Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from GE Healthcare Life Sciences (HyClone; Logan, UT, USA), TRIzol^® reagent was obtained from Thermo Fisher Scientific, Inc. (Invitrogen; Waltham, MA, USA), and the Transcriptor First Strand cDNA Synthesis kit, FastStart Universal SYBR^®-Green Master (Rox) and X-tremeGENE HP DNA transfection reagent were purchased from Roche Diagnostics (Basel, Switzerland). The Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit was obtained from BD Biosciences (BD Pharmingen; Franklin Lakes, NJ, USA). Rabbit monoclonal antibodies against β-catenin (#8480; 1:1,000) and β-actin (#4970; 1:1,000) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA) and horseradish peroxidase-conjugated anti-rabbit secondary antibodies (#7074; 1:2,000) were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Small interfering RNAs (siRNAs) targeting lincRNA-p21 and β-catenin transcripts were purchased from Thermo Fisher Scientific, Inc. The ELISA kit for MIF was purchased from Abcam (#ab7207; Cambridge, UK), and the Mitochondrial Membrane Potential assay kit with JC-1 (#C2006) and Reactive Oxygen Species (ROS) assay kit (#S0033) were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). The Superoxide Dismutase (SOD) Activity Colorimetric assay (#ab211096), Lipid Peroxidation (malondialdehyde; MDA) assay kits (#ab118970) and mouse recombinant MIF were purchased from Abcam.

Bone marrow-derived mesenchymal stem cells (MSCs) were isolated using a standard protocol, as described previously (16). A total of 12 male mice (mean age, 6 months; mean weight, 22.85±2.62 g) were purchased from the Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China). Mice were kept to a 12 h light/12 h dark cycle at 21±2°C with 30-70% relative humidity. Food and water was freely available throughout. All animal procedures were approved by the Wenzhou Medical University Institutional Animal Care and Use Committee (Wenzhou, China). Briefly, bone marrow was isolated from mouse femurs and tibias by flushing with PBS. Adherent MSCs were cultured at 37°C and 5% CO₂ in high-glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Third-passage MSCs were used for experiments.

Apoptosis was induced in vitro by hypoxia and SD to mimic the in vivo conditions of the ischemic myocardium, as previously reported (21). Apoptosis was induced by incubating MSCs in serum-free DMEM in a glove box (model no. 855-AC; Plas Labs, Inc., Lansing, MI, USA) with a regulated atmosphere (anaerobic chamber) to scavenge free oxygen (hypoxia/SD group). For MIF treatment, cells were cultured with DMEM containing 100 ng/ml recombinant MIF and incubated at 37°C for various periods of time, as reported previously (22). Untreated cells were used as the control group throughout.

The effects of MIF on apoptosis were determined by detecting phosphatidylserine exposure on cell plasma membranes using an Annexin V-FITC Apoptosis Detection kit, according to the manufacturer’s protocol. Briefly, cells were harvested (4°C, 5 min, 12,000 × g) and washed in ice-cold PBS, resuspended in 300 μl binding buffer, and incubated with 5 μl Annexin V-FITC solution for 30 min at 4°C in the dark, followed by further incubation in 5 μl propidium iodide for 5 min at 4°C. The cells were then analyzed immediately using bivariate flow cytometry with a BD FACSCanto II equipped with BD FACSDiva software (version 8.0.1; BD Biosciences).

Caspase 3/7/8 activities in MSCs were determined as described previously (23). Briefly, activities of caspases 3/7/8 in cell lysates of MSCs, MSCs under hypoxia/SD conditions and MSCs treated with MIF under hypoxia/SD conditions were measured using a Cell Meter Caspase 3/7/8 Activity Apoptosis assay kit (AAT Bioquest, Sunnyvale, CA, USA) according to the manufacturer’s protocol. Results were read at 520 nm with a microplate reader and expressed as the fold change in caspase 3/7/8 activity compared with the control.

Expression levels of several genes were analyzed using RT-qPCR. Briefly, total cellular RNA was isolated using TRIzol reagent and reverse transcribed using a Transcriptor First Strand cDNA Synthesis kit according to the manufacturer’s protocol. qPCR was performed using the Fast Start Universal SYBR Master and the Applied Biosystems Step One Plus Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. The threshold number of cycles (Cq) was set within the exponential phase of the PCR. The ΔCq value for each target gene was calculated by subtracting the Cq value for GAPDH (internal control) from that of the target gene. Relative gene expression levels were calculated by comparing the ∆Cq values between the control and experimental conditions for each target PCR using the following equation: 2^{−(ΔCq sample−∆Cq control)} (24). The primer pairs used to detect the mRNA levels of target genes are listed in Table I.

MSCs were lysed with ice-cold lysis buffer (Beyotime Institute of Biotechnology) to obtain total protein, then β-catenin and β-actin expression levels were evaluated using western blotting. Cellular extracts were prepared according to the manufacturer’s protocol. Protein samples were quantified and separated by SDS-PAGE. Western blotting was performed as described previously (25). Quantity One software (version 4.5.2; Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for densitometric analysis.

MSCs were transfected using X-treme GENE HP DNA Transfection reagent according to the manufacturer’s protocol. Briefly, MSCs (1×10⁵ cells/well) were cultured in 6-well plates for 24 h and then treated with the transfection reagent (siRNA weight ratio of 3:1) for 20 min. This was followed by the addition of a mixture containing 100 nM siRNA and incubation in 2 ml DMEM for 48 h at 37°C. Scrambled non-targeting siRNA (siRNA-NT) was used as a negative control. The knockdown efficiency was determined by RT-qPCR, as aforementioned. The sequences are listed in Table I.

Adenoviral vectors expressing lincRNA-p21 (Ad-lincRNA-p21 group) and control scrambled sequence (Ad-ctrl group) were designed and synthesized by Shanghai GeneChem Co., Ltd. (Shanghai, China). MSCs were transfected using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at a final concentration of 100 nM.

Cells were cultured in complete DMEM in 96-well microtiter plates at 37°C for 1 day to achieve 1×10⁵ cells/well. The cells were then washed with PBS and incubated with 5 μg/ml JC-1 at 37°C for 15 min. Following two washes with PBS, time-dependent JC-1 fluorescence was recorded using an ELISA plate reader. The fluorescent probe was excited at 490 nm and the emission was read alternately at 530 and 590 nm.

Levels of intracellular ROS were determined using 2,7-dichlorodihydrofluorescein diacetate (Beyotime Institute of Biotechnology), following the manufacturer’s protocol. The fluorescence intensity of the cells was measured using a fluorescence spectrophotometer, with excitation and emission wavelengths of 488 and 525 nm, respectively.

SOD activity in MSCs was determined using a SOD Activity Colorimetric assay according to the manufacturer’s protocol. Briefly, protein was isolated from MSCs using lysis buffer, and SOD activity was measured in 10 μg of total protein extract. Absorbance was measured at 450 nm.

Lipid peroxidation was monitored using an assay kit to measure the formation of MDA, according to the manufacturer’s protocol. Briefly, MSCs (1×10⁶ cells) were homogenized on ice in 300 μl of MDA lysis buffer (with 3 μl of 100X butylated hydroxytoluene), and then centrifuged (4°C, 13,000 × g, 10 min) to remove insoluble material. The supernatant (200 μl) was added to 600 μl of thiobarbituric acid and incubated at 95°C for 60 min. The samples were cooled to room temperature in an ice bath for 10 min, and the absorbance at 532 nm was measured spectrophotometrically.

Data are expressed as the mean ± standard deviation following three repeats. Differences among groups were analyzed using one-way analysis of variance followed by Tukey’s multiple comparisons test, and comparisons between two groups were evaluated by Student’s t-tests using SPSS software (version 19.0; IBM, Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Hypoxia/SD induced MSC injury, with maximal injury at 24 h. To determine if MIF protects MSCs from hypoxia/SD-induced injury, MSCs were exposed to MIF (100 ng/ml) followed by hypoxia/SD for 24 h and apoptosis rates were determined by flow cytometry. MIF demonstrated a significant anti-apoptotic effect of the hypoxia/SD model, as demonstrated by Annexin V-FITCFACS analysis (Fig. 1A and B).

The anti-apoptotic effects of MIF were further examined by measuring the effects of MIF pretreatment on changes in caspases 3/7 and caspase 8 following hypoxia/SD induction. MIF pretreatment significantly reduced the stress-induced increases in caspase activities (Fig. 1C and D).

lincRNA-p21 has been reported to be associated with cellular apoptosis (26). To determine if lincRNA-p21 was involved in hypoxia/SD-induced apoptosis, its expression was evaluated in MSCs exposed to hypoxia/SD for 24 h. lincRNA-p21 was significantly increased in MSCs following hypoxia/SD treatment, as demonstrated by RT-qPCR analysis (Fig. 2A). Furthermore, the hypoxia/SD-induced increase was significantly attenuated by exogenous MIF treatment (Fig. 2A).

The role of MIF-induced inhibition of lincRNA-p21in protecting MSCs from hypoxia/SD-induced apoptosis was further examined. MIF was added to MSCs prior to hypoxia/SD treatment and apoptosis was measured by flow cytometry. In a parallel experiment, MSCs were transfected with Ad-lincRNA-p21 prior to treatment with MIF (Fig. 2B) and cultured under hypoxia/SD conditions. MIF treatment significantly decreased cellular apoptosis (Fig. 2C and D), and reduced caspases 3/7 and caspase 8 activities compared with cells under hypoxia/SD treatment alone (Fig. 2E and F). In addition, these effects were significantly abolished by lincRNA-p21 overexpression (Fig. 2C-F).

The Wnt/β-catenin signaling pathway is a known target of lincRNA-p21, and has been reported to be involved in cellular injury in MSCs (16). In the present study, β-catenin protein expression levels in MSCs were significantly decreased by hypoxia/SD compared with control cells, which was restored following pre-treatment with MIF. Overexpression of lincRNA-p21by Ad-lincRNA-p21 transfection abolished the effect of MIF, while transfection with the control vector had no significant effect (Fig. 3A and B). The mechanism underlying the modulation of lincRNA-p21-Wnt/β-catenin signaling by MIF in hypoxia/SD-associated cellular apoptosis was examined by silencing β-catenin using siRNA. β-catenin mRNA expression levels were significantly reduced in cells transfected with siRNA-β-catenin compared with cells transfected with siRNA-NT control (Fig. 3C). Pretreatment with MIF protected MSCs from apoptosis induced by hypoxia/SD (Fig. 4A and B), and significantly decreased the activities of caspases 3/7 and caspase 8 (Fig. 4C and D). However, these effects were abolished by silencing β-catenin and not by transfection with siRNA-NT (Fig. 4).

Oxidative stress is associated with cellular apoptosis (27), thus, the feedback loop between oxidative stress and the lincRNA-p21-Wnt/β-catenin signaling pathway modulated by MIF was explored. Mitochondrial transmembrane potential, ROS generation, SOD activation and lipid peroxidation were examined (Fig. 5). Hypoxia/SD significantly decreased the mitochondrial transmembrane potential (Fig. 5A) and SOD activation (Fig. 5C), while increasing ROS generation (Fig. 5B) and MDA activation (Fig. 5D). Pretreatment with MIF significantly increased the mitochondrial transmembrane potential and SOD activation, and decreased ROS generation of ROS and MDA activation. These anti-oxidant effects of MIF were abolished by ectopic expression of lincRNA-p21 or by silencing β-catenin (Fig. 5A-D).