The Medical Ethics Committee of the First Affiliated Hospital of Harbin Medical University (Harbin, China) approved the experiments of the present study, and all patients gave their informed consent prior to the study. The current study included paired LA-PV and LAA samples from 16 patients with persistent AF undergoing cardiac surgery. The LA-PV samples were used as the experimental group and the LAA samples were used as the control group. All patients had a history of AF >6 months prior to surgery. AF was diagnosed by evaluation of medical records and 12-lead electrocardiogram observations.

Total RNAs were isolated from atrial samples in the experimental and control groups using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to manufacturer's protocol. The mRNAs were purified from total RNA subsequent to removal of rRNA (mRNA-Only Eukaryotic mRNA Isolation kit; Epicenter; Illumina, Inc., San Diego, CA, USA). Each sample was then and transcribed into cRNA along the entire length of the transcripts without 3′ bias using a SuperScript Double-Strand Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.). The cDNAs were labeled with Cy3 using Quick-Ampl labeling kit (Agilent Technologies, Inc., Santa Clara, CA, USA). Labeled miRNAs were hybrided to the human microarray chip (Human LncRNA Microarray V2.0). Hybridization signals were detected using an Agilent scanner. Images were quantified using the Agilent Feature Extract software, version 11.0 (Agilent Technologies, Inc.). Differential expression of lncRNA between the experimental and control groups was identified by volcano plot. The lncRNAs with ≥2 fold changes between the experimental and control groups were selected.

Pearson correlation analysis was used to determine the association of the lncRNA AK055347 with direct regulated expression of target mRNAs. mRNAs with high Pearson's correlation coefficients (>0.75) were selected as the targets of lncRNA AK055347.

H9C2 cells, a clonal cell line of cardiomyocytes derived from embryonic rat heart tissues, were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin and 100 mg/ml streptomycin. The cells were maintained in a humidified atmosphere with 5% CO2 at 37°C. Cells were subcultured at 1:3 ratio every 3 days.

The rat cDNA sequence was analyzed for potential siRNA target sequences. Three oligonucleotides were analyzed for the inhibition of the expression of AK055347. The siRNAs tested were as follows: siRNA #1, 5′-gaggaucuac uguuaacaga-3′ (sense) and 5′-cuccuagaugacaauuguucu-3′ (antisense); siRNA #2, 5′-cauaccaccaagccuucuu-3′ (sense) and 5′-gua ugg ugg uuc gga aga a-3′ (antisense); siRNA #3, 5′-cguguccucucugcugucucc-3′ and 5′-gca cag gag acg aca gag g-3′. H9C2 cells were transfected with siRNAs using Lipofectamine 2000.

Cell viability was analyzed using the Cell Counting Kit 8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Shanghai, China). Cells were seeded into a 96-well plate at a density of 10⁴ cells/well. Cells were transfected with siRNAs and cultured in 5% CO2 at 37°C for 48 h. CCK-8 solution (10 µl) was added to each well and cultured for an additional 2 h. Absorbance was measured at 490 nm using a MultiSkan 3 microplate reader (Thermo Fisher Scientific, Inc.).

Total RNA was isolated from H9C2 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA was reverse transcribed into cDNA using the reverse transcriptase of Moloney murine leukemia virus (Promega Corporation, Madison, WI, USA). RT-qPCR was performed in a final volume of 20 µl containing 2 µl cDNA, 1 µl of each primer, and 10 µl SYBR Green (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primers used for amplification of AK055347 were 5′-AACTCCTAACACATCTCT-3′ (sense) and 5′-CTAAGGTAGTCAGTCTCA-3′ (antisense). U6 was used as a housekeeping gene. The reaction conditions were as follows: 95°C for 10 min; 95°C for 15 sec, 55°C for 1 min with 40 cycles. Melting curve analyses were performed to verify the amplification specificity. The gene expression ∆Cq values of AK055347 from each sample were calculated by normalizing with internal control U6. The relative expression of AK055347 was calculated using 2⁻∆∆^Cq method (24).

H9C2 cells were homogenized on ice in lysis buffer. Lysates were centrifuged at 13,000 × g at 4°C for 20 min. The supernatants were collected and protein concentrations were determined using a Bicinchoninic Acid Protein Quantitation kit (Abcam, Cambridge, MA, USA). Proteins were resolved using 10–12% SDS-PAGE, and transferred onto polyvinylidene fluoride membranes by electroblotting. Membranes were incubated with primary antibodies against Cyp450 (ab196836; rabbit anti-rat; monoclonal; 1:100; Abcam), adenosine triphosphate (ATP) synthase (ab54880; mouse anti-rat; monoclonal; 1:100; Abcam) and MSS51 (ab63801; mouse anti-rat; monoclonal; 1:100; Abcam). The antibodies were incubated at 4°C overnight. GAPDH (cat. no. BM1623; Wuhan Boster Biological Technology, Ltd., Wuhan, China) was used as a loading control. Membranes were then incubated with horseradish peroxidase-conjugated goat anti-mouse (cat. no. ab6789) or anti-rabbit (cat. no. ab6721) secondary antibodies (1:10,000; Abcam) at room temperature for 40 min. Bands were visualized by exposure to X-ray film (Kodak, Rochester, NY, USA). Images were acquired by scanning the films, and band gray values were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/index.html).

H9C2 cells were grown on glass coverslips in sterile 6-well plates until confluence was reached. Cells were then rinsed with phosphate-buffered saline (PBS) three times, and fixed in 4% paraformaldehyde for 15 min at room temperature. The cells were then permeabilized with 0.5% Triton X-100 for 20 min. Subsequent to three washes with PBS, cells were incubated with primary antibodies against MSS51 (ab165144; mouse anti-rat; polyclonal; 1:100; Abcam) at 4°C overnight. PBS without primary antibodies was used as a negative control. After the primary antibody was removed by washing in PBS, immunoreactivity was detected by incubation in fluorescein isothiocyanate-coupled secondary antibodies (goat anti-mouse IgG; 1:100; cat. no. ab6785; Abcam) at room temperature for 1 h. Cells were counterstained with DAPI. Following washing of the coverslips with PBS, the cells were examined and photographed with a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Analyses were performed using SPSS software, version 13.0 (SPSS, Inc., Chicago, IL, USA). All values are presented as the mean ± standard deviation. One-way analysis of variance followed by Bonferroni's test was used to compare the differences. P<0.05 was considered to indicate a statistically significant difference.

In order to investigate the role of lncRNAs in AF, microarray-based profiling analysis was conducted using LA-PV and LAA tissue samples in patients with AF. By comparing the expression profiles between LA-PV and LAA tissue samples, 94 lncRNAs were identifed that were either significantly upregulated or downregulated (>2 fold change) in LA-PV samples compared with LAA samples (Fig. 1). Table I presents the top 10 lncRNAs including AK055347, AK310040, AK026494, BC010527, AK027294, AB007979, AK091573, UC003qev.1, AK125186 and U9981. AK055347 was selected for further analysis.

The role of lncRNA AK055347 in cell viability in H9C2 cells was investigated using siRNA to knockdown lncRNA AK055347. RT-qPCR results demonstrated that siRNA#1, #2 and #3 significantly downregulated AK055347 expression in H9C2 cells (Fig. 2A). Knockdown of AK055347 significantly reduced cell viability of H9C2 cells (Fig. 2B).

The protein expression of Cyp450 and ATP synthase was measured in H9C2 cells treated with siRNAs against AK055347. Western blotting indicated that knockdown of AK055347 inhibited the expression of Cyp450 and ATP synthases in H9C2 cells (Fig. 3).

Microarray analysis indicated that MSS51 protein was associated with the expression of the lncRNA AK055347 (Table II). It was further investigated whether lncRNA AK055347 regulated the expression of the metabolism-associated protein MSS51 in H9C2 cells treated with siRNAs against lncRNA AK055347, using immunofluorescence and western blotting. Immunofluorescence staining indicated that knockdown of AK055347 reduced the expression of MSS51 in H9C2 cells (Fig. 4A). Consistent with immunofluorescence results, western blotting results indicated that knockdown of AK055347 inhibited the expression of MSS51 in H9C2 cells (Fig. 4B).