Sprague-Dawley rats (age, 5 weeks old; male and female; n=10) weighing between 250 and 300 g and maintained under controlled temperature (20±1°C), humidity (55±10%) and illumination (12 h dark:light), were subjected to limb electrical burns as described previously (14). Subsequent to undergoing anesthesia with 3% pentobarbital (10 mg/kg; Sigma-Aldrich, St. Louis, MO, USA), the rats were maintained in a stationary position on the experimental table in order to shave the hip and right posterior limb. Next, a null electrode was placed on the right limb (0.5 cm proximal to ankle), with a live electrode placed on the ipsilateral gluteus. The electricity (output voltage: 1,000 V) was administered for 0.1 sec to conduct the burns using the YLS-5Q Skin Burning Device model (Biowill Co, Ltd. Shanghai, China). Blood samples were obtained from the tail vein (using 1 ml syringe) at 6 and 24 and 36 h, subsequent to the administration of the electric burns, and then centrifuged at 300 × g, −4°C for 15 min, to obtain the blood serum.

HaCaT cells (Shanghai Bogoo Biotechnology Co., Ltd., Shanghai, China) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) medium supplemented with 10% (v/v) fetal bovine serum, 100 U/ml streptomycin and 100 U/ml penicillin at 37°C in a 5% (v/v) CO₂ humidified atmosphere. DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 0.5 mg/ml collagenase type I was applied at 37°C for 3 h to separate fibroblasts from rat blood serum. These fibroblasts were pelleted by centrifugation at 300 × g, for 7 min, at room temperature, and then cultured in the aforementioned conditions. Cells passaged 2–4 times were used for further experiments.

The full-length wild-type Tm7sf2 coding sequence (alternate names: D14SR, SR-1; RefSeq accession, NP_003264) was sub-cloned into pcDNA3.1⁺ to produce the Tm7sf2 expression vector (pcDNA3.1⁻ Tm7sf2), which was confirmed by sequencing at Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). Tm7sf2-specific siRNA and control siRNA were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used for the cell transfection, according to manufacturer's protocol, and empty construct pcDNA3.1 was also transfected as a control. Stable Tm7sf2 transfectants were selected using G418 (Gibco; Thermo Fisher Scientific, Inc.).

HaCaT cells were divided into four groups and exposed to the following treatments for 20 h: i) Control, 20% normal serum; ii) burn, 20% electrical burned serum; iii) 20% normal serum + siTm7sf2; iv) 20% electrical burned serum + siTm7sf2.

A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) colorimetric assay and Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) invasion chamber assay were used for the determination of cell proliferative and invasive capacities, respectively, as described previously (15,16). Each experiment was performed three times.

Calcein-acetomethyl (25 mg; Molecular Probes; Thermo Fisher Scientific, Inc., in 5 ml dimethyl sulfoxide/ml media; Sigma-Aldrich) was used for labeling the treated HaCaT cells. Subsequent to centrifugation at 300 × g, for 7 min, at room temperature, the cells were washed three times with phosphate-buffered saline (PBS; Sigma-Aldrich) to allow the adherence to a compact monolayer of the EA.hy926 human endothelial cell line (Cell bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) at 37°C for 1 h. Subsequently, the monocytes in the medium were aspirated. A fluorescence microscope (Olympus CX31; Olympus Corporation, Tokyo, Japan) was used for the visualization cell adherence at excitation and emission wavelengths of 488 and 535 nm, respectively. Six fields were selected per well to calculate the number of adherent monocytes using Image J software (version 2; National Institutes of Health, Bethesda, MD, USA).

RT-qPCR was performed as previously described (17). Total mRNA was isolated from cells, and complementary DNA (cDNA) was produced using reverse transcriptase (iScript cDNA Synthesis kit; Bio Rad Laboratories, Inc., Hercules, CA USA). Tm7sf2 expression levels were measured via SYBR Green based qPCR (SYBR-Green Master mix; Thermo Fisher Scientific, Inc.). Reactions were performed with the initial denaturation at 95°C for 5 min and then 40–50 cycles at 95°C for 30 sec, 58–62°C for 30 sec and 72°C for 30 sec, and a final extension at 72°C for 10 sec.

A 10–12% sodium dodecyl sulfate-polyacrylamide gel (Thermo Fisher Scientific, Inc.) was used for protein separation at 100 V for 90 min. Next, the proteins were blotted onto polyvinylidene difluoride membranes, which were blocked with PBST (0.1% Triton X-100; Sigma-Aldrich in PBS), and probed with primary antibodies including anti-Tm7sf2 (cat no. sc-162325) Beclin 1 (cat no. sc-11427) and LC3-II (cat no. sc-28266) all diluted 1:200 in bovine serum albumin) and horseradish peroxidase-conjugated secondary antibodies (sc-391122; diluted 1:5,000) and GAPDH (sc-59540l; diluted 1:1,000). GAPDH was used as a loading control. All antibodies were purchased from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA) unless otherwise stated. Enhanced chemiluminescence (GE Healthcare Life Sciences; Uppsala, Sweden) was utilized to develop the immunoreactive protein bands, which were analyzed by a densitometer (Image Pro Plus, version 1.63; Media Cybernetics, Rockville, MD, USA).

Each sample was processed and measured three times. The data are presented as the mean ± standard deviation. The statistical analysis was performed using SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA) statistical software, one-way analysis of variance, followed by Fisher's Protected Least Significant Difference post-hoc analysis used to determine statistical differences between groups. P<0.05 was considered to indicate a statistically significant difference.

Compared with the control group, Tm7sf2 mRNA levels in rats with electric burns were significantly higher with the increasing time following the administration of the burn (P<0.05). The levels were then reduced after 24 h (Fig. 1A). In the 20% electrical burned serum group, the protein levels of Tm7sf2 were significantly higher than the control group (P<0.05). In the siTm7sf2-transfected cells, Tm7sf2 expression was significantly lower compared with the control group (P<0.05), however was restored to normal with the addition of Tm7sf2 (Fig. 1B; Table I).

The MTT assay demonstrated that the burn serum significantly reduced cell proliferation when compared with the normal serum (P<0.05). Furthermore the proliferation of cells treated with Tm7sf2 siRNA was significantly lower in the burn serum group compared with the control cells (P<0.05; Fig. 2; Table II).

The number of monocytes represent the adhesion strength between the EA.hy926 and HaCaT cells (18). The 24 h treatment with burn serum induced the highest expression levels of Tm7sf2, therefore cell adhesion of the HaCaT cells was detected subsequent to 24 h of treatment. The burn serum treatment significantly enhanced the capacity of monocyte-endothelial cell adhesion, even if the cells had been transfected with siTm7sf2, compared with normal serum treatment (P<0.05; Fig. 3; Table III).

To investigate the influence of the burn serum and siTm7sf2 on autophagic-abilities of HaCaT cells, the autophagy associated Beclin1 and LC3-II protein levels in HaCaT cells were detected using western blot analysis. As indicated in Fig. 4, burn serum (treated for 24 h) increased the expression levels of Beclin1 and LC3-II when compared with control groups (P<0.05). The siTm7sf2 group also presented increased expression levels, while the effect was more evident in the burn serum-treated cells (P<0.01; Table IV).