Animals
All animal experiments were conducted following the Guide for the Use and Care of Laboratory Animals. All experimental protocols were reviewed and approved by the Ethics Committee of Anhui Medical University (Hefei, China). Male Sprague Dawley (SD) rats (220-250 g, 8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The rats were acclimatized to the standard laboratory conditions for one week with normal water and chow available ad libitum after arrived. Afterwards, rats were randomized to normal, DM, DM + insulin and DM + MLT groups (12 rats in per group), and they were received a single injection of streptozotocin (STZ, 55 mg/kg, Sigma‑Aldrich; Merck KGaA, Darmstadt, Germany) except normal group. Animals were considered to be diabetic if the blood glucose levels tested higher than 11.1 mmol/l after 1 week. Diabetic rats were administered via injecting insulin (1u/kg/d within 10 weeks and 2u/kg/d after 10 weeks, Wanbang Biopharmaceuticals, China) and melatonin gavage (10 mg/kg/d, Nanjing Duly Biotech Co., Ltd., China) separately in DM + insulin and DM + MLT groups. During the experiment, all of them could consume the normative water and chow at liberty. At the end of week 16, the rats were anesthetized with a 10% chloral hydrate solution (300 mg/kg). With the heart fetched, cardiac function was immediately evaluated by the way of the Langendorff-perfusion system and then collected the myocardial tissues.
Langendorff-perfusion system
The Langendorff-perfusion system was used to record cardiac function in rats. Briefly, the heart was rapidly excised by thoracotomy under anesthesia, and the aorta was cannulated. The isolated heart was mounted on the Langendorff-perfusion system (Chengdu Techman Software Co., Ltd., China) and perfused with modified Krebs-Henseleit buffer aerated with 95% O2 and 5% CO2, yielding a final pH 7.4 in 37 °C circulating water bath. When stable, the parameters, which is related to cardiac function such as heart rate, were recorded via BL-420S biological function experiment system (Chengdu Techman Software Co., Ltd., China).
Histology
The paraffin‑embedded myocardial tissues were cut into 5‑μm thick sections and stained with H&E (hematoxylin and eosin staining kit, Beyotime, China) or Masson's trichrome staining kit (Nanjing Jiancheng Technology Co., Ltd., China). The discrepant areas were captured by a DMI4000B fluorescence microscope (Leica, USA).
Cell culture
The cell line H9c2 obtained from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences were cultured in low-glucose Dulbecco’s Modified Eagles Media (DMEM) (hyclone, USA) supplemented with 10% fetal bovine serum (FBS) (gbico, Australia) and 100 IU/ml penicillin/streptomycin (Beyotime, China) in 5% CO2 at the temperature of 37 °C. The treatment of insulin (1μM) and melatonin (10μM) was treated 48 h in high glucose concentration.
Cell transfection
H19-shRNA (GenePharm Co., Ltd., Shanghai, China) was used to silence H19 expression. Simultaneously, miR-29c mimics and inhibitors (GenePharm) were used to regulate miR-29c expression. H9c2 cells were transfected with H19-shRNA, or miR-29c mimics, inhibitors, or controls (GenePharm) with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol. A scrambled oligonucleotide (GenePharm) served as a control. Changes in RNA expression were determined by qRT-PCR 24h after transfection, and differences in protein expression were measured via western blotting 48h after transfection.
Western blot analysis
The proteins were separated by SDS–PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Massachusetts, USA) via electroblotting. The membranes were blocked in 5% skim milk for 2 h and incubated overnight at 4 ℃ with primary antibodies against p- JNK (Santa Cruz Biotechnology, USA, Cat# sc-6254), JNK (Santa, Cat# sc-7345), p-ERK (Santa, Cat# sc-7383), ERK (Santa, Cat# sc-135900), p-p38 (Santa, Cat# sc-7975-R), p38 (Santa, Cat# sc-7149), BAX (Santa, Cat# sc-20067), Bcl-2 (Santa, Cat# sc-7382), caspase-3 (Proteintech, Wuhan, China, Cat# 11648-2-AP), caspase-9 (Cell Signaling Technology, USA, Cat# 9508S) and GAPDH (Santa, Cat# sc-32233). The proteins were detected with corresponding horseradish peroxidase-conjugated secondary antibodies coupled with ECL chemiluminescence detection kit (FDbio Science Biotech Co., Ltd., Hangzhou, China).
Hoechst 33258 staining
Cells were seeded in 24-well plates, cultured and treated, and the medium was thrown away. After washing with PBS 3 times, cells were fixed in fixative solution for 10 min. The samples were incubated with Hoechst 33258 (Beyotime, China) staining solution for 5 min in darkness. Then, the plates were kept out light, observed and imaged using a fluorescence microscope.
Luciferase assay
The 3’-UTR regions of lncRNA H19 and MAPK13, including potential miR-29c binding sites, were predicted with TargetScan version 7.11 and amplified by PCR. Subsequently, mutants were constructed by introducing point mutations into the seed binding site for miR-29c. The wild type and mutant fragments were subcloned into the firefly luciferase-expressing vector. H9c2 cells were seeded in 24-well plates and co-transfected with wild-type or mutated luciferase respectively. The Dual Luciferase Reporter Assay System (Promega) was used 48 h after transfection following the manufacturer’s protocol. The relative luciferase activity was calculated according to the ratio of firefly luciferase activity to renilla luciferase activity.
RNA immunoprecipitation (RIP)
We investigated the direct interaction between miR-29c and lncRNA H19 by Argonaute 2 (Ago2)-RNA immunoprecipitation (Ago2-RIP). Anti-Ago2 (Sigma-Aldrich, USA), or control anti-IgG and Dynabeads Protein G (Invitrogen, USA) were pretreated at 4 ℃ with rotation 24 h in advance. Complete RIP lysis buffer, which contained protease inhibitor, phosphatase inhibitor and RNase inhibitor, was used to lyse cells. RNA in Ago2-RIP materials was washed several times with PEB buffer and treated with DNase I and Proteinase K. RNA was extracted with TRIzol (Invitrogen) and precipitated with absolute ethanol overnight at -20 ℃. After the removal of proteins and beads, RT-qPCR analysis of the purified RNA and lncRNA H19 enrichment in Ago2-RIP were put into effect.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
RNA from myocardial tissues and H9c2 cells was isolated using TRIzol reagent and converted to complementary DNA (cDNA) using a First-Strand cDNA Synthesis Kit (Toyobo, Osaka, Japan). Then, the cDNA samples, added with Power SYBR green master mix (Applied Biosystems, Foster City, CA, USA), were subjected to qRT-PCR using a StepOne Real Time PCR system. The H19, miR-29c and mRNA level were respectively standardized by U6 and GAPDH. The amplification results were calculated on the basis of 2 (-ΔΔCt) method. The specific primers used were: H19 forward 5’-ATCGGTGCCTCAGCGTTCGG-3’ and reverse 5’-CTGTCCTCGCCGTCACACCG-3’; MAPK13 forward 5’-GAGAAGGTGGCCATCAAGAA-3’ and reverse 5’-GTCCTCATTCACAGCCAGGT-3; GAPDH forward 5’-GGTGGTCTCCTCTGACTTCAA-3’ and reverse 5’-GTTGCTGTAGCCAAATTCGTTGT-3’; miR-29c, 5’-UAGCACCAUUUGAAAUCAGUGUU-3’; U6, 5’-CGCTTCGGCAGCACATATACTAAAATTGGAAC-3’.
Caspase-3 activity assay
Caspase-3 activity was detected using a caspase-3 activity assay kit (Biomol Research Laboratories, Plymouth Meeting, PA) according to the manufacturer’s protocol. After the cells lysed, total proteins were extracted and quantified using a protein assay kit. Subsequently, the proteins were incubated overnight at 37°C with acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) for the caspase-3 assay. The absorbance of pNA was detected by a microplate reader at 405 nm.
DNA fragment assay
DNA fragments were measured using a Cellular DNA Fragmentation ELISA kit (Roche Applied Science, Greenfield, IN, USA). Cells were seeded in 96-well plates. The medium was changed to serum-free medium after 24 h, and continued to culture cells for an additional 24 h. To label the DNA, the medium was replaced with 10% FBS-DMEM supplemented with 5-bromo-2′-deoxyuridine. Following 24 h incubated, cells were treated with calcitriol for 4 h and MIS for 96 h. Cells were lysed and then soluble DNA fragments were quantified by the Cellular DNA fragmentation ELISA kit according to the manufacturer’s instructions.
Statistical analysis
Statistical analysis was performed using SPSS 20.0. The data were subjected to one-way ANOVA and parametric t-test and presented as means ± standard deviation. All experiments were performed at least in triplicate. The results were considered to denote statistical significance if P<0.05.