Microarray data, identification of DEGs and pathway enrichment analysis
Dataset GSE4745 (Affymetrix platform GPL85) was downloaded from Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE4745). GSE4745 contained global mRNA expression in heart tissues of control and streptozocin induced type I diabetic rats. The rats were sacrificed at 3 days (baseline), 28 days (diastolic dysfunction) and 42 days (both diastolic and systolic dysfunction) after streptozocin injection, respectively. The analyses were carried out using R project (v3.4.1) with packages from Bioconductor website.
The gene expression matrix was obtained from data of CEL format by background correction, quartile data normalization and annotation. The classical t-test was used to identify different expression genes (DEGs) with a cutoff p-value of 0.05.
KEGG is a knowledge base for systematic analysis of gene functions, linking genomic information with higher-order functional information . KEGG pathway analyses were performed using DAVID bioinformatics resources and p < 0.05 was considered statistically significant .
miRNA target prediction
Bioinformatics websites miRBase (http://www.mirbase.org) , TargetScan (http://www.targetscan.org) , PicTar (http://www.pictar.org) , DIANA (http://diana.imis.athena-innovation.gr/DianaTools) , and RNAhybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid)  were used for predicting miRNAs that may target PGC-1β. Intersectional analyses of prediction results from these tools were performed using R project (v3.4.1).
Cell culture medium (DMEM and OPTI-MEM) and fetal bovine serum (FBS) were purchased from GIBCO (Thermo Fisher Scientific, Waltham, MA). The reverse transcriptional and real-time PCR primers of miR-30c and U6, mimics and inhibitor of miR-30c, siRNAs targeting PGC-1β and PPARα, and their negative controls were from RiboBio (Guangzhou, China). Lipofectamine 2000 and Trizol were from Invitrogen (Thermo Fisher Scientific, Waltham, MA). The primers of mRNA real-time PCR were designed and synthesized by BGI (Shenzhen, China). Antibodies against CD36, CPT1B, PDK4, GLUT4 and β-actin were from ABclonal Biotech (Cambridge, MA). Anti-Ago2 antibody was from Novus Biologicals (Littleton, CO). Antibodies against PGC-1β and PPARα were from Abcam (Cambridge, MA). Polyvinylidene difluoride membranes were from Millipore (Merck KGaA, Darmstadt, Germany). The horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence reagents were from Pierce Biotech (Thermo Fisher Scientific, Waltham, MA). pTK-PPREx3-Luc was a kind gift from Dr. Bruce Spiegelman (Addgene plasmid Cat# 1015) . Other reagents were from Sigma-Aldrich (Merck, St. Louis, MO) unless specified description elsewhere.
H9c2 and HEK293T (American Type Culture Collection, Manassas, VA) were routinely cultured in DMEM medium supplemented with 10% FBS. H9c2 cells were transfected with miRNA mimics/inhibitor (100 nM), siRNAs (100 nM) with Lipofectamine 2000 according to manufacturer’s description, respectively, and then subjected to 1 mM palmitate (bound to BSA at a ratio of 5:1) stimulation for 48 h.
Primary cardiomyocytes were prepared from neonatal rats (day 0–3) and identified by immunofluorescent staining with antibody against cardiomyocyte-specific marker ACTN2 as previously described . Primary cells were cultured with DMEM supplemented with 10% FBS and treated with 300 µM saturated FFA palmitate (16:0) for 48 h.
Dual luciferase assays
Luciferase activity was detected using the Dual Luciferase Reporter Assay System (Promega, Beijing, China) as described previously .
To evaluate the binding of miRNA and target mRNA, the sequence containing predicted miR-30c binding site of the human PGC-1β 3′ UTR and corresponding mutant sequence (Additional file 1: Table S1) were synthesized and inserted into pMIR-REPORT luciferase vector (Ambion, Thermo Fisher Scientific, Waltham, MA). To determine whether miR-30c targeting 3′ UTR of PGC-1β, HEK293T cells were co-transfected with appropriate pMIR construct (1 μg/mL), pRL-TK plasmid (0.1 μg/mL) with miR-30c mimics or negative controls (100 nM).
To evaluate the effects of PGC-1β or miR-30c on PPARα transcriptional activity, H9c2 cells were first transfected with PGC-1β siRNA or miR-30c, and then transfected with pTK-PPREx3-Luc plasmid (1 μg/mL), respectively . To determine whether miR-30c regulated PPARα transcriptional activity via PGC-1β, H9c2 cells were transfected with miR-30c and then transfected pTK-PPREx3-Luc plasmid (1 μg/mL) after PGC-1β inhibition by siRNA.
Transfections were done in duplicates and repeated at least three times in independent experiments.
Preparation of rAAV system and animal study
All animal procedures were approved by the Institutional Animal Care and Use Committee of Tongji Medical College of Huazhong University of Science and Technology (HUST), and complied with ARRIVE and the Guide for the Care and Use of Laboratory Animals standards (NIH publication).
The recombinant adeno-associated virus of type-9 (rAAV9) system was a kind gift from Dr. Xiao Xiao (University of North Carolina at Chapel Hill). Synthesized fragments of miR-30c, anti-miR-30c, or miR-random (Additional file 1: Table S2) were inserted into rAAV expression plasmid, respectively. The rAAVs were prepared by triple plasmid co-transfection in human HEK293T cells as described previously .
Male db/db mice with C57BL/Ks background and their control littermates were bred in Model Animal Research Center of Nanjing University and maintained in Laboratory Animal Center of HUST. Db/db mice were randomly assigned to four groups (n = 8–10 for each group). A single tail vein injection of the rAAV-miR-random, rAAV-miR-30c, or rAAV-anti-miR-30c (1 × 1011 vector genomes in 200 μL PBS per mouse)  was performed at the age of 8 weeks in db/db mice, whereas db/db control and C57BL/Ks mice were treated with equal volume of PBS in the same way. At the age of 28 weeks, echocardiographic and hemodynamic analyses were performed and then mice were sacrificed with anaesthetization by the mixture of xylazine and ketamine. Tissue samples were taken for further study.
Echocardiographic and hemodynamic measurements
Echocardiographic measurements were performed under anesthesia with 1.5% isoflurane using a high-resolution imaging system with a 30-MHz high-frequency scanhead (Vevo770, VisualSonics Inc, Toronto, Canada) as previously described . Hemodynamic measurements of left ventricle were performed under anesthesia with intraperitoneal injection of the mixture of ketamine and xylazine using a Millar Catheter System (Millar 1.4F, SPR 835, Millar Instruments Inc, Houston, TX) as described previously .
Western blots analyses
Western blotting was performed using specific antibodies as described previously . The band intensities of target proteins were analyzed by using ImageJ program .
Chromatin immunoprecipitation (ChIP) assays were conducted using a kit from Beyotime (Nanjing, China), following the manufacturer’s protocol . Primers used for ChIP–PCR were listed in Additional file 1: Table S3.
Total RNA was isolated from heart tissues and H9c2 cells with Trizol reagent, and was reverse-transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA). Real-time PCR assays were performed using SYBR rapid quantitative PCR Kit (Kapa Biosystems, Woburn, MA) . The relative miRNA levels were determined by normalizing to U6 level.
To assess the mitochondrial biogenesis, the ratio of mitochondrial to genomic DNA was used. Real-time PCR was carried out with different primers (Additional file 1: Table S4). 18S ribosomal DNA was used to represent genomic DNA; 18S ribosomal DNA and COI was used to represent mitochondrial DNA.
Fresh specimen of mice hearts was fixed with glutaraldehyde in PBS, dehydrated, coated and then observed by electron microscopy analysis (FEI Tecnai G2, Hillsboro, OR) . Lipid droplets were counted in ten images from different fields.
To determine cell apoptosis in myocardial tissue sections, TdT-mediated dUTP nick-end labeling (TUNEL) assay was performed using a kit of R&D Systems (Minneapolis, MN) according to the manufacturer’s instructions . Pictures of slides were obtained by using a microscope (Nikon, Tokyo, Japan) equipped with digital imaging camera.
Staining of heart samples
Neutral lipids accumulating in cultured H9c2 cells was stained with BODIPY 493/503 (Invitrogen, Thermo Fisher Scientific, Waltham, MA) and then imaged by the fluorescence microscopy (Nikon, Tokyo, Japan). Myocardial lipids accumulation in mouse heart frozen sections was stained by oil Red O and then imaged by the ordinary optics microscopy as previously described .
To determine intracellular ROS production in myocardial tissue sections, dihydroethidium (DHE, Beyotime, Nanjing, China) staining was performed according to the manufacturer’s instructions . Pictures of slides were obtained by using a fluorescence microscopy microscope (Nikon, Tokyo, Japan) equipped with digital imaging camera.
Flow cytometry analyses
To evaluate cell apoptosis, H9c2 cells were incubated with Annexin V-FITC/PI (BD Biosciences, Sparks, MD) . To evaluate intracellular ROS production, H9c2 cells were incubated with DCFH-DA (Beyotime, Nanjing, China) . After incubation, cells were analyzed with a FACStar Plus flow cytometer (BD Biosciences, Sparks, MD) as previously described .
ATP level determination
The ATP level was determined using an Enhanced ATP Assay Kit (Beyotime, Nanjing, China) according to the manufacturer’s instructions. Briefly, cells were lysed with ATP lysis buffer on ice and centrifuged. The supernatant was then transferred to the ATP working solution and mixed quickly. A luminometer (Promega, Beijing, China) was used to measure the luminescence immediately. The luminescence data were normalized by the total protein levels of individual samples.
Detection of mitochondrial membrane potential
The mitochondrial membrane potential (ΔΨm) was assessed using a JC-1 kit from Invitrogen (Thermo Fisher Scientific, Waltham, MA) according to manufacturer’s description.
All data were shown as mean ± SEM unless otherwise noted. Differences between groups were evaluated using Student’s t-test of unpaired data or one-way analysis of variance (ANOVA) and Bonferroni post-test. p < 0.05 was considered statistically significant. All statistical tests were performed using GraphPad Prism (v5.0) and SPSS (v18.0) unless otherwise stated.