1. Volume 25, Issue 12, Pages 2676-2688 (December 2017)
MicroRNA-218-5p as a Potential Target for the Treatment of Human Osteoarthritis 
Jun Lu, Ming-liang Ji, Xue-jun Zhang, Pei-liang Shi, Hao Wu, Chen Wang, Hee-Jeong Im 
Molecular Therapy 
Volume 25, Issue 12, Pages (December 2017)
DOI: /j.ymthe
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2. Figure 1
Identification of Differentially Expressed miRNAs in OA Cartilage Tissues
(A) Hierarchical clustering performed with average linkage and uncentered correlation showed a distinguishable miRNA expression profile between OA and NC. Red represents higher and green represents lower expression relative to the mean intensity value (black) across all samples (paired t test). (B) Volcano plot illustrating the biological and statistical significances of differential miRNA expressions between OA and NC. The red points in the plot represent the differentially expressed miRNA with statistical significance (≥2-fold or ≤ 0.5-fold, Benjamini-Hochberg-corrected P). (C) Compared with controls, miR-218-5p expression level was higher in moderate (22) and severe (58) OA patients. ***p < by one-way ANOVA test followed by post hoc SNK test. (D–F) ISH analysis of miR-218-5p expression patterns from control (D), moderate OA (E), and severe OA (F) (scale bars, 50 μm). The cartilage was obtained from the middle zone. (G) The miR-218-5p expression level was positively correlated with the Mankin scale (r = 0.746, p < 0.001). NC, normal control.
Molecular Therapy  , DOI: ( /j.ymthe )
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3. Figure 2 In Vitro Study of miR-218-5p
(A) miR-218-5p transfecting primary chondrocytes, as confirmed by Cy5-oligonucleotides. (B) 50 nM miR-control, miR-218-5p mimic, or inhibitor was transfected into primary human chondrocytes. 48 hr after transfection, the cells were used for the following experiments. Transfection efficiency of miR-218-5p was analyzed using qRT-PCR (n = 6 replicates per group). ***p < by one-way ANOVA test followed by Tukey’s multiple comparisons post-test. (C) Chondrocyte proliferation was investigated in miR-218-5p mimic or inhibitor transfected primary human chondrocytes at 24, 48, 72, and 96 hr, respectively (n = 3 replicates per group). ***p < by two-way ANOVA test. (D–F) Analysis of chondrocyte apoptosis in blank (D), miR-218-5p inhibitor (E), or mimic (F) -transfected primary human chondrocytes (n = 3 replicates per group). (G) The expressions of Col II, aggrecan, MMP13, and ADAMTS-5 were detected by western blot. (H) Type II collagen expression of transfected chondrocytes was assessed using laser scanning confocal microscopy under a 100 objective.
Molecular Therapy  , DOI: ( /j.ymthe )
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4. Figure 3 In Vivo Study of miR-218-5p
(A) Compared with the SS group, the OA group showed a higher level of miR-218-5p (n = 15 per group) at 8 weeks post-operation. ***p < by Student’s unpaired t test. (B) OA model was established. qRT-PCR analysis of mRNA expression of COL2A1, ACAN, MMP13, and ADAMTS-5 in cartilage tissue from the OA model treated with the intra-articular delivery of LV-miR-control or LV-miR-218-5p inhibitor. OA groups, treated with the intra-articular delivery of saline; OA + miR-control group, treated with the intra-articular delivery of LV-miR-control; OA + miR-218-5p inhibitor group, treated with the intra-articular delivery of LV-miR-218-5p inhibitor. Compared with the OA and OA + miR-control groups, the OA + miR-218-5p inhibitor group demonstrated increased levels of COL2A1 and ACAN and decreased MMP13 and ADAMTS-5 levels at 8 weeks post-operation (n = 15 per group). The OA and OA + miR control groups showed higher levels of MMP13 and ADAMTS-5 and lower levels of COL2A1 and ACAN. ***p < by post hoc SNK test. (C) To investigate whether miR-218-5p can penetrate into the cartilage, sections of mice knees were examined after the injection of GFP-tagged LV (lentivirus)-miR-218-5p inhibitor and LV-Scr (scramble). GFP was observed in the cartilage and synovium, indicating that miR-218-5p could penetrate both the synovium and cartilage. The expression of miR-218-5p can penetrate into the whole layer of cartilage. Scale bars, 50 μm. (D) At 5 and 8 weeks post-operation, cartilage tissue was obtained for analysis. Safranin O-fast green stained sections showed that mice treated with the miR-218-5p inhibitor exhibited significant protection from OA at 5 and 8 weeks post OA associated with reduced cartilage degradation/fibrillation, proteoglycan loss, and reduced loss of articular chondrocyte cellularity. Scale bars, 400 μm (top); 100 μm (bottom). (E) The quantification of articular chondrocyte cellularity. ***p < by post hoc SNK test. (F) A significant reduction in the OARSI scale was observed at both the medial tibial plateau and medial femoral chondyle in the OA + miR-218-5p inhibitor group compared with OA, OA + miR control, and OA + miR-218-5p mimic (5 weeks, n = 6 per group; 8 weeks, n = 6 per group). ***p < by post hoc SNK test.
Molecular Therapy  , DOI: ( /j.ymthe )
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5. Figure 4 Identification of PIK3C2A as a Target of miR-218-5p
(A) Venn diagram displaying miR-218-5p computationally predicted to target PIK3C2A by different algorithms. (B) Sequence alignment of a putative miR-218-5p binding site within the 3′ UTR of PIK3C2A mRNA shows a high level of sequence conservation and complementarity with miR-218-5p. (C) The wild or mutant type PIK3C2A 3′ UTR reporter plasmid was co-transfected with miR-control, miR-218-5p mimic, inhibitor control, or miR-218-5p inhibitor into human OA chondrocytes, SW1353 cells, and C28/I2 cells. 48 hr after transfection, luciferase activity was measured (n = 3 replicates per group). ***p < by one-way ANOVA test followed by Tukey’s post hoc analysis. (D and E) In SW1353 cells (D) and C28/I2 cells (E) transfected with miR-control, miR-218-5p mimic, inhibitor control, or miR-218-5p inhibitor, PIK3C2A expression level was measured (n = 6 replicates per group). (F and G) ***p < by one-way ANOVA test followed by Tukey’s post hoc analysis, which was confirmed by western blot using SW1353 (F) and C28/I2 cells (G). (H) To confirm that miR-218-5p mimic induced inhibition in Col II and Aggrecan is due to the decreased level of PIK3C2A in SW1353 cells, we performed a rescue experiment by transiently transfecting a vector encoding the human PIK3C2A cDNA in the SW1353 cells, which stably expressed miR-218-5p-GFP. miR-218-5p overexpression in SW1353 cells downregulates PIK3C2A, which in turn inhibits the Col II and Aggrecan expressions. Furthermore, these miR-218-5p-inhibited Col II and Aggrecan expressions were rescued by partial restoration of PIK3C2A expression. CDS, coding sequence; Poly (A), poly A tail.
Molecular Therapy  , DOI: ( /j.ymthe )
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6. Figure 5
The Modulation of miR-218-5p on the PI3K/Akt/mTOR Signaling Pathway
(A) SW1353 cells were transfected with miR-218-5p mimic, miR-218-5p inhibitor, their negative control, control siRNA, or PIK3C2A siRNA for 72 hr, and then the levels of Akt, p-Akt, mTOR, p-mTOR, S6, p-S6, 4EBP1, and p-4EBP1 were measured by western blotting analysis. (B) Schematic model of a putative mechanism for miR-218-5p regulation.
Molecular Therapy  , DOI: ( /j.ymthe )
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7. Figure 6
The Expression of the PI3K/Akt/mTOR Signaling Pathway in Clinical Samples
(A) The expression levels of PIK3C2A, Akt, mTOR, S6, and 4EBP1 were detected by qRT-PCR (n = 18 per group) (***p < by Mann-Whitney U test), which was further validated by immunostaining. (B) Images (middle zone from cartilage) were obtained using a 100 objective. (C and D) PIK3C2A expression level was negatively correlated with miR-218-5p level (r = −0.919, p < ) (C) and a modified Mankin scale (r = −0.809, p < ) (D).
Molecular Therapy  , DOI: ( /j.ymthe )
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