2.1 Ethics statement
All the experimental procedures were in consistency with the guidelines issued by the National Institutes of Health and with approval of the Institutional Animal Care and Use Committee of Henan Provincial People's Hospital (HN-20190225). Adequate measures were taken to minimize the suffering of the included animals.
2.2 Establishment of GDM-CI rat models
The 12-week-old female rats (purchased from Beijing Union-Genius Pharmaceutical Technology Co. Ltd., the certificate number is SYXK (Beijing) 2020-0021) were randomly divided into two groups: the blank group (rats were instilled conventional feed) and the GDM-CI group (rats were instilled a high-glucose and high-saturated fat compound feed). The fasting blood glucose value of rats in the GDM-CI group was 150–250 mg/dl, which was considered to be diabetic, thus indicative of successful model establishment, while the fasting blood glucose value of the control rats was less than 100 mg/dl. The rats in the blank and GDM-CI groups were mated with the normal adult rats. The appearance of mating plugs was an indicator of successful mating, which was defined as the first day of successful pregnancy (10 rats per group).
2.3 Cell culture
After 21 days of rearing, the rats were euthanized. The heart tissues of rats were divided into small pieces, detached continuously with 0.1% trypsin for 30 minutes, treated at 37°C with 0.2% type II collagenase in Dulbecco's modified Eagle's medium (DMEM, Gibco, Gaithersburg, MD, USA) for 10 hours, and then filtered using a nylon mesh (pore size: 70 mm, BD Falcon, Bedford, MA, USA). The separated cells were cultured using DMEM-containing 10% fetal bovine serum (FBS), and under saturated conditions of 37℃ and 5% CO2. The medium was replaced every 48 hours.
2.4 Immunofluorescence assay
The well-cultured cells were rinsed 3 times with preheated phosphate buffered saline (PBS), fixed using 4% paraformaldehyde for 15 minutes, then permeabilized by Triton-X-100 (2 mL/L) for 3 minutes, and incubated with the bovine serum albumin (BSA, 50 g/L) at room temperature for 30 minutes. Next, the cells were cultured with anti-a-SM-actin (1:100, ab124964, Abcam, Cambridge, MA, USA) at 4°C overnight, heated at 37°C for 30 minutes the following day and incubated at 37°C for 1 hour with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG H&L (1:1000, ab6785, Abcam) under conditions devoid of light. The cell nuclei were stained with 4,6-diamino-2-phenylindole (DAPI) (Solarbio Science and Technology Co., Beijing, China), and then observed under a confocal microscope (Zeiss LSM 510 META, Carl Zeiss AG, Germany) to analyze the results.
2.5 Cell transfection and grouping
The cultured cells at the second and third passage were divided into the GDM-CI group and the blank group. The cell transfection was implemented in strict accordance with the provided instructions of Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Cells were transfected with the si-PNPase and si-negative control (NC) plasmids (siRNA/transfection reagent = 12.5 pmoles/µL) to establish the PNPase expression inhibition group (GDM-CI + si-PNPase group) and the NC group (GDM-CI + si-NC group). Cells were transfected with miR-26a mimic (Genecopoeia, Guangzhou, China) to establish the miR-26a overexpression group (GDM-CI + miR-26a mimic group) and the corresponding NC group (GDM-CI + mimic-NC group). Next, miR-26a inhibitor was utilized for treatment of cells in the GDM-CI + si-PNPase group to establish the GDM-CI + si-PNPase + miR-26a inhibitor group and the GDM-CI + si-PNPase + inhibitor-NC group.
2.6 Quantitative polymerase chain reaction (qPCR)
The total RNA content was separated using the TRIzol reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA). Roche Light-Cycler480 real-time qPCR system was adopted to detect the transcription level. Next, qPCR was performed with the SYBR Green PCR Master Mix (total reaction volume, 20 µL). The 2−ΔΔCT method was adopted for subsequent quantification, and the expression level of the detected gene was normalized to U6 or glyceraldehyde phosphate dehydrogenase (GAPDH) to determine the relative quantification. The primer sequence is shown in Table 1.
Table 1
The primer sequence for genes
Gene
|
Sequence
|
miR-26a
|
F: 5’-CGTCCTTCAAGTAATCCAGGA-3’
|
R: 5’-GCAGGGTCCGAGGTATTC-3’
|
U6
|
F: 5’- TGCGGGTGCTCGCTTCGCAGC-3’
|
R: 5’-CCAGTGCAGGGTCCGAGGT-3’
|
PTEN
|
F: 5’-TGGAAAGGGACGAACTGGTG-3’
|
R: 5′’-CATAGCGCCTCTGACTGGGA-3’
|
GAPDH
|
F: 5’-AGGTCGGTGTGAACGGATTTG-3’
|
R: 5’-TGTAGACCATGTAGTTGAGGTCA-3’
|
Note: F, forward; R, reverse; miR, microRNA; PTEN, phosphatase and tensin homolog deleted on chromosome 10; GAPDH, glyceraldehyde phosphate dehydrogenase. |
2.7 Cell counting kit (CCK)-8 assay
Cell suspension was prepared in conformity with the provided instructions of the CCK-8 reagent (Signalway Antibody, P002, College Park, USA), and the number of cells in each group was adjusted to 1 × 105 cells/well. Cells (100 µL per well) were seeded in a 96-well culture plate and incubated at 37°C with 5% CO2. Each group was set with 3–5 duplicated wells. With 24, 48, and 72 hours of incubation, 100 µL of the CCK-8 solution was added to each well and then incubated for 90 minutes. A microplate reader (Infinite M200; Tecan Austria, GmbH, Grödig, Austria) was adopted to measure the absorbance value at wavelength of 450 nm to assess the cell proliferation activity.
2.8 Flow cytometry
Cell apoptosis was determined in strict accordance with the Annexin V-FITC Apoptosis Detection Kit (Abcam). Briefly, the cells were seeded onto 6-well plates at a concentration of 1.0 × 106 cells/well, after which the cells were incubated for 10 minutes with 5 µL of Annexin V-FITC and incubated for 15 minutes with 5 µL of propidium iodide at room temperature under conditions devoid of light. Cell apoptosis was measured using a FACScan flow cytometer (Beckman Coulter Inc., Brea, CA, USA).
2.9 Western blot analysis
Cells collected from each group were rinsed with pre-cooled PBS, and the protein content was extracted by radioimmunoprecipitation assay lysis buffer (Thermo Scientific). The protein samples (50 µg each group) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotting onto polyvinylidene fluoride membranes, incubation at 4°C overnight with the primary antibody and subsequent incubation for 2 hours at room temperature with the horseradish peroxidase (HRP)-labeled secondary antibody. The enhanced chemiluminescence kit (Thermo Scientific) was used to visualize the immune complexes. The included antibodies were as follows: PNPT1 (1:1000, ab157109), IgG H&L (HRP) (1:2000, ab205718) and β-actin (1:5000, ab6276) (all from Abcam).
2.10 Dual luciferase reporter gene assay
The PTEN 3'-untranslated region (UTR) containing the miR-26a wild-type (Wt) and mutant type (Mut) binding sites were amplified and cloned into the pGL3 vector (Promega Corporation, Madison, WI, USA) to construct pGL3-Wt-PTEN-3'-UTR and pGL3-Mut-PTEN-3'-UTR. The luciferase reporter vector was co-transfected with miR-26a mimic or the corresponding NC into the 293T cells (American Type culture collection, Manassas, VA, USA). The PRL-TK plasmid (Promega) was used as a control. After 48 hours, the Dual-Luciferase Reporter Assay System (Promega) was used to measure the two luciferase activities individually.
2.11 Hematoxylin and eosin (HE) staining and transmission electron microscope (TEM) observation
The pathological examination was performed according to defined laboratory standards. The heart tissues were preserved in 2.5% glutaraldehyde for 3 hours, rinsed with 0.1M PBS, embedded in 2% agarose gel, and then finally fixed in 4% osmium oxide solution for 1 hour. Subsequently, the tissues were rinsed under distilled water, stained with 0.5% uranyl acetate for 1 hour, and ultimately embedded in epoxy resin. The resin was polymerized at 60°C for 48 hours, after which the ultrathin sections were stained with 5% uranyl acetate and 2% lead citrate aqueous solution. The ultrastructure was observed under a TEM (JEOL JEM2100, Tokyo, Japan).
The heart sections were rinsed with PBS, deparaffinized with xylene, and hydrated with gradient ethanol. Then, the sections were stained using hematoxylin and eosin (Beyotime, Shanghai, China). The images were documented under a microscope (IX73, Olympus, Japan) in bright field mode.
2.12 Scratch test
The cells were seeded onto 6-well culture plates with 10% FBS-contained DMEM. Upon attaining 80% cell confluence, the fused monolayer membrane was scratched using the tip of a 200 µL pipette. Next, the cells were sustained in a stable and suitable environment for 72 hours. At each time point, a digital camera connected to a phase contrast microscope was used to document cell findings until the wound healed and closed. The wound gap area was measured by the IMAGE-PRO PLUS software.
2.13 Statistical analysis
All statistical data analyses were processed using the SPSS 22.0 software (IBM, Chicago, IL, USA) and GraphPad Prism 8.0. The measurement data were depicted as mean ± standard deviation. Firstly, a normality and variance homogeneity test was conducted and the data in compliance with the assumption of normality and homogeneity of variance were compared using the t-test (two groups) or one-way or two-way analysis of variance (ANOVA). The t test or Tukey's multiple comparisons test was employed as the post hoc test. A value of P < 0.05 was considered to be statistically significant.