2.1 Cell culture and treatment
The H9C2 and 293T cells used in this study were provided by Wan Lei Biotechnology Co., Ltd. (Shenyang, China). In the control group, high glucose (4.5g/L) Dulbecco's modified Eagle's medium (DMEM; Gibco; ThermoFisher Scientific, Inc., Waltham, MA, USA) was supplemented with 10% fetal bovine serum (FBS; EverGreen, Zhejiang Tianhang Biological Technology Co., Ltd. China) at 37ºC and 5% CO2. To simulate ischemia and hypoxia, the cells underwent the culture in hypoglycemia (1.0g/L) DMEM (Gibco; ThermoFisher Scientific, Inc. USA), and then they were placed in a hypoxic incubation chamber with 1% O2, 94%N2 and 5% CO2 for 6 h.
2.2 Transfection
H9C2 cells were plated in 6-well plates (1x105 per well) and subsequently incubated at 37 ℃ for 24 h under 5% CO2. By complying with the manufacturer's protocol, the cells underwent the transient transfection with a final 20 nM dose of overexpression plasmid of LncRNA-NR_027324 (Lncm), empty plasmid/overexpression plasmid control (EPC), LncRNA small interfering RNA (si-Lnc), LncRNA small interfering RNA control (si-Lnc-NC), miR-103-3p mimics (miRm) or miR-103-3p mimics control (miR-NC) with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After the cells were incubated for 24 h, the different cell groups were subsequently analyzed.
2.3 Dual luciferase reporter assay.
293T cells were plated in 12-well plates (2.5×105 per well) at 37 ℃ for 24 h with 5% CO2. NR_027324 was ligated into pmirGLO dual luciferase reporter vector (wt); the mutated NR_027324 was classified as the control(mut). Subsequently, miR-103-3p-mimic, miR-103-3p-mimic-NC and the mentioned recombinant plasmids were co-transfected into 293T cells with Lipofectamine2000 (Invitrogen; Thermo Fisher Scientific, Inc. USA). After the cells were incubated for 48 h, the luciferase activity was measured with the dual luciferase reporter gene assay kit (cat. No. KGAF040, Dual Luciferase Reporter Gene Assay Kit, KeyGEN BioTEH, China) following the manufacturer's protocol. Such measurement was performed three times for each per sample.
2.4 Reverse transcriptional quantitative polymerase chain reaction (RT-qPCR) analysis
Total RNA was extracted with TRIpure reagent (Cat.No.RP1001;BioTeke Corporation, Beijing, China). The concentration and purity of RNA were measured with Nanodrop2000 system (ThermoFisher Scientific, Inc.). In accordance with the manufacturer's protocol, complementary DNAs (cDNAs) were synthesized with Super M-MLV reverse transcriptase (Cat.No.PR6502; BioTeke Corporation, Beijing, China). Complying with the manufacturer's protocol, SYBRGREEN mastermix (Cat.No.SY1020; Solarbio technology co.LTD, Beijing, China) was adopted to amplify the cDNA samples. Besides, the fluorescence quantitative analysis was conducted with the ExicyclerTM96 fluorescence quantitative instrument produced by BIONEER company in Korea. In brief, the PCR reaction conditions of mRNA and LncRNA included 94 °C for 5 min, followed by 40 cycles of 94 °C for 10 sec and 60 °C for 20 sec. The PCR reaction conditions of miRNA included 94 °C for 2 min, followed by 40 cycles of 94 °C for 15 sec and 60 °C for 15 sec. Moreover, ddH2O was used as a non-template control for each plate. 5S was used to normalize the expression levels of miR-103-3p. β-actin was employed for normalizing the expression levels of NR_027324 and Atg5. The relative expression was quantified by 2-△△CT method, which was repeated three times. The median of the three results was obtained to calculate the relative expression level. The sequence list of PCR reaction primers is presented in Table 1.
2.5 Western blot analysis
The total protein was lysed with a whole protein extraction kit (Cat.No.WLA019; wanleibio Co., Ltd., Shenyang, China). The BCA protein concentration determination kit (Cat.No. WLA004; wanleibio Co., Ltd.) was employed to measure the concentration and purity of the protein. 40 μg of protein samples per lane were separated on SDS-PAGE gels (10%) and subsequently transferred to polyvinylidene difluoride membranes (EMDMillipore, Bedford, MA, USA). At ambient temperature, the membrane was blocked in 5% skim milk for 2 h; subsequently, it underwent the incubation with the primary antibodies (Atg5, Cat.No. WL02411, 1:500; Bax, Cat.No. WL01637, 1:400; Bcl-2, Cat.No. WL01556,1:1000;cleavedcaspase-3, Cat.No. WL02117,1:500;cleavedcaspase-9, Cat.No. WL03421,1:500;βactin, Cat.No. WL01845,1:1000; Wanleibio Co., Ltd., Shenyang, China) overnight at 4°C. Next, the membrane was washed 3 times with TBST and incubated with horseradish peroxidase-labeled secondary antibody (Cat.No. WLA023, 1:5,000; wanleibio Co., Ltd., Shenyang, China) at 37°C for 45 min; afterwards, it was further washed 6 times with TBST. Besides, the ECL chemiluminescence kit (Cat.No. WLA003; wanleibio Co., Ltd., Shenyang, China) was employed to detect the blot, and the gel image processing system (Gel-Pro-Analyzer software) was adopted to analyze the optical density of the target band value.
2.6 Immunofluorescence
1-2 drops of cell suspension were dropped on the glass slides that were washed in advance, sterilized by pure alcohol and sterilized under high pressures, and the cell adhesion was observed the next day. After the cells were adhered to the wall and exhibited the required density, they were washed with 1x phosphate buffer (PBS) for 5min x 3 times and then fixed in 4% paraformaldehyde for 15 min. Next, the cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min. The samples were blocked with goat serum at ambient temperature for 15 min and subsequently incubated with primary antibody (LC3-I/II) overnight at 4 ℃. On the following day, the samples were incubated with secondary antibodies for 1 h at ambient temperature in the dark. Lastly, the cells were stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich; Merck KGaA) at ambient temperature for 5 min. After each step, the slides were washed with PBS for 5min x 3 times. The anti-fluorescence quenching agent was dropped on the glass slide, and the slide upside down on the glass slide dripped with anti-fluorescence quenching agent was blocked. Olympus BX53 fluorescence microscope (Olympus corporation, Tokyo, Japan) was used to capture images.
2.7 Lactate dehydrogenase (LDH) assay
H9C2 cells were cultured with 10% fetal calf serum and were inoculated with 24-well plates (1x105 per well). After the cells were transfected, the LDH leakage assay was performed to determine cell injury with the LDH cytotoxicity assay kit (Cat.No. WLA072; wanleibio Co.,Ltd., Shenyang, China) by complying with the manufacturer's protocol. The absorbance was measured at 490nm enzyme-linked immunosorbent assay (ELX-800, BIOTEK, USA), which was repeated three times in each group.
2.8 MTT assay
The H9C2 cells were plated in 96-well plates (5×103 per well). In each group, 5 repeats were set, and a blank control was set with the culture medium only. After the cells were adhered to the wall, the cells received the transfection and culture at 37 ℃ for 24 h. The cells were administrated with hypoglycemia and hypoxia according to different groups. After reaching the time point, the culture medium was removed; 20 μl of MTT reagent was added to each well and then incubated at 37 ℃ and 5%CO2 for 4 h; the supernatant was rigorously discarded; afterwards, 150 μl dimethyl sulfoxide (DMSO) was added and maintained in the dark for 10 min. The absorbance of each well was measured with a microplate reader at 570nm, and the average value of 5 wells was determined.
2.9 Statistical analysis
Data were analyzed with SPSS version 16.0 (IBM corp., Armonk., NY, USA). All data are expressed as the mean ± standard deviation. Significant differences were identified by Student's t-test or one-way analysis of variance followed by Tukey's test. P<0.05 was considered to exhibit a statistically significant difference.