Research Paper Volume 12, Issue 2 pp 1159—1170

Circular RNA HIPK3 downregulation mediates hydrogen peroxide-induced cytotoxicity in human osteoblasts

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 (BI, N and Q). * P < 0.05 vs. “Vec” cells (O). #P < 0.05 vs. H2O2 treatment of “miRi-C” cells (CH); * P < 0.05 vs. “miR-C” cells (J-M). #P < 0.05 (N and P). Experiments were repeated three times, with similar results obtained.