An engineered miRNA PS-OMe miR130 inhibits acute lung injury by targeting eCIRP in sepsis

Experimental animals

Adult male Sprague–Dawley rats (300 g) and C57BL/6 mice (20–25 g) were purchased from Charles River Laboratories (Wilmington, MA), and housed in a temperature-controlled room on a 12-h light-dark cycle and fed a standard rodent chow diet. Animals were acclimated to the environment for 5–7 days. Every attempt was made to limit the number of animals used. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Feinstein Institutes for Medical Research and were performed in accordance with the National Institutes of Health and the Guide for the Care and Use of Laboratory Animals.

PS-OMe miR130 synthesis

Single stranded PS-OMe miR130 (5′-mC*mA*mG*mUmGmCmAmAmUmGmAmUmGmAmAmAmGmGmG*mC*mA*mU-3′) was synthesized by Integrated DNA Technologies (Coralville, Iowa) and provided as a lyophilized powder. * Indicates a PS bond and “m” indicates 2′OMe modification. The powder was then resuspended in nuclease free PBS for the desired concentration. A cy-3 labeled PS-OMe miR130 was synthesized and labeled at the 3′ end for the in vivo half-life experiment.

Treatment of macrophages with eCIRP and PS-OMe miR130

Mouse macrophage cell line RAW264.7 cells were purchased from American Type Culture Collection (ATCC). Peritoneal macrophages were isolated from C57BL6 mice. All cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Life Technologies Corporation, Grand Island, NY) with 10% of heat-inactivated fetal bovine serum (FBS; MP Biomedicals, Santa Ana, CA), 100 U/ml of each penicillin and streptomycin (Thermo Fisher Scientific, Waltham, MA), and 5% glutamine (Life Technologies Corporation). Prior to experiments, the medium was changed to OPTI-MEM (Life Technologies Corporation) for a total of 1 h. Cells were treated for 24 h with 1 µg/ml of eCIRP with or without 10 nM PS-OMe miR130 combined 30 min prior to stimulation of RAW 264.7 cells or peritoneal macrophages. Cell culture supernatant was collected for analysis. eCIRP was produced in house and quality control assays were performed as previously described (Qiang et al. 2013).

In vitro stability of PS-OMe miR130

8 µM of the PS-OMe miR130 was incubated in DMEM containing 10% non-heat-inactivated FBS, which has nucleases activities including RNAses, for different time points including 0, 6, 24, 48, and 72 h to measure the stability of PS-OMe miR130, similar to other groups (Barragan-Iglesias et al. 2018; Lennox et al. 2010). The control sample was 8 µM of PS-OMe miR130 in DMEM without the 10% non-heat-inactivated FBS. At each time point, an equal volume of 2X TBE-urea sample buffer (Invitrogen, Thermo Fisher Scientific) was added to the sample, and then flash frozen over dry ice. Samples were thawed and heated to 70 °C for 3 min and subsequently run on a 15% polyacrylamide TBE-urea gel (Invitrogen, Thermo Fisher Scientific) for 75 min at 180 V. After, the gel was stained with ethidium bromide (2 µg/ml) for 20 min (Sigma Aldrich, St Louis, MO), and washed with nuclease free water. The gel was imaged on a Bio-Rad gel reader.

In vivo half-life of PS-OMe miR130

Sprague-Dawley rats (n = 4) underwent induction of anesthesia with 2–4% inhalation isoflurane after which the bilateral groins were shaved and disinfected by swabbing with Betadine alternated two times with 70% alcohol. At this time, the right femoral artery and left femoral vein were cannulated with PE-50 polyethylene tubing (BD, Sparks, MD) containing a small amount of heparin (2 IU/ml) in normal saline solution. At time 0, 125 µl of 100 µM cy-3 labeled PS-OMe miR130 diluted in 375 µl of normal saline was injected via the femoral vein. At different time points, which included every 3 min for the first 15 min, and then every 15 min until 180 min, 200 µl of blood was withdrawn from the femoral artery. Animals were resuscitated at each time point with 200 µl of normal saline via the femoral vein. Serum was collected and fluorescence (550 nm excitation, 570 nm emission) was measured for each sample. The β-half-life (elimination half-life) was then calculated.

Surface plasmon resonance (SPR) analysis for eCIRP and PS-OMe miR130 interaction

eCIRP was immobilized on the surface of sensor as a ligand and PS-OMe miR130 was injected as an analyte. Binding reactions were performed in PBS 0.05% P20, pH7.4. Carboxyl sensors were used for the experiments. The sensor was first cleaned by injection 10 mM HCl 150 µl, followed by injection of 150 µl of the mixture of 1 aliquot of N-ethyl-N′-[3-diethylaminopropyl]-carbodiimide (EDC) and 1 aliquot of N-hydroxysuccinimide (NHS) to activate the sensor surface. An aliquot of 200 µl of 50 µg/ml of the ligand diluted in 10 mM sodium acetate (pH 5) was injected into channel-2 of the sensor for immobilization. Next, 150 µl of 1 M ethanolamine (pH 8.5) was injected to deactivate the remaining active sites on channel 1&2. The channel-1 was used as a control to evaluate nonspecific binding. The binding analyses were performed at a flow rate of 40 µl per min at 20 °C. To evaluate the binding, the analyte ranging from 10 to 300 nM were injected into channel-1 & 2, and the real-time interaction data were analyzed by TraceDrawer (Nicoya). The signals from the control channel-1 were subtracted from the channel coated with the ligand-2 for all samples. Data were globally fitted for 1:1 binding (one-to-one model).

Interaction between TLR4 and PS-OMe miR130 bound eCIRP

To determine the effect of PS-OMe miR130 on eCIRP binding to TLR4 or the TLR4/MD2 complex, NTA sensors were used. Human TLR4 & human TLR4/MD2 were purchased (R&D systems). The NTA sensor was first cleaned by injection 10mM HCL 150 µl and followed by injection of 150 µl of EDTA. Then the surface was activated by an injection of 40 mM NiCl2. Human TLR4 or the human TLR4/MD2 was immobilized in the running buffer at concentration 50 µg/ml to channel 2; eCIRP was injected as an analyte in concentrations of 125 nM to 1 µM. For effect of PS-OMe miR130, eCIRP was preincubated with PS-OMe miR130 with different concentration for 30 min at room temperature and then the complex was injected to channel 1 and 2. Binding reactions were carried out at 10 mM HEPES buffer, 150 mM NaCl, 3 mM EDTA, 0.05% P20, pH 7.4 at a flow rate of 40 µl/min at 20 °C. The channel-1 was used as a control to evaluate nonspecific binding, and the real-time interaction data were analyzed by TraceDrawer (Nicoya). The signals from the channel-1 were subtracted from the channel-2 coated with the ligand for all samples. Data were globally fitted for 1:1 binding (one-to-one model).

Three-dimensional virtual modelingStructure modeling

The nucleotide sequence of miRNA 130b-3p was derived from miRbase database (Kozomara et al. 2019). PS-OMe miR130 was modelled using PyMOL builder (PyMOL 2021). First, the three 5′ and 3′ terminal base in the phosphodiester linkage were replaced with sulfur giving rise to terminal phosphorothioate bonds. Second, 2′O-methyl ribose bases were incorporated throughout the miRNA.

Docking studies

The docking of CIRP and PS-OMe miR130 was performed using NPDock (Tuszynska et al. 2015), which combines GRAMM program to perform a rigid body global search, ranking, and scoring of best decoys using statistical potentials, clustering of best decoys. Finally, a Monte Carlo simulated annealing procedure (involving protein and nucleic acid molecules as rigid bodies) to optimize the protein-nucleic acid interactions in the representative clusters. The CIRP-PS-OMe miR130 and TLR4 receptor structure were docked using HDock (Yan et al. 2017), the FFT based translational search algorithm, which is optimized by iterative knowledge based scoring function, which is used both for protein-DNA and protein-RNA interactions and Patchdock (Schneidman-Duhovny et al. 2005), the algorithm that uses object recognition and image segmentation techniques. The interactions of CIRP and PS-OMe miR130 and the CIRP- PS-OMe miR130 and TLR4 structure were analyzed using PDBePISA tool (Krissinel et al. 2007). All the protein-microRNA structure complexes were visualized using PyMOL and Chimera tools (PyMOL 2021; Pettersen et al. 2004).

Mouse model of cecal ligation and puncture (CLP)

CLP was performed as previously described (Denning et al. 2020; Gurien et al. 2020). Briefly, mice were anesthetized with 2–4% inhalation isoflurane and placed in the supine position. The ventral abdomen was shaved and then disinfected by swabbing with Betadine alternated two times with 70% alcohol. A 2 cm incision was made, and the cecum was exposed. The cecum was ligated with 4-0 silk suture 1 cm proximal to the distal end of the cecum. The cecum was then punctured twice with a 22-guage needle and a small amount of cecal contents was extruded from each puncture. The cecum was then placed back in the peritoneal cavity and the incision was closed in two layers; a subcutaneous bolus of 1 ml of normal saline, and a subcutaneous dose of buprenorphine (0.05 mg/kg) were given. Mice were allowed to recover from surgery and anesthesia and then returned to their home cages. After 20 h, mice were sacrificed, and blood and lung tissue were collected and stored at − 80 °C for quantitative analysis. A section of the right lower lobe of the lung was stored in 10% formalin for histologic analysis. Mice were randomly assigned to create a total of 3 group: sham, CLP + vehicle, and CLP + treatment.

In vivo administration of PS-OMe miR130

After closure of the abdomen, vehicle (PBS) or PS-OMe miR130 at a dose of 12.5 nmol/mouse was injected intravenously via retroorbital injection using a 28G needle. The dose was determined from our previous study investigating the unmodified miRNA 130b-3p mimic in eCIRP induced inflammation and polymicrobial sepsis (Gurien et al. 2020).

Measurement of organ injury markers

Whole blood samples were centrifuged at 3000×g for 10 min to collect serum, which was then stored at − 80 °C prior to use. Serum levels of LDH was determined using specific colorimetric enzymatic assays (Pointe Scientific, Canton, MI) according to manufacturer’s instructions.

Cytokine measurements by enzyme-linked immunosorbent assay (ELISA)

Cell culture supernatant or mouse serum was analyzed by ELISA kits specific for IL-6, TNF-α (BD Biosciences, San Jose, CA), and IL-1β (Invitrogen, Thermofisher Scientific) according to manufacturer’s instructions.

Histological evaluation of lung injury

Lung tissues were collected from the right lower lobe and were fixed in 10% formalin before being embedded in paraffin. Tissues were cut in 5 μm cuts and stained with hematoxylin-eosin. Slides were evaluated under light microscopy to evaluate the degree of lung injury. Scoring was performed using a system created by the American Thoracic Society (Matute-Bello et al. 2011). Scores ranged from 0 to 1 and were based on neutrophils in the alveolar space, neutrophils in the interstitial space, hyaline membranes, proteinaceous debris filling the airspaces, and alveolar septal thickening.

Lung myeloperoxidase (MPO) assessment

Lung tissue was homogenized by sonication in 500 µl of potassium phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide. Two freeze–thaw cycles were performed over dry ice. Samples were centrifuged to collect the supernatant. The protein concentration of the supernatant was determined. The reaction was then carried out in a 96-well plate by adding samples into phosphate buffer containing O-dianisidine hydrochloride and H2O2. Light absorbance was read at 460 nm over a period of 5 min. Results were normalized to the protein concentration of each sample. MPO activity (1 unit was equal to the change in absorbance per min) was expressed as units per gram of protein.

Measurement of cytokines and chemokines by reverse transcription-quantitative (RT-qPCR) analysis

Total RNA was extracted from ischemic portions of the liver by TRIzol reagent (Invitrogen, Thermo Fisher Scientific Inc.) and was reverse transcribed into cDNA with reverse transcriptase (Applied Biosystems, Thermo Fisher Scientific Inc.). PCR reactions were carried out in 20 µl of a final volume of 0.08 µM of each forward and reverse primer, cDNA, water, and SYBR Green master mix (Applied Biosystems, Thermo Fisher Scientific Inc.). Amplification and analysis were conducted in a Step One Plus real-time PCR machine (Applied Biosystems, Thermo Fisher Scientific Inc.). Mouse β-actin mRNA was used as an internal control for amplification, and relative gene expression levels were calculated using 2−ΔΔCt method. Relative expression of mRNA was expressed as a fold change in comparison with sham tissues. The sequence for the primers used are as follows:

IL-6, 5′-CCGGAGAGGAGACTTCACAG-3′ (forward), 5′-CAGAATTGCCATTGCACAAC-3′ (reverse);

TNF-α, 5′-AGACCCTCACACTCAGATCATCTTC-3′ (forward), and 5′-TTGCTACGACGTGGGCTACA-3′ (reverse);

IL-1β, 5′-CAGGATGAGGACATGAGCACC-3′ (forward), and 5′-CTCGCAGACTCAAACTCCAC-3′ (reverse);

KC, 5′-GCTGGGATTCACCTCAAGAA-3′ (forward), and 5′-ACAGGTGCCATCAGAGCAGT-3′ (reverse);

MIP-2, 5′-CCCTGGTTCAGAAAATCATCCA-3′ (forward), and 5′-GCTCCTC-CTTTCCAGGTCAGT-3′ (reverse);

β-Actin, 5′-CGTGAAAAGATGACCCAGATCA-3′ (forward), and 3′-TGGTACGACCAGAGGCATACAG-3′ (reverse).

TUNEL assay

Apoptosis was assessed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. For TUNEL staining, fluorescence staining was performed using a commercially available In Situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN). The assay was conducted according to the manufacturer’s instructions. 4′,6-Diamidino-2 phenylindole (DAPI) was used as a nuclear counterstain. TUNEL positive cells were counted using ImageJ software.

Survival study

Animals also underwent a 10-day survival study as previously described (Denning et al. 2020). For the survival experiments, mice underwent CLP with a single puncture using 22G needle and were given 500 µl of the antibiotic imipenem (0.5 µg/kg, Merck) and 500 µl of normal saline subcutaneously at the time of laparotomy. The mice were then given a one-time dose of vehicle or PS-OMe miR130 (12.5 nmol/mouse) via retroorbital injection. Mice were then monitored twice daily for 10 days for their survival rates.

Statistical analysis

Data represented in the figures are expressed as mean ± SEM, was checked for normality using Kolmogorov–Smirnov test. Normally distributed data was compared by using one-way analysis of variance (ANOVA) using Student–Newman–Keuls (SNK) post hoc analysis for multiple groups. When appropriate, non-normally distributed data was compared using nonparametric 1-way comparison among multiple groups using Kruskal–Wallis test with Dunn’s multiple-comparisons test. The specific tests used for each graph are identified in the figure legends. Survival rates were analyzed by the Kaplan–Meier estimator and compared using a log-rank test. Differences in values were considered significant if p ≤ 0.05. Data analysis was carried out using GraphPad Prism graphing and statistical software (GraphPad Software).

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