A Novel Rac1-GSPT1 Signaling Pathway Controls Astrogliosis Following Central Nervous System Injury^*

Generation of Rac-KO Mice in Astrocytes

Among the three members of Rac, only Rac1 mRNA was detected in astrocytes by RT-PCR (Fig. 1A). Based on this result, we generated Rac1-KO mice in astrocytes using GFAP-Cre transgenic mice (23) and Rac1^flox/flox mice (24), hereafter referred to as GFAP-Cre;Rac1^flox/flox (Rac1-KO) mice. KO of Rac1 in astrocytes was confirmed by immunoblotting using primary astrocytes obtained from Rac1-KO mice (Fig. 1B). Moreover, the pattern of GFAP-Cre-driven recombination was assessed using GFAP-Cre;Rac1^flox/+;tdTomato mice generated by the intercrossing of GFAP-Cre;Rac1^flox/flox and CAG-STOP^flox;tdTomato mice (Fig. 1, C and D). Fluorescence for tdTomato expression was positive in GFAP-positive astrocytes within the spinal cords of GFAP-Cre;Rac1^flox/+;tdTomato mice but not control (Rac1^flox/+;tdTomato) mice (Fig. 1E).

Better Recovery of Rac1-KO Mice after SCI

We developed SCI in Rac1-KO mice via a contusion injury and evaluated the locomotor capabilities of their hind limbs at 18 time points (1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35 days post-injury) over 35 days using 2 scoring systems: the 0–8-point Basso Mouse Scale (BMS) score (25) and the 0–4-point Body Support Scale (BSS) score (26). The BMS and BSS evaluate hind limb movement and body support, respectively. Recovery of locomotor capability after SCI was significantly better in Rac1-KO mice compared with control mice for both scoring systems. Better recovery in Rac1-KO mice was observed from 7 days after SCI (but not significant for BMS, p = 0.124). Recovery was statistically improved for both scores from 9 days after SCI until 35 days after SCI (Fig. 2A).

To examine histological differences between Rac1-KO mice and control mice, the spinal cord was fixed at 35 days after SCI. GFAP immunoreactivity around the injury sites (100 μm from the lesion) was significantly weaker in Rac1-KO mice compared with control mice (Fig. 2B). As an alternative approach to demonstrate GFAP immunoreactivity in Rac1-KO mice after CNS injury, we applied microplanar beam irradiation (100-μm width at 550 Gy with 400-μm gaps (center to center distance)) supplied from a Spring-8 synchrotron radiation facility (27) to the cerebellar cortex and brainstem. The injury caused by high energy X-rays is limited to a narrow region (27, 28). GFAP-positive immunoreactivity in a linear band surrounding the irradiated lesions was weaker in the brainstem of Rac1-KO mice compared with control mice (Fig. 2C). Together these results strongly suggest that immunoreactivity to GFAP, namely astrogliosis, after CNS injury was reduced by Rac1-KO in astrocytes.

Reduced Proliferation and Migration in Rac1-KD and Rac1-KO Astrocytes

We hypothesized that reduced GFAP immunoreactivity in two different CNS-injury models is due to the reduced proliferation and/or migration following Rac1-KO in astrocytes, thereby leading to reduced astrogliosis. To examine the contribution of Rac1 to cell cycle progression and cell migration, we used the Fucci system (29) and scratch wound assay, respectively, under a long term, time lapse live-imaging system.

Rac1-KD in LN229 cells, a cell line derived from a human glioblastoma, was achieved using a verified plasmid expressing shRNA for Rac1 (6) (Fig. 3A). Cell cycle progression, defined as the time from one cytokinesis to another cytokinesis, was significantly longer in Rac1-KD LN229 cells than in control cells (29.95 ± 0.66 h versus 36.58 ± 1.56 h; Fig. 3B). the G₁ phase was significantly extended by Rac1-KD (14.60 ± 1.09 h versus 19.64 ± 1.87 h); however, the S-M phase, defined by the green fluorescence of Venus-tagged hGeminin, was not changed by Rac1-KD (Fig. 3B).

Cell migration, evaluated using the scratch wound assay, was also significantly reduced in Rac1-KD LN229 cells compared with control cells (168.7 ± 21.8 μm versus 40.6 ± 7.3 μm) (Fig. 3C). Both delayed cell cycle progression and reduced cell migration were also observed in Rac1-KD primary astrocytes (cell cycle: 24.00 ± 1.04 h versus 31.03 ± 1.72 h, cell migration: 187.8 ± 14.7 μm versus 102.9 ± 7.4 μm) and in primary astrocytes obtained from Rac1-KO mice (cell cycle: 20.11 ± 0.77 h versus 27.59 ± 1.12 h, cell migration: 349.1 ± 12.9 μm versus 172.3 ± 11.4 μm) (Fig. 4, A–C). The reduced cell migration in Rac1-KO primary astrocytes was confirmed using a CytoSelect cell migration assay kit (Fig. 4D).

GSPT1 Is a Downstream Effector of Rac1 Signaling

LPS has been reported to induce Rac1-mediated up-regulation of various proteins, including the pro-inflammatory cytokine IL-1β (30). IL-1β is a key driver of inflammatory response and astrogliosis induced by brain damage, including SCI (31) and ischemic brain injury (32). First, we confirmed increased protein levels of IL-1β following LPS treatment in primary astrocytes from WT mice, as well as reduced IL-1β expression following LPS treatment in primary astrocytes from Rac1-KO mice. These findings indicate the existence of a Rac1-dependent transcriptional pathway enhanced by LPS treatment (Fig. 5A). Second, to identify novel downstream molecules in Rac1 signaling associated with the cell cycle and cell migration, we performed a DNA microarray using control and Rac1-KD LN229 cells treated with LPS (Fig. 5B), from which reduced levels of GSPT1 in Rac1-KD cells were detected compared with control cells. Decreased protein levels of GSPT1 were confirmed in LN229 cells using two different siRNAs for Rac1, as well as from primary astrocytes from Rac1-KO mice (Fig. 5C). In addition, GSPT1 was increased after treatment with LPS, and the decreased expression of GSPT1 following LPS treatment was observed in Rac1-KD LN229 cells (Fig. 5C). Furthermore, the increase in GSPT1 levels following LPS treatment was inhibited by a JNK inhibitor (JNK-IN-8), an ERK inhibitor (U0126), and an NF-κB inhibitor (BAY 11-7085), but not by a p38 MAP kinase inhibitor (SB203580) (Fig. 5E). These results suggest that GSPT1 is transcriptionally regulated by Rac1 through the activation of at least JNK, ERK, and NF-κB and that GSPT1 is up-regulated and continues to function after CNS injury, which induces inflammation.

The Rac1-GSPT1 Signaling Axis Is Involved in the Cell Cycle but Not Cell Migration

To examine the role of Rac1 and GSPT1 in cell cycle progression, we analyzed the cell cycle under long term, time lapse live-imaging in HeLa cells (another cell line rather than LN229). The cell cycle of HeLa cells was significantly prolonged following KD using two different siRNAs for GSPT1 (control: 18.47 ± 0.36 h, si620: 22.93 ± 0.40 h, and si1374: 23.25 ± 0.61 h) (Fig. 6, A and B). Furthermore, the prolonged cell cycle induced by Rac1-KD (2.5 nm: 22.05 ± 0.38 h, 5 nm: 22.38 ± 0.44 h) in HeLa cells was ameliorated by overexpression of GFP-tagged GSPT1 (GFP-GSPT1) (2.5 nm: 19.73 ± 0.48 h, 5 nm: 19.76 ± 0.41 h) (Fig. 6, C and D). The delayed cell cycle progression was also observed in GSPT1-KD primary astrocytes (20 nm; control: 24.02 ± 0.82h, si620m: 28.74 ± 0.86 h; Fig. 6, E and F).

To determine whether GSPT1 controls cell migration, we employed a scratch wound assay. Cell migration was not affected by two siRNAs for GSPT1 in LN229 cells (Fig. 7, A and B). The absence of an effect of GSPT1-KD on cell migration in primary astrocytes was also confirmed using a CytoSelect cell migration assay kit (20 nm of si-cont and siGSTP1–620m; Figs. 7, C and D). These results suggest that GSPT1 is a downstream effector in Rac1 signaling and that the Rac1-GSPT1 signaling axis is involved in cell cycle regulation but not cell migration.

Animals

All animal experiments were conducted in accordance with Kobe University and University of Toyama guidelines. GFAP-Cre mice (23) and Rac1^flox mice (24) have been described previously. GFAP-Cre;Rac1^flox/+ progeny of Rac1^flox/flox and GFAP-Cre mice were back-crossed with Rac1^flox/flox to obtain GFAP-Cre;Rac1^flox/flox (hereafter referred to as Rac1 KO) mice, which were backcrossed to Rac1^flox/flox to generate the GFAP-Cre;Rac1^flox/flox and Rac1^flox/flox experimental mice. CAG-STOP^flox–tdTomato (Ai9) mice, in which the ROSA26 region was used for transgene insertion, were purchased from The Jackson Laboratory (Bar Harbor, ME). GFAP-Cre;Rac1^flox/+;tdTomato progeny of GFAP-Cre;Rac1^flox/flox and CAG-STOP^flox-tdTomato mice were used to examine the efficacy of the GFAP promoter in astrocytes of the spinal cord. The GFAP-Cre;Rac1^flox/+;tdTomato mice were back-crossed with Rac1^flox/flox to obtain GFAP-Cre;Rac1^flox/flox;tdTomato mice, which were then backcrossed to Rac1^flox/flox to generate the GFAP-Cre;Rac1^flox/flox;tdTomato and Rac1^flox/flox;tdTomato experimental mice. Astrocytes obtained from GFAP-Cre;Rac1^flox/flox;tdTomato mice are labeled with tdTomato fluorescence, which is a marker of Rac1-KO. Control astrocytes obtained from Rac1^flox/flox;tdTomato mice are negative for tdTomato fluorescence and are thus not Rac1-KO cells. Offspring were genotyped by PCR using the following primers: 5′-ACTCCTTCATAAAGCCCTCG-3′ and 5′-ATCACTCGTTGCATCGACCG-3′ for GFAP-Cre; 5′-ATTTTCTAGATTCCACTTGTGAAC-3′ and 5′-ATCCCTACTTCCTTCCAACTC-3′ for Rac1^flox; 5′-GGCATTAAAGCAGCGTATCC-3′ and 5′-CTGTTCCTGTACGGCATGG-3′ for tdTomato. WT C57BL/6 mice were purchased from Clea Japan.

SCI Model Experiments

14–20-week-old Rac1 KO mice and control mice were used. The mice were anesthetized via the administration of trichloroacetaldehyde monohydrate (500 mg/kg, i.p.). After the mice had completely lost their righting reflex, surgical procedures to produce SCI were performed, as described previously (26) with slight modifications. Contusion injuries were produced by dropping a 6.5-g weight from a height of 7 mm once onto the exposed dura mater of the lumbar L1 level of the spinal cord using a stereotaxic instrument (Narishige, Tokyo, Japan). The mice were allowed to recover for 35 days. For behavioral scoring, the mice were placed individually in an open field (23.5 cm × 16.5 cm × 12.5 cm) and observed for 5 min. Open field locomotion focused on each hind limb was evaluated using the 0–8-point BMS locomotion scale (25) and the 0–4-point BSS locomotion scale (26).

Microplanar Microbeam Irradiation at SPring-8

8–12-week-old Rac1-KO mice and control mice were used. The SPring-8 synchrotron facility (Japan Synchrotron Radiation Research Institute, RIKEN, Sayo, Japan) was used to supply microplanar beam irradiation. The radiation beam traveled in a vacuum transport tube with minimized air scattering of the primary beam. X-rays were emitted from the vacuum tube into the atmosphere after first passing through a beryllium vacuum window and then into a 2.0-m helium beam path consisting of an aluminum tube and a thin aluminum helium window located 42 m from the synchrotron radiation output. The sample positioning system was placed 2.5 m from the thin aluminum window. This beamline produces nearly parallel X-rays, and the mice were irradiated at a position 2.5 m from the thin aluminum window. White beam X-rays with an energy level of ∼100 keV were derived through 3-mm Cu absorbance. The mice were irradiated with a single slit collimator at the same beamline, with multiple horizontal microplanar beams 100-μm thick at an extremely high dose of 550 Gy with 400-μm gaps between beams on the brain. Anesthetized mice were positioned horizontally in front of the horizontally propagating beams, with the right brain aligned perpendicular to the direction of the beam. The multislit collimator, which produces 10 peak dose areas composed of 100-μm width with 400-μm gaps between them to process the microplanar beam, was set downstream of the output of the beamline hatch. The details of this multislit irradiation system have been described previously (28).

Chemicals and Antibodies

LPS was obtained from InvivoGen (San Diego, CA). JNK-IN-8 (Millipore, Billerica, MA), a JNK inhibitor; SB203580 (Cell Signaling Technology, Danvers, MA), a p38 MAP kinase inhibitor; U0126 (Cell Signaling Technology), an ERK inhibitor; and BAY 11-7085 (Wako Pure Chemical Industries, Japan), an NF-κB inhibitor were purchased from the respective vendors. The following specific antibodies were used (polyclonal unless indicated): Rac1 monoclonal (23A8, Millipore); GFAP (DAKO, Carpinteria, CA); IL-1β (H-153, Santa Cruz Biosciences, Santa Cruz, CA), and GSPT1 (10763-1-AP; Proteintech, Rosemont, IL) and eRF3 (antibody against GSPT, 14980; Cell Signaling Technology). Alexa 488-conjugated secondary antibodies were obtained from Invitrogen. HRP-conjugated GAPDH or tubulin-α antibodies were obtained from MBL International.

Plasmids and siRNA

Venus-tagged hGeminin plasmid, which is an indicator of the S-M phase and one of two components of the Fucci system (29), was provided as a kind gift from Dr. Miyawaki (RIKEN, Wako, Japan). Human GSPT1 cDNAs were obtained from RIKEN (Tsukuba, Japan) and cloned into pEGFP-C3 (Clontech, Mountain View, CA) and p3×FLAG-CMV-10 (Sigma-Aldrich). Myc-tagged human Rac1 plasmid and a validated target sequence of human/mouse Rac1 for RNAi (human nucleotides 618–636 from ATG: CCTTTGTACGCTTTGCTCA) were developed as previously described (6). shRNA expression plasmids for Rac1 containing the validated sequence were prepared using pSUPER, pSUPER(gfp) (OrigoEngine, Seattle, WA), and pSUPER(rfp), in which gfp of pSUPER(gfp) was replaced by mCherry. The plasmids were named pSUPER(618) (55), shRac1(618gfp), and shRac1(618rfp), respectively.

The target sequences for RNAi were CCTTTGTACGCTTTGCTCA and GCCACTACAACAGAATTTT for human RAC1 (618–636 and 1977–1995 from ATG, named siRac1(618) (100% conserved in mouse) and siRac1–1977, respectively), CTAAGAAAGAGCATGTAAA and GGAATCAGGATCTATTTGT for human GSPT1 (620–638 and 1374–1392 from ATG, named siGSPT1–620 and siGSPT1–1374, respectively), and CCAAGAAGGAACATGTAAA for mouse GSPT1 (620–638 from ATG, named siGSPT1(620m)). siRNAs and control siRNA (MISSION Universal Negative Control) were purchased from Sigma-Aldrich.

siRNA and shRNA plasmids were transfected into LN229 and HeLa cells using Lipofectamine 3000 (Invitrogen) or RNAiMAX (Invitrogen). siRNAs were transfected into primary astrocytes using a NEPA21 electroporator (Nepa Gene Co., Ltd., Japan). Compared with lipofection, electroporation has been reported to require a higher concentration of siRNAs (56). Thus, 20 nm siRNA was used for electroporation.

Cells

LN229 astrocytic cells and HeLa cells were maintained in DMEM (Wako) containing 10% FBS (Nichirei Biosciences, Japan). Primary astrocyte cultures were prepared from mouse cerebral cortex at postnatal day 1 or 2. Dissected cerebral cortexes were dissociated in Eagle's minimal essential medium (Wako) supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin and were cultured in 25-cm² flasks (2 brains/flask) (Corning Inc., Corning, NY). After 5–7 days, the flasks were subjected to 2 h of continuous shaking to obtain purified astrocytes. Trypsinized cells and cell lysates were used for the experiments, as indicated (only one trypsinization step was used for the primary astrocytes). The percentage of primary astrocytes obtained from GFAP-Cre;Rac1^flox/flox;tdTomato mice with tdTomato fluorescence was 80–90%. All cells were maintained in a 5% CO₂ humidified incubator at 37 °C.

RT-PCR

RT-PCR was performed with 1 μg of total RNA obtained from the primary astrocytes of WT mice using SuperScript III reverse transcriptase (Invitrogen) and random primers. The following primer pairs were used for PCR (30 cycles): 5′-GCAGACAGACGTGTTCTTAATTTGC-3′ and 5′-TGTAACAAAAACTTGGCATCAAATGCG-3′ for Rac1, 5′-GGAGGACTATGACCGCCTC-3′ and 5′-AAATAGGATGTGGCCTATGAACATCC-3′ for Rac2, and 5′-CCCACACACACCCATCCTTC-3′ and 5′-TGGAGCTATATCCCAGAAAAAGGAG-3′ for Rac3.

Cell Cycle Analysis

LN229 and HeLa cells were cultured on 35-mm glass-bottomed dishes (gbd; MatTek, Ashland, MA). LN229 cells were transfected with Venus-tagged hGeminin + shRNA expression plasmid (pSUPER(rfp) or shRac1(618rfp)) using Lipofectamine 3000. HeLa cells were transfected with Venus-tagged hGeminin + siRNA (control or siGSPT1). For rescue experiments, HeLa cells with transfection of siRac1(618) were simultaneously transfected with pEGFP-C1 or GFP-GSPT1 using Lipofectamine 3000. Starting at 24 h after transfection, the cells were imaged every 20 min for 72 h at 37 °C in 5% CO₂ using a computer-assisted incubator fluorescence microscope system (LCV110; Olympus). This system enabled ultra long term imaging of living cells without removal of the cells from the culture conditions. LN229 cells with RFP fluorescence and HeLa cells with Venus or GFP fluorescence were analyzed as cells with shRNA or siRNA. Cell cycle (doubling time) was defined as the time from one cytokinesis to the next cytokinesis. The experiments were performed in duplicate, and at least three independent transfection experiments were conducted.

siRNA (control, siRac1(618) or siGSPT1(620m)) was electroporated into WT primary astrocytes in combination with the pEGFP-C1 plasmid. Primary astrocytes obtained from GFAP-Cre;Rac1^flox/flox;tdTomato (Rac1-KO) mice and control (Rac1^flox/flox;tdTomato) mice or primary astrocytes subjected to electroporation were cultured on 35-mm gbd. Starting at 48 h after plating on gbd, the cells were imaged every 20 min for 72 h using a live imaging LCV110 system, as described above. Astrocytes with tdTomato fluorescence and GFP fluorescence were considered to be Rac1-KO cells and cells containing siRNA, respectively.

Scratch Wound (Wound Healing) Assay

LN229 cells were transfected with shRNA expression plasmid (pSUPER(gfp) or shRac1(618gfp)), or pEGFP(C1) + siRNA (control or siGSPT1) using Lipofectamine 3000. At 24 h after transfection, culture media were changed to serum-free media, and cells were grown for an additional 24 h. Forty-eight hours after transfection, an approximately 1,000-μm-wide section of the cells was scratched using a sterilized 1,000-μl filter tip, and imaged every 20 min for 48 h using a live imaging LCV110 system (see the “cell cycle analysis” section for details). Cells with GFP fluorescence were analyzed as cells with shRNA or siRNA. Cells whose cell bodies has translocated were considered to be migrated cells. Cell migration was defined as the distance from the leading edge of the cell at the starting time point to the same point on the cell at the ending time point. Experiments were performed in duplicate, and at least three independent transfections were conducted.

siRNA (control, siRac1(618), or siGSPT1(620m)) was electroporated into WT primary astrocytes in combination with the pEGFP(C1) plasmid. Primary astrocytes obtained from GFAP-Cre;Rac1^flox/flox;tdTomato (Rac1-KO) mice and control mice or primary astrocytes treated with electroporation were cultured on 35-mm gbd. Forty-eight hours after plating on gbd, the cells were imaged every 20 min for 72 h using a live imaging LCV110 system. Astrocytes with tdTomato fluorescence and GFP fluorescence were considered to be Rac1-KO cells and cells containing siRNA, respectively.

Cell Migration Assay

Migration of primary astrocytes was evaluated using the CytoSelect cell migration assay kit (12 μm pore size, colorimetric format; Cell Biolabs, Inc., San Diego, CA) according to the manufacturer's protocol. Briefly, 2.0 × 10⁵ primary astrocytes obtained from GFAP-Cre;Rac1^flox/flox;tdTomato (Rac1-KO) mice and control (Rac1^flox/flox;tdTomato) mice or 5.0 × 10⁵ WT primary astrocytes treated with electroporation (pEGFP-C3 + 20 nm of siRNA (control or siGSPT1(620m)) suspended in serum-free DMEM were placed in 24-well insets, and 500 μl of DMEM containing 10% FBS was added to the lower wells of the 24-well plate. After 24 h (Rac1-KO) or 32 h (GSPT1-KD), non-migrating cells on the interior surface of the inset were removed, and migrated cells on the exterior surface of the inset were stained using stain solution. After extraction of the migrated cells in extraction solution, the A value at 560 nm was obtained using a plate reader (Multiskan GO; Thermo Fisher Scientific). The data are shown as percentages of control.

Section Preparation and Immunohistochemistry

At 35 days after SCI and 21 days after microbeam irradiation injury, the animals were deeply anesthetized using pentobarbital and transcardially perfused with ice-cold 0.9% saline solution and then with 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4) (57). Spinal cords and brains were dissected, post-fixed overnight in the same fresh fixative, and then embedded in paraffin. Three-μm sagittal sections of the spinal cord and cerebellum with brainstem were obtained (from 5-mm rostral and caudal to the injury site in the case of SCI). After deparaffinization and permeabilization with PBS containing 0.3% Triton X-100 (PBS-0.3T), spinal cord sections were incubated overnight with a GFAP antibody in PBS containing 0.03% Triton X-100 (PBS-0.03T) at 4 °C, followed by incubation with Alexa 488-conjugated secondary antibodies for 1 h at 24 °C. The cerebellum with brainstem sections were incubated overnight with a GFAP antibody in PBS containing 0.03% Triton X-100 (PBS-0.03T) at 4 °C, followed by diaminobenzidine staining using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and counterstaining using Cresyl violet solution (Muto Pure Chemicals, Tokyo, Japan). Quantification of GFAP immunoreactivity was performed as follows. For SCI cases, fluorescent images were captured using a fluorescence microscopy system (Biozero, Keyence, Japan). Immunoreactivity in a 100-μm area from the lesion edge was measured using ImageJ software (National Institutes of Health, Bethesda, MD). Areas that appeared brighter than the background were defined as GFAP-positive areas. The number of pixels were calculated, and the GFAP-positive area was defined as a percentage of the number of pixels for the entire area. For microbeam irradiation injury cases, diaminobenzidine stainings were photographed using a light microscope (Axioplan II; Carl Zeiss) with a DP26 camera (Olympus). Immunoreactivity areas that appeared brighter than the background (as analyzed by ImageJ software) were defined as GFAP-positive areas, which were defined in a linear band. The region of interest (ROI) was defined as a 400-μm square with its center in the linear immunoreactivity band of the GFAP-positive area, and the GFAP-positive immunoreactivity area was calculated as a percentage of the total area.

Immunoblotting

The cells were lysed in homogenizing buffer (58) by sonication in the presence of protease inhibitor mixture, protein phosphatase inhibitor mixture (Nacalai Tesque, Tokyo, Japan), and 1% Triton X-100. Total lysates were centrifuged at 800 × g for 5 min at 4 °C, and the supernatants were subjected to SDS-PAGE, followed by immunoblotting for 2 h at 24 °C using primary antibodies diluted in PBS-0.03T. The bound primary antibodies were detected with secondary Ab-HRP conjugates using the ECL detection system (GE Healthcare).

DNA Microarray

LN229 cells were transfected withpSUPER or shRac(618). At 24 h after transfection, LN229 cells were treated with LPS (0.5 μg/ml) for 24 h, and then total RNAs were extracted using TRIzol (Invitrogen). The quality and quantity of RNA were determined using the Agilent 2100 BioAnalyzer. Gene expression profiles were examined using the SurePrint G3 Mouse Gene Expression 8 × 60K microarray kit (Agilent Technologies, Lexington, MA).

Statistical Analysis

All data are presented as the means ± S.E. For comparisons of two groups, unpaired Student's t tests were used. For comparisons of more than two groups, one-way analysis of variance (ANOVA) or repeated measures two-way ANOVA was performed and followed by Bonferroni post hoc test of pairwise group differences. Statistical analyses were performed using Prism 6.0 software (GraphPad, La Jolla, CA); p < 0.05 was considered statistically significant.