A total of 155 healthy Sprague-Dawley (SD) rats (specific-pathogen-free; weight, 200–250 g; male) were purchased from Chengdu Dashuo Biotech Co. (Chengdu, China). The mice were maintained in standard conditions at room temperature with 50% humidity, under 12 h/12 h light/dark cycle and with free access to food and water. All animal experiments were conducted according to the ethical guidelines of the First Hospital of Tsinghua University (Beijing, China).

TRIzol Total RNA Extraction kit was purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). PrimeScript RT Reagent kit and SYBR PrimeScript RT-PCR kit were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). The plasmids of shRNA-normal control (NC), shRNA-RREB1, agomiR-NC and agomiR-145 were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). EntransterTM-in vivo transfection reagent was purchased from Engreen Biosystem Co., Ltd. (Beijing, China). Goat anti-rat RREB1 (St. Louis Park, MN, USA), rabbit anti-rat p-AKT (Cell Signaling Technology, Inc., Danvers, MA, USA), mouse anti-rat GAPDH and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Abcam (Burlingame, CA, USA). Dual Luciferase Reporter Assay kit was purchased from Promega Corporation (Madison, WI, USA).

A total of 125 rats were randomly divided into the control, sham, CCI model, agomiR-145 and agomiR-NC groups, with 25 rats in each group. For the CCI group, the left sciatic nerve of rats was exposed by surgery and ligated. For the sham group, the left sciatic nerve of rats was exposed by surgery, but without ligation. Following the establishment of the CCI model, intrathecal injection of agomiR-145 was performed in the agomiR-145 group once daily for 7 consecutive days. For the agomiR-NC group, intrathecal injection of agomiR-NC was performed once daily for 7 consecutive days. For the control group, no surgery or treatment was performed.

Following the establishment of the CCI model, an additional 30 healthy male SD rats were randomly divided into shRNA-NC and shRNA-RREB1 groups. In vivo transfection of shRNA-RREB1 and shRNA-NC plasmids was performed by EntransterTM-in vivo transfection reagent, according to the manufacturer's instructions. The transfection was performed once daily for 7 consecutive days following the establishment of CCI.

At 1 day prior to, and 1, 3, 5 and 7 days following surgery, the PWTL and PWMT of rats in each group were detected. At each time point, 5 rats in each group were used. In addition, at each time point, 5 rats in each group were sacrificed by cervical dislocation following anesthesia using 1% pentobarbital (EMD Millipore, Billerica, MA, USA). The lumbar spinal cord tissues were collected from these rats for miRNA and protein detection.

Rats were placed on a 7370 Planter Test machine (Ugo Basile S.R.L., Varese, Italy) and allowed to acclimatize for 3 days prior to testing. A radiant thermal stimulator was focused onto the plantar surface of the hind paw for 15 sec. The nociceptive endpoints in the radiant heat test were the characteristic lifting or licking of the hind paw, and the time to the endpoint was recorded as the PWTL. To avoid tissue damage, a cut-off time of 30 sec was used. There were 3 repeats of the trial per rat, and 5 min intervals between trials. The mean PWTL was obtained from the three recordings.

A 2390 Electronic von-Frey Anesthesiometer (Naturegene Life Science, Cranbury, NJ, USA) was used. PWMT was performed according to the method described by Hargreaves (18). Brisk withdrawal or paw flinching were considered as positive responses. There were 3 repeats of the trial per rat, and 5 min intervals between trials. The mean PWMT was calculated from the three recordings.

Total RNA was extracted from lumbar spinal cord tissues using the TRIzol Total RNA Extraction kit, according to the manufacturer's instructions. RNA was reverse transcribed into cDNA using a PrimeScript RT Reagent kit. The expression level of miR-145 was detected by RT-qPCR. The forward primer sequence for miR-145 was 5′-CCAGTTTTCCCAGGAATC-3′; the reverse primer sequence was a universal primer provided by the kit. RT-qPCR was performed using a SYBR Green PrimeScript RT-PCR kit, according to the manufacturer's instructions. The volume of the PCR reaction system was 20 µl, which consisted of 10 µl qRT-PCR mix, 0.5 µl forward primer and 0.5 µl reverse primer, 2 µl cDNA and 7 µl ddH₂O. The following thermal cycling conditions were used: Pre-denaturation at 95°C for 1 min; 40 cycles of 95°C for 15 sec; and 60°C for 30 sec. The 2^−ΔΔCt method was used to calculate the relative expression levels of miR-145 (19).

Tissues were ground into a powder in liquid nitrogen, and were lysed on ice in radioimmunoprecipitation assay buffer (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Beijing, China) with 1 mM PMSF for 30 min. Total proteins were extracted by centrifugation at 14,000 × g for 10 min at 4°C. Total proteins were extracted (15 µg) from lumbar spinal cord tissues and separated by a 10% SDS-PAGE. Next, proteins were transferred onto a polyvinylidene fluoride membrane. After blocking with non-fat milk, the membrane was incubated with the following rabbit primary antibodies at 4°C overnight: Anti-RREB1 (ab64168, 1:2,000), anti-p-AKT (ab38449, 1:2,000) and anti-GAPDH (ab8245, 1:5,000, all Abcam). After washing, the membrane using 0.1% phosphate-buffered saline and Tween 20 for 5 mins each time and in total three times, the membrane was incubated with HRP-conjugated secondary antibodies [goat anti-rabbit IgG (ab6721), and goat anti-mouse IgG H&L (ab6789, both 1:10,000, both Abcam)] at room temperature for 1 h. Finally, the membrane was developed by enhanced chemiluminescence plus reagent (Hanbio Biotechnology Co. Ltd., Shanghai, China). The developed film was scanned using AlphaImager gel imaging systems (AlphaImager, Santa Clara, CUA, USA), and the western blot images were analyzed using Quantity One software version 4.62 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). GAPDH was used as an internal control.

The dual luciferase reporter assay was performed using a Dual Luciferase Reporter Assay kit. Briefly, the wild type and mutant 3′-untranslated region (UTR) of RREB1 mRNA were cloned into a pMIR-REPORT vector. Then, these plasmids, together with miR-145 mimics (100 nM), were co-transfected into 293T cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China). After incubation at 37°C for 24 h, cells were lysed by washing with cold PBS, the cells were incubated with RIPA lysis buffer with 1% PMSF on ice for 20 min and fluorescence was detected using GloMax 20/20 Luminometer (Promega Corporation). Cells without miR-145 mimic transfection were used as control cells. The fluorescence intensity of Renilla was used as an internal reference.

Data was expressed as the mean ± standard deviation and was analyzed using SPSS statistical analysis software (version 17.0; SPSS, Inc., Chicago, IL, USA). A paired t-test was used to analyze the difference between groups. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effect of miR-145 on the mechanical threshold of rats, PWMT was performed 1 day prior to, and 1, 3, 5 and 7 days after CCI. As shown in Fig. 1, at 1 day prior to CCI, there was no significant difference in mechanical threshold among the 5 groups. In the control and sham group, the mechanical threshold was not significantly different at any time points. However, the mechanical threshold in the CCI and agomiR-NC group at 1, 3, 5 and 7 days after CCI was significantly decreased compared with 1 day prior to CCI (CCI: 1 day, P=0.034; 3 day, P=0.024; 5 day, P=0.042; 7 day, P=0.020 and agomiR-NC: 1 day, P=0.023; 3 day, P=0.029; 5 day, P=0.022; 7 day, P=0.019). At 5 and 7 days after CCI, the mechanical threshold in the agomiR-145 group was significantly increased compared with the CCI group (5 day, P=0.033; 7 day, P=0.027). No significant difference was identified between the CCI and agomiR-NC groups at any time points. These results suggest that miR-145 treatment alleviates the mechanical threshold decrease induced by CCI.

To determine the effect of miR-145 on thermal latency, PWTL was performed 1 day prior to, and 1, 3, 5 and 7 days after CCI. Similar to PWMT, no significant difference was found 1 day prior to CCI among the 5 groups (Fig. 2). In the control and sham group, there was no significant difference in thermal latency at any time points. However, at 1, 3, 5 and 7 days after CCI, the thermal latency in the CCI and agomiR-NC groups was significantly lower compared with the thermal latency 1 day prior to CCI (CCI: 1 day, P=0.036; 3 day, P=0.023; 5 day, P=0.040; 7 day, P=0.022 and agomiR-NC: 1 day, P=0.032; 3 day, P=0.027; 5 day, P=0.037; 7 day, P=0.025). After miR-145 treatment, the thermal latency was elevated in the agomiR-145 group. Compared with the CCI group, the thermal latency in the agomiR-145 group was significantly higher at 3, 5 and 7 days after CCI (3 day, P=0.039; 5 day, P=0.027; 7 day, P=0.017). There was no significant difference in the thermal latency between the CCI and agomiR-NC groups at any time points. This data indicates that miR-145 treatment improves the thermal latency decrease induced by CCI.

To detect the expression of miR-145 in lumbar spinal cord tissues, RT-qPCR was conducted 1 day prior to, and 1, 3, 5 and 7 days after CCI. Results are presented in Fig. 3. There was no significant difference in miR-145 expression levels between the control and sham groups, or between the CCI and agomiR-NC groups, at any time points. Compared with the control group, miR-145 expression levels in the CCI group was significantly lower at 1, 3, 5 and 7 days after CCI (1 day, P=0.043; 3 day. P=0.038; 5 day, P=0.002; 7 day, P=0.001). After treatment with agomiR-145, miR-145 expression was increased in the agomiR-145 group. The expression level of miR-145 1, 3, 5 and 7 days after CCI was 2.18±0.43, 5.34±0.57, 6.18±0.81 and 6.56±1.16, respectively, in the agomiR-145 group, which was significantly higher compared with the CCI group (1 day, P=0.027; 3 day, P=0.007; 5 day, P=0.001; 7 day, P=0.001). Thus, the results demonstrate that miR-145 expression is decreased in CCI rats, and that agomiR-145 treatment can increase miR-145 expression levels in CCI rats.

To measure the expression of RREB1 and p-AKT in the lumbar spinal cord tissue of CCI rats 1 day prior to, and 1, 3, 5 and 7 days after CCI, western blot analysis was performed; representative and quantitative western blot results are presented in Fig. 4. In the control group, there was no significant difference in the expression of RREB1 or p-AKT at any time points (Fig. 4A). In the CCI group, the expression levels of RREB1 and p-AKT were elevated (Fig. 4B). As shown in Fig. 4C, following treatment with agomiR-145, the expression levels of RREB1 and p-AKT in the agomiR-145 group were decreased at 3, 5 and 7 days after CCI. Statistically, the expression levels of RREB1 (1 day; P=0.039; 3 day, P=0.021; 5 day, P=0.027; 7 day, P=0.036; Fig. 4D) and p-AKT (1 day; P=0.031; 3 day, P=0.027; 5 day, P=0.021; 7 day, P=0.033; Fig. 4E) in the CCI group were significantly higher compared with the control group at 1, 3, 5 and 7 days after CCI. In addition, at 1, 3, 5 and 7 days after CCI, the agomiR-145 group had significantly lower expression levels of RREB1 (1 day; P=0.048; 3 day, P=0.032; 5 day, P=0.015; 7 day, P=0.018; Fig. 4D) and p-AKT (1 day; P=0.046; 3 day, P=0.016; 5 day, P=0.011; 7 day, P=0.023; Fig. 4E) compared with the CCI group. The changes in expression levels of RREB1 and p-AKT in the sham and agomiR-NC groups were similar to those in the control and CCI groups, respectively (data not shown). These results indicate that CCI increases the expression levels of RREB1 and p-AKT in lumbar spinal cord tissues, whereas miR-145 decreases the increase induced by CCI.

To determine the effect of RREB1 in CCI rat models, in vivo transfection of shRNA-RREB1 and shRNA-NC plasmids was performed following the establishment of CCI in rats. The pain behavior of PWMT and PWTL, protein expression of RREB1 and p-AKT, and expression level of miR-145 were detected. As shown in Fig. 5A, the mechanical threshold of CCI rats in the shRNA-NC group 1, 3, 5 and 7 days after CCI was decreased compared with that at 1 day prior to CCI. However, in the shRNA-RREB1 group, the mechanical threshold of CCI rats was increased compared with the shRNA-NC group; this difference was significant at 3, 5 and 7 days after CCI (3 day, P=0.032; 5 day, P=0.370; 7 day, P=0.041). Similar results were observed in the thermal latency of CCI rats (Fig. 5B). Thermal latency in the shRNA-RREB1 group was significantly increased compared with the shRNA-NC group 1, 3, 5 and 7 days after CCI (1 day, P=0.021; 3 day, P=0.026; 5 day, P=0.031; 7 day, P=0.043). As shown in Fig. 5C, the expression levels of RREB1 and p-AKT 7 days after CCI were decreased following shRNA-RREB1 transfection. In addition, compared with the shRNA-NC group, the shRNA-RREB1 group had significantly higher levels of miR-145 7 days after CCI (P=0.023; Fig. 5D). Collectively, these results suggest that downregulation of RREB1 protein expression levels in CCI rats may improve pain behavior, decrease p-AKT protein expression and increase miR-145 expression levels.

To determine whether RREB1 mRNA is one of the targets of miR-145, a dual luciferase reporter assay was performed following transfection of miR-145 mimics and wild type or mutant RREB1 mRNA. Cells without miR-145 mimic transfection were used as control cells. The sequences of the wild type and mutant 3′-UTR region of RREB1 mRNA are presented in Fig. 6A. The luciferase intensity is presented in Fig. 6B. In cells transfected with miR-145 mimics and wild type RREB1, the luciferase intensity was significantly decreased compared with control cells (P=0.017). However, there was no significant difference in luciferase intensity between control cells and cells transfected with miR-145 mimics and mutant RREB1. This data suggests that RREB1 mRNA is a target of miR-145.