Materials
Human renal cortex proximal convoluted tubule epithelial cells HK-2 (Procell Life Science & Technology Co., Ltd, Wuhan, China); DMEM-12 medium (KGM12500S, KeyGen bioTECH); Lipofectamine Reagent™3000Transfection(L3000015, Invitrogen™); Cell Counting Kit-8 (KGA317, KAIJI Biotech); Reporter plasmid pmirGLO, Plasmid extraction kit (DP103-02, TIANGEN Biotech); Annexin V-FITC/PI Apoptosis Kit (AP101-100-kit, MULTI SCIENCES); Internal reference antibody: rabbit anti-β-actin (AF7018, affinity); target primary antibodies: anti-p-PI3K (rabbit, AF3241, affinity), anti-p-AKT (rabbit, AF0832, affinity), anti-Bax (rabbit, AF0120, affinity), anti-Bcl-2 (rabbit, AF6139, affinity), anti-Cleaved Caspase-3 (rabbit, AF7022, affinity).
Cell culture and cell transfection
HK-2 cells were divided into 5 groups as follows: normal control (Control group), high glucose model group (HG 60mM group), Model with siRNA-NC treatment group (HG 60mM + NC group), Model with HOTAIR siRNA treatment group (HG 60mM + siRNA group), Model with HOTAIR siRNA and miR-126-5p inhibitor treatment group (HG 60mM + siRNA + inhibitor group). Hk-2 cells were cultured using the medium containing the treatments mentioned above.
In the progression of siRNA transfection, HK-2 cells were initially cultured with a serum-free medium. Two mixtures later, 125 µL Opti-MEM + 5 µL Lipofectamine 3000 and 125 µL Opti-MEM + 0.25 nmol siRNA, were added respectively in two tubes and incubated for 5 min. Then, mix the two reagents, wait for another 15 minutes, and add them to the medium. 4-6h later, 1 mL 20% serum-supplemented culture medium was added to the medium.
CCK-8
Cell Counting Kit-8 (KGA317, KAIJI Biotech) was used to test cell viability. HK-2 cells (3×103 cells/well) were seeded in a 96-well plate. when HK-2 cells adhered to the dish, 100 µL/well high-glucose mediums of different concentrations (30, 45, 60, 75, 90 mM) were added to the medium. 72 h later, 10 µL CCK-8 reagent was added to each well for another 1h of incubation. Finally, detecting the OD values at 450 nm with a microplate reader.
Flow cytometry
HK-2 cells (1×106) were resuspended by 300 µL Binding Buffer, prepared by diluting 5×Binding Buffer with double distilled water. Subsequently, 5 µL Annexin V-FITC and 10 µL PI were added into wells and incubated in for 10 min at room temperature, protected from light exposure. Finally, 200 µL pre-cooled 1×Binding Buffer was added and the mixed sample was processed for flow cytometry assay.
RT-qPCR analysis
RNA isolation was performed using a DNA/RNA Extraction Kit (DNA/RNA Extraction Kit, China). A TaqMan RNA Reverse Transcription Kit (Thermo Fisher, USA) was used for the RNA reverse transcription of cDNA. qPCR was performed on a 7500 Real-Time PCR System. β-actin served as the internal control. The relative expression of the target gene was calculated with the 2−△△Ct method. The Primers were listed in Table 1.
Table 1
| Name | Sequence(5’-3’) |
| β-actin F | TGGCACCCAGCACAATGAA |
| β-actin R | CTAAGTCATAGTCCGCCTAGAAGCA |
| HOTAIR F | ATAGGCAAATGTCAGAGGGTT |
| HOTAIR R | ATTCTTAAATTGGGCTGGGTC |
| Bax F | GGATGCGTCCACCAAGAA |
| Bax R | AAAGTAGAAAAGGGCGACAAC |
| Bcl-2 F | GAGGATTGTGGCCTTCTTTG |
| Bcl-2 R | GCCGGTTCAGGTACTCAGTC |
| Caspase-3 F | AGCGAATCAATGGACTCTGG |
| Caspase-3 R | GACTTCTACAACGATCCCCTCT |
| U6 F | CTCGCTTCGGCAGCACA |
| U6 R | AACGCTTCACGAATTTGCGT |
| miR-126-5p F | GCGCGCATTATTACTTTTGG |
| miR-126-5p R | AGTGCAGGGTCCGAGGTATT |
Western blot
Scraping the HK-2 cells with a cell scraper into a marked tube by a pipette and then removing the deposit at 2,000 rpm for 10 min. Total proteins were collected from the supernatant and the protein concentration was determined by a BCA Protein Assay Kit. The proteins were denatured, loaded, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 2 h. Then transferring the separated proteins to a PVDF membrane at 300 mA for 80 min. incubating with Antibody at 4°C overnight, followed by hybridization with the secondary antibodies for 2 h. The PVDF membrane was exposed to ECL reagents and protein bands were detected on the Gel Imaging System. Grey values of the bands were measured by Image-J software.
Luciferase reporter assay
HOTAIR and mutant HOTAIR 3’-UTR reporter vectors were purchased from BoYuan (Shanghai, China). Luciferase reporter vectors were delivered into 1×106 HK-2 cells at 70% confluence, along with the treatment of five groups. After 2 days, the cells were collected to analyze the luciferase signals using a dual-luciferase assay system (Promega, USA)
Statistical analysis
The data were presented as mean value ± S. D. (standard deviation). One-way ANOVA analysis and unpaired t-test were adopted for multiple-group comparisons, with P < 0.05 considered to be statistically significant. Data processing of the study was performed using Graph prism 9.0.