each group).(C) The Mankin scoring system was used to evaluate articular cartilage of mice from each group (n = 4).(D) The proliferation of chondrocytes in mouse cartilage tissues was detected using Ki67 immunohistochemistry. Upper panel: representative images of Ki67 staining (scale bar, 100 µm). Inset boxes indicate the areas of the articular cartilage. Lower panel: the percentage of Ki67-positive chondrocytes in mouse articular cartilage of each group (n = 4).(E) Apoptosis of chondrocytes in mouse cartilage tissue was evaluated using the TUNEL assay. Left panel: representative images of TUNEL staining (scale bar, 100 µm). Dotted lines indicate the area of the articular cartilages. Right panel: the quantification of TUNEL-positive cells in the articular cartilages from each group (n = 4).(F) The expressions of COL2A1 and MMP13 in knee articular cartilage were detected by IHC staining (scale bar, 100 µm). Inset boxes indicate the area of articular cartilage (n = 4 for each group).(G) The expression levels of HS3ST3B1-IT1 in the knee articular cartilage were detected by qRT-PCR. Data were presented as the mean ± SD (n = 3). Statistical differences were determined using an unpaired Student’s two-tailed t test (C–-E and G). Saf-O/FG, safranin-O/fast green; TB, toluidine blue staining; AAV, adeno-associated virus; IT1, HS3ST3B1-IT1; MIA, monosodium iodoacetate; NC, negative control; ns, no significant difference.

Fig 5: LncRNA HS3ST3B1-IT1 and HS3ST3B1 are downregulated in human osteoarthritic cartilage(A and B) The expression levels of (A) HS3ST3B1-IT1 and (B) HS3ST3B1 in normal and osteoarthritic cartilage were evaluated by qRT-PCR. ß-actin served as an internal control. n = 50 normal cartilage tissues from 50 individuals with no prior medical history of OA and undergoing hip arthroplasty surgery, and n = 50 OA cartilages from 50 patients with OA undergoing total knee replacement surgery. Each dot represented one donor as calculated by log2 transform. The horizontal lines indicated the mean ± SD from different donors per group.(C) The expressions of HS3ST3B1 in normal and osteoarthritic cartilage were determined by IHC assays. Representative images from three independent experiments were shown in the left panel (scale bar, 200 µm). The quantification of cells positive for HS3ST3B1 was shown in the right panel. Values were shown as mean ± SD of n = 3 from three individual donors.(D) Associations between HS3ST3B1-IT1 and HS3ST3B1 in osteoarthritic cartilage (n = 50) were assessed by Pearson correlation analysis. Statistical analysis was performed using an unpaired Student’s two-tailed t test (A–C). OA, osteoarthritis.