Ethical oversight
The Committee for Protection of Animal Care Committee at Nanjing Medical University approved all animal studies, which were consistent with the NIH guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). And all methods were reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments.
Animal Experiments
C57BL/6 mice (male, 6 weeks old) from the Animal Research Center of Nanjing Medical University were randomized into normal diet (ND) and high-fat diet (HFD) groups. Animals in the HFD group were administered a diet in which 45% of the calories were derived from fat, whereas ND animals were fed standard chow. All mice were housed in a climate-controlled facility (22 ± 2°C, 55 ± 5% humidity, 12 h light/dark cycle). Animals were fed the indicated diets for 12 weeks during which food intake and body weight values were recorded weekly. Murine oral glucose tolerance test (OGTT) analyses were performed by fasting mice for 12 h overnight followed by oral gavage with dextrose (1 g/kg). Blood samples were then collected from the tip of the tail after 0, 30, 60, 90, and 120 min to assess blood glucose levels. Following a 6 h fast, insulin tolerance testing (ITT) was performed by injecting mice with insulin (0.5 IU/kg) and then measuring blood glucose after 0, 30, 60, 90, or 120 min. Any mice in the HFD group that exhibited normal OGTT were omitted from these analyses. Samples of skeletal muscle and visceral adipose tissue were harvested from these animals at appropriate time points and snap frozen in liquid nitrogen for subsequent analyses. Samples of blood were obtained from the orbital venous plexus for subsequent exosome isolation and associated analyses.
Cell Culture
The murine C2C12 myoblast and 3T3-L1 pre-adipocyte fibroblast cell lines were cultured based on provided directions. The differentiation of 3T3-L1 cells was achieved by growing these cells to 100% confluence and then incubating then for 10 days in DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 250 nM insulin (Sigma, USA), 1 mM dexamethasone (Sigma, USA), and 0.5 mM 3-isobutyl-1-methylxanthine (Sigma, USA), replacing media every other day until lipids had accumulated. C2C12 cells were routinely cultured in DMEM containing 10% FBS and penicillin/streptomycin, with differentiation being induced by replacing this media with DMEM containing 2% horse serum (Gibco) for 5 days. Following differentiation, these C2C12 cells were treated with palmitate (0.5 mM) to promote IR.
Sirna-mediated Knockdown
C2C12 cells were grown until 60% confluent, at which time they were transfected with an siRNA pool (50 nM) specific for the lncRNA AK018453 (Supplementary Table S1) through the use of Lipofectamine 2000 (Invitrogen) in OptiMEM (Thermo Fisher Scientific) based on provided directions. A Stealth RNAi Negative Control (GenePharma, China) was used for control cell transfection, and qPCR was used to confirm the efficiency of knockdown.
In Vivo Sirna Delivery
For in vivo siRNA studies, appropriate siRNA constructs were administered via the tail vein to 8-week-old mice, with control animals instead being administered empty liposomes (FormuMax).
Exosome Isolation
Circulating exosomes were isolated from murine blood samples immediately following their collection. Briefly, samples were centrifuged for 10 min at 3,000 xg at 4℃ to eliminate remaining cells and associated debris, after which supernatants were centrifuged for 30 min at 12,000 xg at 4℃. These supernatants were then passed through a 0.22 µm filter, followed by ultracentrifugation for 180 min at 100,000 xg at 4℃. The isolated exosomes were then used for transmission electron microscopy (TEM) (USA, FEI Company, FEI Tecnai T20), RNA extraction, or analysis with a Nano Sight Nano ZS instrument (Malvern Instruments, United Kingdom).
Microarray Analyses
A Serum/Plasma Kit (QIAGEN, Germany) was used to isolate total RNA from exosomal samples, after which an Agilent Bioanalyzer 2100 (Agilent Technologies, USA) was used based on provided directions to assess the integrity of these RNA samples. A Low Input Quick Amp Labeling Kit (Agilent Technologies) was used based on provided directions to amplify this RNA, after which an Arraystar lncRNA Array analysis (Huada, China) was performed. Raw data were extracted using the Feature Extraction software 12.0 (Agilent Technologies), normalized using a Quantile algorithm in the R limma package, and compared using hierarchical clustering and volcano plots to detect differentially expressed lncRNAs in the HFD and ND samples using the following criteria (student’s t-test: |log2 fold change (FC)| ≥ 2 and P < 0.05).
Qpcr
The Power SYBR Green Master Mix (Applied Biosystems, USA) and a Life Tech-ViiA7 instrument (Applied Biosystems) were used for qPCR analyses of mRNA and lncRNA expression. The 2-ΔΔCT method was used to assess relative expression, with GAPDH as a reference control. Data were analyzed in triplicate. Primers used for this study are compiled in Table S2.
Functional Enrichment Analyses
Gene Set Enrichment Analysis (GSEA) of target mRNAs of interest were identified using the default settings of the online GSEA tool. Differentially expressed lncRNAs were used for target gene predictions, with the DAVID Bioinformatics Resources 6.8 tool being used to assess gene functions related to these lncRNAs. Gene ontology (GO) analyses of these genes were used to evaluate their enrichment in particular biological processes, cellular components, and molecular functions, with KEGG pathway enrichment also being assessed.
Assessment of interactions between lncRNAs and miRNAs
miRNAs (miRNAs) are small ncRNAs that are ubiquitously expressed in eukaryotes, controlling the translation of target mRNAs [13]. Exosomal lncRNAs have been shown to control the ability of specific miRNAs to regulate gene expression through their ability to function as competing endogenous RNAs (ceRNAs) that readily sequester these miRNAs [14, 15]. The miRanda (v3.3a) database was used to predict lncRNA/miRNA interactions based on the presence of miRNA response elements (MREs) within lncRNAs with selection being based upon seed-match sequences.
Exosome Labeling
The fluorescent PKH26 (Umibio, UR52302) dye was used to label exosomes based on provided directions. C2C12 cells were seeded in 12-well plates for 12 h, then combined with these PKH26-labeled exosomal solutions
Statistical Analyses
GraphPad Prism 5.0 and SPSS 18.0 were used for data analyses. Data are means ± SD and were compared via Student’s t-tests. P < 0.05 was the significance threshold.