Mir 30a 5p inhibitor

HK-2 cells has been assigned to 10 groups and treated as follows: Experiment 1: i) control group (cells without any treatment); ii) H/R group (cells exposed to H/R); experiment 2: iii) H/R + negative control 1 (NC1) group (cells treated with Mimic NC before H/R was induced); iv) H/R + mimic group (cells treated with miR-30a-5p mimic before H/R was induced; v) H/R + NC2 group (cells treated with inhibitor NC before H/R was induced); vi) H/R + inhibitor group (cells treated with miR-30a-5p inhibitor before H/R was induced); experiment 3: vii) H/R + NC2 + si-control group (cells treated with inhibitor NC and siRNA NC to GLUD1 before H/R was induced); viii) H/R + inhibitor + si control group (cells treated with miR-30a-5p inhibitor and siRNA NC to GLUD1 before H/R was induced); ix) H/R + NC2 + si-GLUD1 (cells treated with inhibitor NC and siRNA to GLUD1 before H/R was induced); and x) H/R + inhibitor + si-GLUD1 (cells treated with miR-30a-5p inhibitor and siRNA to GLUD1 before H/R was induced).
Mimic NC, inhibitor NC, miR-30a-5p mimic, miR-30a-5p inhibitor, siRNA NC and siRNA GLUD1 were provided by GenePharma Co., Ltd. Cell transfection was conducted following the instructions in the Lipofectamine 2000 transfection kit (Thermo Fisher Scientific, Ltd.). Forty-eight hours after transfection, HK-2 cells were subjected to H/R treatment as mentioned above.

Conditionally immortalized mouse podocytes (MPC-5) and human HEK293 cells (h243) were obtained from iCell Bioscience Inc. (Shanghai, China). Podocytes and HEK293 cells were grown in Dulbecco’s modification of Eagle’s medium (DMEM) (GIBCO, New York, USA) containing 10% fetal bovine serum (GIBCO), 1% penicillin-streptomycin, and incubated at 37°C with 5% CO
₂. After cell cultures reached a confluence of 70%--80%, podocytes were treated with 80 μM Hcy (Sigma-Aldrich, Darmstadt, Germany) for 48 h. For transfection experiments, miR-30a-5p inhibitor, miR-30a-5p mimic, miRNA negative control (miR-neg), and small interfering RNAs (siRNAs) specifically targeting DNA methyltransferase enzyme 1 (si-DNMT1) were obtained from Gene Pharma (Shanghai, China). Recombinant adenoviruses expressing DNMT1 (Ad-DNMT1) were purchased from HANBIO (Shanghai, China). Then, the cells were seeded in 6-well plates and transfected according to the supplier′s instructions. The siRNA sequences of DNMT1 are as follows: si-NC: 5′-UUCUCCGAACGUGUCACGUTT-3′; si-DNMT1-1: 5′-GCAAAGAGUAUGAGCCAAUTT-3′; si-DNMT1-2: 5′-GCUGGUCUAUCAGAUCUUUTT-3′, and si-DNMT1-3: 5′-CCGAGGCCUUUACUUUCAATT-3′.