A total of 22 adult patients with high-risk diffuse large B-cell lymphoma (DLBCL) between July 2013 and February 2014 in Guangdong General Hospital, China were enrolled in this study. Positive human immunodeficiency virus (HIV) status and pregnancy were exclusive criteria. All patients received a dose of 75 mg/m2 epirubicin (a kind of Dox) i.v. every 3 weeks for 6 times. Two mL peripheral blood was collected for detection of concerned plasma miRNAs at 3 time points, including before initiating treatment, 9 weeks and 16 weeks after initiating treatment. This study protocol was performed conform the declaration of Helsinki and has been approved by the ethics committees of Guangdong General Hospital. All patients provided informed consent to allow the use of their blood samples for research purposes.
Male sprague-dawley (SD) rats weighing 250 ± 9 g, license number SCXK (YUE) 2004–0011 (Department of Experimental Animal Research Center, Sun Yat-sen Medical College, Sun Yat-sen University, Guangzhou, China) were used. All animals were housed under pathogen-free conditions and kept on standard mouse chow with free access to tap water. This study conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (8th Edition, National Research Council, 2011). All methods and experimental protocols in the present program were also approved by the research ethics committee of Guangdong General Hospital (the approval number: No. GDREC2010093A). In the present study, SD rats received Dox intravenously (IV) via tail vein injections twice a week for 4, or 8 weeks at a dose of 2 mg/kg (cumulative dose: 16 mg/kg or 32 mg/kg) to achieve cardiotoxicity. Animals in the Dox + DEX groups were given an intraperitoneal (IP) injection of 20 mg/kg DEX followed (30 minutes later) by an IV injection of 2 mg/kg DOX twice a week for 4, or 8 weeks.
Left ventricular (LV) function variables were assessed by transthoracic echocardiography. After the induction of light general anesthesia, the rats underwent transthoracic twodimensional (2D) guided M-mode echocardiography with an 8.5-MHz transducer (Acuson, Mountain View, CA). From the cardiac short axis (papillary level), the LV anterior wall enddiastolic thickness (LVAWd), the systolic LV anterior wall thickness(LVAWs), the LV internal dimension at end-diastole (LVIDd), the LV internal dimension at end-systole(LVIDs), the LV posterior wall end-diastolic thickness (LVPWd), the LV posterior wall end-systolic thickness(LVPWs), the ejection fraction (EF) and fractional shortening (FS) were measured. Echocardiographic measurements were averaged from at least three separate cardiac cycles.
Levels of plasma cTnT and MDA, and Ca-Mg-ATP enzyme activity were detected according to the protocols of the corresponding assay kit instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Rats were sacrificed with an intraperitoneal injection of 2 mL of pentobarbital. The heart was excised, and the LV myocardium fixed overnight in 10% formalin. Samples were embedded in paraffin and cut into 4 μm thick sections. They were mounted on normal glass slides and stained with Masson trichrome for histological examination. For the collagen volume fraction (CVF) analysis, eight separate views (magnification = original × 400) were selected and assessment of CVF used the following formula: CVF = collagen area/total area.
MiRNA expression analysis was performed on total RNA extracted from a pool of 5 to 8 myocardium or plasma samples of rats received Dox or Dox + DEX treatment by using Trizol reagent and Trizol LS reagent (Invitrogen, Carlsbad, CA, USA), respectively. Microarray procedures and data analysis were performed at Bioassay Laboratory of CapitalBio Corporation (Beijing, China). Briefly, 200 ng of total RNA extracted from myocardium or plasma samples was fluorescently labeled with Cyanine3-pCp using miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, CA, USA). The labeled samples were then concentrated and hybridized with the Hybridization Chamber gasket slides (Agilent, USA). Arrays were scanned on Agilent chip scanner (G2565CA), and image analysis was performed using Agilent Feature Extraction(v10.7) software (Agilent, USA), followed with data normalization using Agilent GeneSpring software (Agilent, USA).
Rat cardiomyocyte H9c2 cells were grown to confluency on 10-cm cell culture dishes in growth media (DMEM/LG 10% FBS, 1% penicillin and 1% streptomycin) at 37 °C in humid air with 5% CO2. H9c2 cells were incubated with 1 to 5 μM Dox for 24 h to induce the apoptotic phenotype. Cells were treated with NF-κB inhibitor JSH23 (10 μM) or QNZ (5 nM). Cells were transfected with 50 nM scramble, miR-34a-5p mimic, Sirt1 siRNA and NF-κBP65 by oligofectamine reagent (Invitrogen, Carlsbad, CA), and were transfected with pcDNA3-Sirt1 by lipofectamine 2000 reagent (Invitrogen, CA).
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed as in our previous report18. Briefly, H9c2 cells were cultured on coverslips and were fixed in 4% paraformaldehyde after experimental treatments, followed with permeabilization with 0.2% Triton X-100. Cy5-dUTP (Amersham, Piscataway, NJ, USA) was used to label DNA strand breaks in the apoptotic cells. The level of TUNEL-positive cells was detected by fluorescence microscopy, about 200 cells per field in five different visual fields were counted in the present study.
At 24 h post-transfection of miR-34a-5p mimic or scramble control, H9c2 cells were washed thrice with PBS (pH 7.2, 1 mL), then incubated for 15 min with 3 μM rhodamine 123 (Molecular Probes, USA) in PBS. Cell suspensions were incubated for 15 min at 37 °C. Cells were subsequently analyzed with a flow cytometer (Beckman, USA). Results were expressed as the proportion of cells exhibiting low mitochondrial membrane potential estimated by reduced uptake of rhodamine 123.
For detection of mRNA expression of coding genes, first-strand cDNA was generated from 1.5 μg total RNA using a mixture of oligo (dT)15 and random primers with superscript reverse transcriptase (Invitrogen, Carlsbad, CA). Expression of miR-34a-3p in rat myocardium and H9c2 cells was detected by RT-qPCR as previous report19. Determination of miR-34a-5p in the supernatant of H9c2 cells or in the plasma of rats and DLBCL patients were performed by using the poly(A) method20 as follows. In brief, 300 μl cell supernatant or plasma was incubated with 1 ml Trizol LS reagent (Invitrogen), followed with total RNA extraction with 200 μl phenol/chloroform. Ten μl RNase-free water was used to re-suspend the RNA pellet, and 4 μl RNA solution could be used for one cDNA synthesis reaction. After polyadenylation, the reverse transcription of miRNA was carried out using a universal RT primer that contains degenerate nucleotides at 3′ end followed by an oligo(dT) and universal reverse primer sequence. The cDNA was amplified with specific forward and universal reverse primers. To normalize RNA content, the exogenous cel-miR-54 was used for plasma miR-34a-5p template normalization. The amplification products were detected by SYBR Green I. PCR and analyses were performed with a ViiA7 Quantitative PCR System (Applied Biosystems, Carlsbad, CA). The 2−∆∆Ct method was used to calculate relative expression levels of coding genes and miR-34a-5p between treatments. PCR primers for coding genes are shown in Supplementary Table 1.
The amount of 40 μg protein prepared from rat myocardium or H9c2 cells was used in a standard Western blot analysis. The polyvinylidene fluoride (PVDF) membrane binding sample protein was incubated with a high affinity anti-Bax antibody (1:2000 dilution), anti-Bcl-2 antibody (1:1000) (Abcam, Cambridge, MA), anti-caspase-3 antibody (1:2000), anti-Sirt1 antibody (1:1000) and anti-p66shc antibody (1:2000) (Cell Signaling Technology, Danvers, MA), respectively. An anti-GAPDH antibody (1:2000) (Santa Cruz Biotechnology, Santa Cruz, CA) was used to detect level of GAPDH as an internal control. Proteins were visualized using the ECL Plus detection system (GE Healthcare, Waukesha, WI).
As in our previous report21, the recombinant luciferase reporter plasmids containing sequences of potential miR-34a-5p binding sites in 3ʹ UTR of Sirt1 gene were constructed. Using a site-directed mutagenesis kit (TransGen, Beijing, China), miR-34a-5p complementary binding sequence ACUGCC was replaced with AGACGC to construct recombinant luciferase reporter plasmids containing the mutant potential miR-34a-5p binding sequences.
Human embryonic kidney (HEK) 293 cells (3 × 105 cells per well in 12-well plate) were co-transfected with 200 ng of recombinant luciferase reporter plasmid, 50 nM miR-34a-5p mimic, and 20 ng of pRL-TK as an internal control (Promega, Madison, WI). Activities of firefly luciferase (FL) and Renilla luciferase (RL) were measured 24 hr after transfection, and the relative ratio of the FL/RL was used to indicate the miR-34a-5p-mediated knockdown of Sirt1.
The data are presented as the means ± s.e.m. In each experiment, all determinations were performed at least in triplicate. Statistical significance between two measurements was determined by the two tailed unpaired Student’s t test, and among groups, it was determined by one-way ANOVA. A value of p < 0.05 was considered to be significant.
