Patients and controls

This study included a total of 60 patients with H/R (H/R group, 30 males and 30 females) and 60 healthy controls (30 males and 30 females) at The Second Xiangya Hospital, Central South University from May 2019 to May 2020. All participants were at the age of 38 to 66 years, with an average of 52.1±6.7 years for patients and 52.0±6.8 years for controls. All patients were diagnosed based on the ACC/AHA/ESC/WHF practice guidelines. Table 1 lists patients’ detailed clinical information. H/R in the 60 patients was caused by the formation of blood clots in arteries. The patients were divided into 5 stage groups according to the time after infarction, namely, <4 h, 4-6 h, 6-8 h, 8-10 h, and 10-12 h. The number of H/R patients in each group was 11, 12, 11, 16, and 10, respectively. The patients with chest pain of more than 12 h and those with cardiac injury caused by factors other than H/R were excluded. All healthy controls received systemic physiological exams, and all physiological functions were within normal range. Patients with other severe diseases, such as cancers and diabetes, were excluded. This study was approved by the Ethics Committee of the Second Xiangya Hospital, Central South University. All patients and controls signed informed consent.

Table 1 Clinical parameters of the study subjects

Plasma and cells

To exclude the effect of dietary on ciRs-126 and miR-21 levels, blood (3 ml) was extracted into EDTA tubes from all patients and controls after they were fasted for more than 8 h and prepared as plasma samples by centrifugation for 15 min at 1200 g. The plasma samples were kept in liquid nitrogen prior to use.

Primary cardiomyocytes (C-12,810, Sigma-Aldrich, USA) were cultured in Myocyte Growth Medium (Sigma-Aldrich) in an incubator with 5% CO2 at 37 ºC following the instructions provided by the vendor. Cells at passages 3 to 5 were used for all experiments.

Cell transfections

A vector expressing ciRs-126 was constructed using pcDNA3.1(+) CircRNA Mini Vector (Addgene) as the backbone. MiR-21 mimic and negative control (NC) miRNAs were purchased from Sigma-Aldrich (USA). Cardiomyocytes were transfected with 40 nM miRNA or 1 µg expression vector using Lipofectamine 2000 (Invitrogen). To perform NC experiments, cells were transfected with empty vector or NC miRNA. Control (C) cells were untransfected cells. Cells were incubated with transfection mixtures for 6 h and in fresh medium for 48 h prior to the subsequent assays.

H/R model

After transfections (including C and NC groups), cardiomyocytes were cultured under hypoxic conditions (1% O2, 95% N2 and 5% CO2) for 2 h at 37 °C and in normoxic culture medium for 3 h at 37 °C to achieve reoxygenation.

RNA preparations

RNAs from plasma and cardiomyocytes were extracted using RNAzol (Sigma-Aldrich). After being treated with DNase I (Invitrogen) for 2 h at 37 °C to remove genomic DNAs, RNA purity and integrity were examined by OD260/280 ratios and 6% urea-PAGE.

RT-qPCRs

To synthesize cDNA samples, RNA samples with satisfactory quality were subjected to reverse transcriptions (RTs) using SS-III-RT system (Invitrogen). CiRs-126 expression was determined by qPCRs using SYBR Green Master Mix (Bio-Rad) with GAPDH, 18 S rRNA, or β-actin as the internal controls. Mature miR-21 level was determined using All-in-One™ miRNA qRT-PCR reagent kit (GeneCopoeia). To perform the reverse transcription effectively, addition of poly (A) to mature miRNAs was performed (polyA tailing method), followed by miRNA RTs and qPCRs using RPL30 and 18 S rRNA as the internal controls. All steps were completed following the manufacturer’s instructions. It is worth noting similar results were obtained using different internal endogenous controls. QPCRs were performed at 95 ºC for 1 min followed by 40 cycles of 10 s at 95 ºC and 48 s at 58 ºC.

All qPCRs were performed with three technical replicates. Ct values of target genes were used normalized to the internal controls using the 2−ΔΔCt method. Supplemental Table 1 lists all primer sequences.

Dual-luciferase assays

Dual-GloR Luciferase Assay System (Promega, USA) was used to conduct the dual-luciferase assays. In brief, miR-21 promoter was amplified using primer pair Fr-TGTAAAACGACGGCCAGT and Re-CAGGAAACAGCTATGACC and cloned into the pmirGLO vector. Cardiomyocytes were transfected with the reporter plasmid and with/without ciRs-126. After 2 days, cells were washed, digested with trypsin, and collected by centrifugation for 3 min at 5000 g. The cellular proteins were then extracted using 100 µL of lysis buffer. 50 µL of protein supernatants were added to a 96-well measuring plate and mixed in turn with 50 µL of Luciferase Reagent and Stop & Glo Reagent. The firefly luciferase activities were measured on Centro LB960 with renilla luciferase as the internal reference to correct the transfection efficiency.

Western blot analysis

Total proteins were extracted from transfected cells. After determining protein concentration using BCA Protein Assay Kit, the same amount of proteins were separated by SDS-PAGE and transferred onto membranes. Protein levels were detected using antibodies against caspase-3 (1:1000; sc-65,497), Bax (1:1000; sc-7480), Bcl-2 (1:1000; sc-7382) and GAPDH (TA-09) (Yatai hengxin. Beijing, China), and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:1000; sc-2004, Santa Cruz, CA).

Cell apoptosis assay

Following cell transfections and H/R modeling, cardiomyocytes were collected, washed with ice-cold PBS, and resuspended in Annexin binding buffer to reach a final density of 105 cells per ml (Dojindo). After that, cells were stained with Annexin V-FITC and PI (Dojindo) in the dark for 12 min. Apoptotic cells were then analyzed by flow cytometry. In each experiment, three biological replicates were included.

Statistical analysis

Gene expression levels in plasma samples were expressed as average values of three technical replicates and compared using unpaired t test. Data of three biological replicates of cell transfection groups were expressed as mean±SD values and compared by ANOVA Tukey’s test. P<0.05 was considered statistically significant.

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