Research Paper Volume 12, Issue 2 pp 1159—1170

Figure 4. miR-124 inhibition attenuates H₂O₂-induced cytotoxicity in human osteoblasts. OB-6 human osteoblastic cells were transfected with circHIPK3-expressing lentivirus (“LV-circHIPK3”) or control lentivirus (with empty vector, “Vec”), as well as lentiviral circHIPK3 shRNA (“sh-circHIPK3-a”) or control shRNA lentivirus (“sh-C”), expression of listed microRNAs (miR-124, miR-152 and miR-338) was tested by qPCR (A). OB-6 cells were treated with hydrogen peroxide (H₂O₂, 250 μM) and cultured for 24h, expression of listed microRNAs (miR-124, miR-152 and miR-338) was tested by qPCR (B). OB-6 cells were transfected with miR-124 inhibitor (“miR-124i”, 500 nM), miR-152 inhibitor (“miR-152i”, 500 nM), miR-338 (“miR-338i”, 500 nM) or the non-sense control miRNA inhibitor (“miRi-C”) for 24h, followed by hydrogen peroxide (H₂O₂, 250 μM) treatment, cell viability and apoptosis were tested by MTT (C) and TUNEL staining (D), respectively. Mitochondrial depolarization was tested by JC-1 assay (E). The primary human osteoblasts were transfected with 500 nM of miR-124i or the miRi-C for 24h, followed by hydrogen peroxide (H₂O₂, 250 μM) treatment for indicated time periods, relative miR-124 expression (F), cell viability (G), cell death (H) and apoptosis (I) were tested by qPCR, MTT, LDH release and TUNEL staining assays, respectively. OB-6 cells or the primary human osteoblasts (“Osteoblasts”) were transfected with 500 nM of the miR-124 mimic or miR non-sense control (“miR-C”) for 48h, relative miR-124 expression (J), cell viability (K), cell death (L) and apoptosis (M) were tested similarly. OB-6 cells were transfected with the miR-124 inhibitor (“miR-124i”, 500 nM) for 24h, followed by H₂O₂ (250 μM) treatment for 16h, expression of listed genes was shown (N); The mRNA and protein expression of CDK6 and ROCK1 in stable OB-6 osteoblastic cells, with circHIPK3-expressing lentivirus (“OE-circHIPK3-L1/2”) or control lentivirus (with empty vector, “Vec”), was shown (O); OB-6 cells were transfected with 500 nM of the miR-124 mimic or miR non-sense control (“miR-C”) for 48h, with expression of listed genes examined (P). The primary human osteoblasts with or without H₂O₂ (250 μM, 16h) treatment were examined for the listed genes (Q). Quantified values were mean ± standard deviation (SD, n=5). * P < 0.05 vs. “sh-C” cells (A); * P < 0.05 vs. “Veh” treatment (B–I, N and Q). * P < 0.05 vs. “Vec” cells (O). ^# P < 0.05 vs. H₂O₂ treatment of “miRi-C” cells (C–H); * P < 0.05 vs. “miR-C” cells (J-M). ^# P < 0.05 (N and P). Experiments were repeated three times, with similar results obtained.