Singleminded-2s (Sim2s) Promotes Delayed Involution of the Mouse Mammary Gland through Suppression of Stat3 and NFκB

Mammary gland involution is delayed in MMTV-Sim2s mice through suppression of STAT3 and NFκB signaling pathways.

Abstract

Postlactational involution of the mammary gland provides a unique model to study breast cancer susceptibility and metastasis. We have shown that the short isoform of Singleminded-2s (Sim2s), a basic helix loop helix/PAS transcription factor, plays a role in promoting lactogenic differentiation, as well as maintaining mammary epithelial differentiation and malignancy. Sim2s is dynamically expressed during mammary gland development, with expression peaking during lactation, and decreasing in early involution. To determine the role of SIM2S in involution, we used transgenic mice expressing SIM2S under the mouse mammary tumor virus-Sim2s promoter. Overexpression of Sim2s in the mouse mammary gland resulted in delayed involution, indicated by a lower proportion of cleaved caspase-3-positive cells and slower reestablishment of the mammary fat pad. Immunohistochemical and quantitative RNA analysis showed a decrease in apoptotic markers and inflammatory response genes, and an increase in antiapoptotic genes, which were accompanied by inhibition of signal transducer and activator of transcription 3 activity. Microarray analysis confirmed that genes in the signal transducer and activator of transcription 3 signaling pathway were repressed by SIM2S expression, along with nuclear factor-κB and other key pathways involved in mammary gland development. Multiparous mouse mammary tumor virus-Sim2s females displayed a more differentiated phenotype compared with wild-type controls, characterized by enhanced β-casein expression and alveolar structures. Together, these results suggest a role for SIM2S in the normal involuting gland and identify potential downstream pathways regulated by SIM2S.

Mammary gland involution is the regression of a lactating mammary gland to its quiescent state after weaning and is characterized by a decrease in milk protein, collapse of alveolar structures, apoptosis of epithelial cells, and reestablishment of the fat pad (1–5). Involution has been shown to proceed in two separate phases: first an acute response phase characterized by a decrease in milk protein synthesis, and epithelial cell apoptosis (1, 2, 6–12). This initial phase is reversible and occurs 1–3 d after weaning. Acute phase involution is characterized by a drop in signal transducer and activator of transcription (Stat)5, followed by an increase in Stat3 signaling. The second, irreversible, phase begins at 72 h after pup removal and is typified by a collapse in the alveoli, extensive epithelial apoptosis, and breakdown of the basement membrane by matrix metalloproteinases (13). Involution is a unique process with a wound-healing signature and controlled inflammation, both of which are associated with breast cancer progression, metastasis, and survival. Studies investigating differences in gene expression, extracellular matrix composition, and signaling associated with involution and breast cancers have shown that metastatic breast cancer shares characteristics of the involuting mammary gland (7, 8, 14–22). Therefore, defining the mechanisms regulating involution will identify potential pathways involved in breast cancer progression.

Singleminded-2s (Sim2s) is a member of the basic helix loop helix/PAS family of transcription factors that has been implicated in normal mammary gland development and is frequently lost or reduced in primary human breast tumors. We have shown that Sim2s is expressed in mouse luminal mammary epithelial cells and is developmentally regulated with highest expression observed in midlactation (23, 24). To further define the role of SIM2S in mammary gland development, we generated a transgenic mouse expressing Sim2s under control of the mouse mammary tumor virus promoter (MMTV-Sim2s) (24). Analysis of mammary glands from staged virgin MMTV-Sim2s and wild-type (WT) mice showed that mRNA levels of Csn2 and Wap were significantly increased, implying that SIM2S expression is associated with enhanced differentiation (24). Although SIM2S is expressed in mammary epithelial cells, it is down-regulated in a majority of breast tumors and breast cancer cell lines, and forced expression of Sim2s in invasive breast cancer cells inhibits growth and motility. In addition, loss of Sim2s in the mouse mammary gland and in normal human breast epithelial cells results in an epithelial-mesenchymal transition, inducing an aggressive basal-like phenotype (23, 25, 26). Mammary glands from Sim2s-null mice show impaired development because the epithelial ducts do not properly form and differentiate (26).

To further elucidate the function of SIM2S in breast cancer progression and metastasis, as well as the function of SIM2S in normal mammary development, we analyzed the effect of Sim2s overexpression on involution after forced weaning (24). We found that mammary gland involution is disrupted in MMTV-Sim2s mice as demonstrated by a delay in the reoccurrence of the fat pad and decrease in apoptosis. Furthermore, overexpression of Sim2s inhibited STAT3-mediated signaling and other pathways involved in the acute phase response. These studies extend the role of SIM2S in mammary gland development and identify SIM2S-regulated gene expression signatures that correlate with involution.

Results

Constitutively active SIM2S delays mouse mammary gland involution

To determine the effect of SIM2S on mammary involution, we analyzed glands from MMTV-Sim2s transgenic mice force weaned at lactation d 10 and harvested at 24, 48, and 72 h after pup removal. Histological analysis showed clear differences starting at 24 h after weaning in the number of apoptotic cells shedding into the alveolar lumen (Fig. 1, A–D). Transgenic Sim2s mammary glands maintained distinct alveolar structures with copious amounts of milk at 72 h of involution (Fig. 1, K and L), whereas the WT glands had larger adipocytes and breakdown of luminal structures (Fig. 1, I and J). Consistent with the observation that SIM2S promotes alveolar differentiation (24), we also observed an increase in β-casein (Csn2) and whey acidic protein (Wap) gene expression 24 h after pup removal as compared with controls (Fig. 1, M and N). However, we observed no changes in phospho-STAT5A staining (data not shown), suggesting that STAT5A signaling is not involved in mediating this phenotype. Immunohistochemical staining of involuting mammary glands with antiperilipin revealed distinct differences in adipocyte size between the transgenic and WT glands 48 and 72 h after pup removal. Figure 2 shows both fewer and smaller adipocytes in the fat pads of MMTV-Sim2s mice compared with WT mice, indicating a delay in adipocyte lipid synthesis and repopulation of the mammary fat pad resulting from Sim2s overexpression.

Fig. 1.

Involution is delayed and milk protein mRNA levels are increased in MMTV-Sim2s transgenic mice. Normal (FVB) and MMTV-Sim2s mice were harvested at 24-, 48-, and 72-h involution (I24, I48, I72). A, B, E, F, I, and J, H&E-stained mammary glands from WT FVB mice. C, D, G, H, K, and L, H&E-stained mammary glands from MMTV-Sim2s transgenic mice. Transgenic mammary glands show less apoptotic cell shedding, slow fat pad reoccurrence, and slower alveolar regression (indicated by arrows). A–D, Involution at 24 h (I24). E–H, Involution at 48 h (I48). I–L, Involution at 72 h (I72). Magnification bar represents 100 μm on all images. Images are representative of at least 60% of samples collected. M, qPCR analysis of involuting mammary gland β-casein (Csn2). N, qPCR analysis of involuting mammary gland whey acidic protein (Wap). Transgenic mammary glands have an increased trend of milk protein mRNA expression throughout involution when normalized to an epithelial specific control gene (Claudin 7).

Fig. 2.

Delayed fat pad regeneration in MMTV-Sim2s mice during involution. Perilipin staining of WT FVB and transgenic MMTV-Sim2s mice. A–D, Mammary gland involution at 48 h (I48). E–H, Mammary gland involution at 72 h (I72). A, B, E, and F, WT FVB mammary glands. C, D, F, and H, MMTV-Sim2s mammary. Transgenic mammary glands show less fat pad regeneration after forced weaning. Magnification bars, 100 μm. Images are representative of at least 60% of samples.

The acute phase of involution is largely characterized by shedding of apoptotic cells into the lumen of mammary gland alveoli. To evaluate differences in apoptosis between transgenic and WT involuting mammary glands, we used immunohistochemistry to evaluate cleaved caspase-3, which is normally confined to those cells that have undergone apoptosis and shed into the lumen. The results showed a significant decrease in caspase-3-positive cells in involuting glands from MMTV-Sim2s mice at 24, 48, and 72 h compared with controls (Fig. 3).

Fig. 3.

MMTV-Sim2s transgenic mice have significantly lower levels of apoptotic cells during involution. A–L, WT and MMTV-Sim2s mammary glands stained for cleaved caspase 3. A–D, Involution at 24 h (I24). E–H, Involution at 48 h (I48). I–L, Involution at 72 h (I72). A, B, E, F, I, and J, WT FVB mammary glands. C, D, G, H, K, and L, MMTV-Sim2s mammary glands. Magnification bars, 100 μm in all images. Images are representative of at least 60% of samples. M, Quantification of cleaved caspase 3 images, percent positive cells taken from four images, counted and averaged. MMTV-Sim2s transgenic mammary glands have significantly lower levels of cleaved caspase 3-positive cells. *, P < 0.05.

Multiparous MMTV-Sim2s mammary glands exhibit alveolar structure and milk protein expression

To determine the effect of SIM2S overexpression on multiple rounds of involution, mammary histology of transgenic and WT mice that had gone through at least three pregnancies each was evaluated. Analysis of hematoxylin and eosin (H&E) staining shows a distinct alveolar phenotype in the MMTV-Sim2s mammary glands with a more differentiated morphology and lipid accumulation (Fig. 4, B, C, E, and F) as compared with control animals (Fig. 4, A and D). Furthermore, immunohistochemical analysis showed that WT glands have little β-casein expression, whereas the MMTV-Sim2s gland has distinct staining within the alveolar structures (Fig. 4, H–M).

Fig. 4.

Active STAT3 and STAT3 target genes are significantly lower in MMTV-Sim2s mice during involution. A–L, Immunohistochemical staining of phosphorylated STAT3 in WT and MMTV-Sim2s mice. A–D, Involution at 24 h (I24). E–H, Involution at 48 h (I48). I–L, Involution at 72 h (I72). A, B, E, F, I, and J, WT mammary glands have very high levels of PSTAT3, especially in the epithelial secretory cells. C, D, G, H, K, and L, MMTV-Sim2s transgenic mammary glands exhibit significantly lower levels of PSTAT3 staining. Heterogeneous mixture of PSTAT3 staining is consistent with heterogeneous expression of MMTV-long terminal repeat. No difference was seen in pan STAT3 staining (Supplemental Fig. 1). Magnification bars, 100 μm. Images are representative of at least 60% of samples. M, Quantification of PSTAT3 staining; four images were taken for each gland harvested and counts were averaged. N, mRNA expression levels of pan Stat3 in involuting mammary glands. O and P, STAT3 target genes C/EBPδ and IGFBP5 have downward trends in mRNA expression in transgenic mice as shown by qPCR. *, P < 0.05.

SIM2S expression inhibits activation of STAT3

STAT3 signaling plays an important part in regulating mammary gland involution by inducing genes involved in inflammation and the acute phase response. Activation of STAT3 through phosphorylation promotes translocation into the nucleus, where phospho-STAT3 (PSTAT3) functions as a transcriptional regulator. Analysis of PSTAT3 staining in WT and MMTV-Sim2s mice showed chimeric staining patterns throughout the mammary epithelium, but glands from transgenic mice appeared to have far fewer PSTAT3-positive cells than WT glands (Fig. 5, A–L). Quantification of PSTAT3-positive cells revealed a significant decrease in Sim2s glands at 48 and 72 h of involution (Fig. 5M). Pan-Stat3 immunohistochemistry was also performed to ensure that PSTAT3 changes seen are due to altered activation of STAT3. No changes are seen between WT and transgenic mammary glands in levels of STAT3 (Supplemental Fig. 1, A–L, published on The Endocrine Society's Journals Online web site at http://mend.endojournals.org). To determine whether the reduction of active STAT3 seen in MMTV-Sim2s mice correlated with a decrease in downstream STAT3 target genes, we performed quantitative PCR (qPCR) analysis for CCAAT enhancer-binding protein (C/EBPδ) and IGF-binding protein 5 (IGFBP5) and found a distinct downward trend in gene expression in Sim2s transgenic mice, with the greatest difference at 72 h of involution (Fig. 5, N and O). Together, these results suggest that the delayed involution observed in mammary glands from MMTV-Sim2s mice is mediated, in part, by SIM2S-dependent suppression of the STAT3 pathway.

Fig. 5.

Multiparous MMTV-Sim2s females have more alveolar structures in the nonlactating gland, and higher β-casein expression. A–F, H&E staining for multiparous (+1 litter) WT and MMTV-Sim2s mice. A and D, WT multiparous, nonlactating mammary glands have small ductal branching similar to the virgin gland. B, C, E, and F, MMTV-Sim2s multiparous, nonlactating mammary glands have larger alveolar glands indicating an incomplete involution (indicated with arrows). Magnification bars, 100 μm. G–L, β-Casein immunohistochemical staining of multiparous WT and MMTV-Sim2s mice. G and J, WT multiparous, nonlactating mammary glands show little to no detectable levels of β-casein. H, I, K, and L, MMTV-Sim2s multiparous, nonlactating mammary glands show much higher levels of β-casein in the alveolar structures still present in the quiescent gland. Magnification bars represent 100 μm. Images are representative of at least 60% of samples.

Microarray analysis revealed changes in pathways regulating involution, specifically Stat3 and NFκB

Microarray analysis was performed using mammary glands from WT and MMTV-Sim2s transgenic females harvested 72 h after force weaning to identify additional pathways regulated by SIM2S during involution. Data were analyzed using the Ingenuity Pathway Analysis Software. The significance of the association between the data set and the canonical pathway was measured by taking a ratio of the number of molecules from the data set that map to the pathway divided by the total number of molecules that map to the canonical pathway displayed. Fisher's exact test was used to calculate a P value to determine the probability that the association between the genes in the data set and the canonical pathway is explained by chance alone. As expected, one of the most significantly affected pathways identified in the involuting MMTV-Sim2s mammary gland was the Jak/Stat signaling pathway, which is involved in regulating mammary gland growth, differentiation, migration, and apoptosis. Other pathways identified, including NOTCH, WNT/β-catenin, phosphatidylinositol 3-kinase/AKT, and NFκB, also play roles in involution and breast cancer progression. Changes in multiple genes involved in nuclear factor-κB (NFκB) signaling, identified in the microarray studies, were verified using qPCR (Fig. 6, B and C). In transgenic mammary glands, Ikk2, an activator of NFκB, showed reduced expression compared with WT glands throughout the time series. NFκB2 expression was gradually up-regulated in WT glands, showing maximal expression by 72 h; however, the levels remained low in glands from transgenic females. These data suggest that SIM2S regulates multiple pathways involved in normal mammary gland function and malignancy.

Fig. 6.

Canonical pathway analysis of 72-h involuting glands reveals multiple pathways that are affected by MMTV-Sim2s over expression. A, Canonical pathway analysis was performed using Ingenuity Pathway Analysis Software. Statistically significant data (P < 0.05) from the Codelink Microarray was analyzed to determine what pathways were significantly altered by SIM2S expression. B–E, qPCR analysis of Ikk2, NFκB1, NFκB2, and RelA show downward trends in MMTV-Sim2s mice. AMPK, AMP activated protein kinase; FGF, fibroblast growth factor; PI3K, phosphatidylinositol 3-kinase.

Discussion

We have shown that SIM2S promotes mammary lactogenic differentiation when overexpressed in virgin mice (24). The objective of this study was to evaluate the effect of Sim2s overexpression on mammary involution after forced weaning. Here, we demonstrate that MMTV-Sim2s transgenic mice experience delayed involution, characterized by reduced epithelial cell apoptosis and lower STAT3 activation. Microarray studies performed on tissue from involuting glands revealed that SIM2S inhibited pathways associated with inflammation and apoptosis, both of which are hallmarks of involution and cancer progression. Previously, our laboratory has shown that SIM2S is required for proper development and differentiation of the mammary ductal tree. Additionally, down-regulation of Sim2s in MCF7 breast cancer cells and MCF10A breast epithelial cells results in the loss of epithelial characteristics and the acquisition of a basal phenotype (24–26). The results from this study support a role for SIM2S in maintaining epithelial differentiation and suppressing involuting pathways.

Recent studies have used the involuting mouse mammary gland as a model to identify pathways involved in breast cancer progression (7, 27–36). Special interest has focused on the regulation of apoptosis during the acute phase of involution, and on controlled inflammation, which are part of the wound-healing signature. BAX, AKT, IL6, STAT3, and other proapoptotic genes associated with involution have been conditionally deleted in the mammary gland to determine their roles in involution (6, 16, 27, 29, 30, 33, 35–44). Gene expression analysis of the involution signature shows that STAT3 and NFκB pathways are induced during involution, whereas the STAT5 pathway is inhibited. From these results, we know that a substantial number of factors up-regulated during involution play various roles in inflammation and the acute phase of involution. These pathways have also been analyzed in conjunction with various breast cancer subtypes and correlate with an increase in metastasis and poor prognoses (1, 6–7, 11, 17). In addition, the microenvironment of the involuting mammary gland has been shown to promote metastasis due to active basement membrane degradation, controlled inflammatory signaling, activation of fibroblasts, and other signals also involved with a wound-healing signature (1, 7, 8, 15, 18, 19, 21, 45).

The acute phase of involution, which occurs during the first 72 h after weaning, is characterized by an increase in apoptosis, an enhanced immune response, and decreased milk protein synthesis (7, 29, 39, 41, 46–49). Many of these processes are dependent on STAT3, which mediates apoptosis of secretory epithelial cells, as well as their removal from the gland during involution. STAT3 is also the key regulator of the controlled inflammatory response observed in the involuting mammary gland and is constitutively active in many breast cancer cell lines and primary tumors (21, 48–52). STAT3 regulates both pro- and antiinflammatory responses, based on leukemia inhibitory factor and Oncostatin M signaling, and is often activated by tumor cytokines, oncogenes, and growth factors. In STAT3-null mice, involution is delayed and the secretory alveolar epithelial cells maintain functional integrity up to 6 d after weaning in the absence of lactogenic stimuli (21, 29, 36, 42, 44, 53). We observed a similar phenotype in our MMTV-Sim2s mice with a significant inhibition of PSTAT3 activity and reduced expression of downstream target genes IGFBP5 and C/EBPδ (42).

STAT3 and NFκB are known to interact at multiple levels, and similar to STAT3, NFκB is constitutively active in cancers (51). NFκB expression is high during the acute phase of mammary gland involution and has been shown to play a role in milk clearance (54). Conditional deletion of IKK2, a NFκB activator, resulted in delayed involution and a decrease in cleaved-caspase 3-positive apoptotic cells (55). These studies indicate that NFκB plays a role in proapoptotic signaling, in addition to the commonly described antiapoptotic regulation of BCL2 and BCLX_L. Transgenic mice with a doxycycline-inducible NFκB construct undergo rapid loss of milk and alveolar collapse shortly after pup weaning, in addition to an increase in cleaved-caspase 3-positive cells compared with WT counterparts (54). Similarly, induction of NFκB expression during lactation resulted in decreased milk protein levels and alveolar collapse. NFκB has been implicated in tumorigenesis, because constitutive activation results in increased proliferation, inhibition of apoptosis, increased metastasis, and angiogenesis (21, 31, 51, 56–61). A proinflammatory target of NFκB, IL-6, is a known activator of STAT3, and forms a positive feedback loop that is a major regulator of inflammation (61). IL-6-null mice also have delayed involution and decreased epithelial cell death; however, STAT3 remains activated in these mice, indicating that STAT3 is activated through multiple cytokines (44). STAT3 also inhibits antitumorigenic pathways that are typically activated by NFκB REL, allowing only the expression of pro-oncogenic REL A (51). In tumors, this results in a reciprocal relationship between STAT3 and NFκB because many cytokines and growth factors encoded by RELA activate STAT3. Similar to STAT3, active NFκB signaling has also been indicated in maintenance of mammary stem cells (60, 61).

We have shown that mammary gland involution is delayed in MMTV-Sim2s mice and that SIM2S inhibits gene signatures associated with involution including STAT3 and NFκB signaling pathways. This suggests that SIM2S expression in breast cancer cell lines might suppress the pro-oncogenic activities of these transcription factors. Further studies into the inhibition of these pathways by SIM2S may significantly contribute to the understanding and therapeutic treatment of metastatic breast cancer. Increasing evidence indicates that the differentiation status of a tumor correlates with its aggressiveness, thus showing the importance of finding factors that induce terminal differentiation not only in the mammary gland, but also in primary tumors. Based on the studies shown here, and our previous work showing that SIM2S promotes mammary alveolar differentiation, we hypothesize that overexpression of Sim2s will inhibit tumor progression, metastasis, and render breast tumors more susceptible to therapeutics, leading to a better prognosis.

Materials and Methods

MMTV-Sim2s mice

The transgenic mice used in this study were described previously (24). An MMTV-KCR cassette was used with the Sim2s coding sequence to overexpress SIM2S in the mammary gland of FVB mice. All procedures were approved and followed the guidelines set forth by the Texas A&M University Animal Use and Care Committee.

Animals

Pups were removed from both WT and MMTV-Sim2s mice at lactation d 10. Tissue was harvested at 24, 48, and 72 h after weaning. The fourth inguinal mammary glands were used for histological sectioning, RNA isolation, and protein isolation. Litters were normalized to eight pups at parturition. All animals were housed in solitary caging after parturition (with litter) under a standard 12-h photoperiod. The animals were given access to food and water ad libitum. Five mice were analyzed for each time point, and transgene expression was confirmed before further experimentation, producing an n = 3. Procedures were approved by the University Laboratory Animal Care Committee at Texas A&M University.

Immunostaining

Immunostaining was carried out as previously described (23). Samples were incubated in blocking solution for 1 h, followed by incubation in primary antibody overnight at 4 C. Antibodies used include SIM2 (Millipore Corp., Bedford, MA), cleaved caspase 3 (Cell Signaling Technology, Beverly, MA), phospho-STAT3 (Cell Signaling), Perilipin (Cell Signaling), and β-casein (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Tissue preparation and hematoxylin and eosin (H&E) staining were done by the Histology Core Facility at Texas A&M University College of Veterinary Medicine & Biomedical Sciences. Statistical analysis of positive staining was performed using the ImageJ (National Institutes of Health, Bethesda, MD) cell-counting feature.

RNA isolation and reverse transcription

RNA was isolated from tissues using Trizol Reagent (Invitrogen, Carlsbad, CA), followed by purification using a QIAGEN RNEasy Mini Kit (QIAGEN, Valencia, CA) as previously described (23). Total RNA (2 μg) was reverse-transcribed using oligo(dT) and Superscript II Reverse transcriptase (Invitrogen). Reverse transcription reactions were performed on tissues as previously described (24). Sim2s overexpression of samples was confirmed via qPCR before immunostaining and other qPCR analysis.

Quantitative PCR

Quantitative PCR was performed as previously described (23). qPCR was performed using SYBR Green Master Mix (Applied Biosystems, Foster City, CA). Primers for analysis of Sim2s, Cldn7, Wap, and Csn2 mRNA levels have been previously described (24). Primers used for further mRNA analysis were Stat3 [forward primer (FP): 5′-GGA-GTA-CGT-GCA-GAA-GAC-ACT-GA-3′, reverse primer (RP): 5′-TCC-GAT-GCA-CGC-GAT-CT-3′), C/EBPδ (FP: 5′-CGC-CGC-AAC-CAG-AT-3′, RP: 5′-GCT-GAT-GCA-GCT-TCT-CGT-TCT-3′), IGFBP5(FP: 5′-GAT-GAG-ACA-GGA-ATC-CGA-ACA-AG-3′, RP: 5′-TTG-AAC-TCC-TGG-AGG-GAA-GCT-3′), Ikk2 (FP: 5′-CAG-CGA-GCA-GCC-ATG-ATG-3′, RP: 5′-GGA-GGC-CAT-GGC-GTT-CT-3′), NFκB1(FP: 5′ GCC-GTG-GAG-TAC-GAC-AAC-ATC-3′, RP: 5′-TGT-CCA-CGT-GGG-CAT-CAC −3′), NFκB2 (FP: 5′ GGG-CAG-ACT-GGT-GTC-ATT-GA −3′, RP: 5′-GGT-TGA-TGA-CGC-CGA-GGT-A-3′), and RelA (FP: 5′-GCC-CAT-GGA-GTT-CCA-GTA-CTT-G-3′, RP: 5′-GTC-CTT-TTG-CGC-TTC-TCT-TCA-3′)]. Claudin7 (Cldn7) was used as the normalizing gene, and data were analyzed using the ΔΔC_T method (62).

Gene expression microarray

The Whole Mouse Genome CodeLink Bioarray (Amersham, GE Healthcare, Piscataway, NJ) was used to determine differential gene expression between WT and MMTV-Sim2s transgenic females at 72 h of involution (GEO accession no. GSE27012). Sample preparation and hybridization were performed according to the manufacturer's protocols and as previously described (63). RNA samples were confirmed for Sim2s overexpression via qPCR before microarray analysis.

Microarray analysis

Raw microarray data were initially normalized and analyzed using CodeLink Expression Analysis Software version 4.1.0.29054 (GE Healthcare). A median normalization method was used, with a 20% threshold trim percentage. Microarray data were analyzed, and functionally analyses were generated through the use of Ingenuity Pathways Analysis (Ingenuity Systems, www.ingenuity.com). A data set containing gene identifiers and corresponding expression values was uploaded into the application. The identifiers were mapped to their corresponding objects in Ingenuity's Knowledge Base. A P value cutoff of 0.05 was set to identify molecule the expression of which was significantly differentially regulated. These molecules were integrated into a molecular network developed from information in Ingenuity's Knowledge Base. The data were then analyzed for the biological functions and/or diseases that were most significant.