A total of 2 recombinant E. coli-derived NRG1α1 (rNRG1α, RP-317-P0A; Thermo Fisher Scientific, Inc.) and NRG1β1 (rNRG1β, RP-318-P0A; Thermo Fisher Scientific, Inc.) isoforms, corresponding to the extracellular domains of the 2 molecules were used. A rat anti-human CHL1 antibody specifically targeting the extracellular domain of CHL1 was purchased from R&D Systems, Inc. (1:200 for both immunohistochemistry and immunuofluorescence, and 1:500 for western blot analysis; cat. no. MAB2126) and an anti-rabbit phosphorylated(p)-Neu antibody (1:200; cat. no. sc-101695) was purchased from Santa Cruz Biotechnology, Inc. A donkey anti-rat secondary antibody conjugated to Alexa Fluor 488 (1:500; cat. no. 712-545-153), a donkey anti-rat secondary antibody conjugated to Dylight™ 594 (1:1,000; cat. no. 712-515-150), a donkey anti-mouse secondary antibody conjugated to Dylight™ 488 (1:500; cat. no. 715-485-150) and a donkey anti-rabbit secondary antibody conjugated to Dylight™ 594 (1:1,000; cat. no. 711-515-152) were all purchased from Jackson ImmunoResearch Laboratories, Inc. A mouse monoclonal anti-NRG1 antibody (1:500; cat. no. MA5-12896; Thermo Fisher Scientific, Inc.), a mouse monoclonal anti-PCNA antibody (1:200; cat. no. sc-25280; Santa Cruz Biotechnology, Inc.), a mouse monoclonal anti-ERK1/2 antibody (1:1,000; cat. no. sc-135900; Santa Cruz Biotechnology, Inc.), a mouse monoclonal anti-p-ERK1/2 antibody (1:1,000; cat. no. sc-7383; Santa Cruz Biotechnology, Inc.), a mouse monoclonal anti-GAPDH antibody (1:1,000; cat. no. sc-3650620; Santa Cruz Biotechnology, Inc.) and horseradish peroxidase-conjugated goat anti-mouse (1:1,000; cat. no. BA1050; Wuhan Boster Biological Technology, Ltd.) and rabbit anti-rat secondary antibodies (1:1,000; cat. no. BA1058; Wuhan Boster Biological Technology, Ltd.) were used. Dulbecco's modified Eagle's medium (HyClone; Thermo Fisher Scientific, Inc.), penicillin/streptomycin mixture (Beijing Solarbio Science and Technology Co., Ltd.), 10% FBS (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.), 10% normal goat serum and 10% normal donkey serum (Beijing Solarbio Science and Technology Co., Ltd.) were used. A human glioma microarray, containing 184 glioma tissues, which can be graded from I to IV and 18 cancer adjacent normal (CAN) tissues, was purchased from US Biomax, Inc. (cat. no. GL 2083). The tumors were graded by a licensed pathologist on a scale of I to IV according to their degree of malignancy as defined by the World Health Organization classification of tumors of the central nervous system (23): I, Pilocytic astrocytoma; II, fairly differentiated astrocytoma; III, anaplastic astrocytoma; and IV, glioblastoma.

Immunohistochemical staining of paraffinized sections was performed as previously described (22). Human glioma tissue microarray sections (4-µm thick and deparaffinized) were rehydrated using a graded ethanol series (from 100, 90, 80, 70, 60 to 50%) that culminated with PBS. Antigen retrieval was performed using 10 mM citrate buffer (pH 6.0) at 99°C for 1 h and endogenous peroxidase was cleared by incubation in 3% H₂O₂ at room temperature for 20 min. The sections were then blocked with 10% normal goat serum in PBS at room temperature for 30 min, and the samples were incubated at 4°C overnight with a rat-anti-human primary antibody targeting human CHL1 (1:100). The antigen-antibody complexes were visualized using the avidin-biotin-peroxidase complex method using an AEC kit (cat. no. ZLI-9036, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.). For H&E staining, tissues were stained with eosin solution (cat. no. BSBA-4022, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) for 5 min at room temperature. Counterstaining was performed with Mayer's hematoxylin (cat. no. BSBA-4021A, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) for 1 min at room temperature. Images of the H&E and immunohistochemical staining results were captured using a light microscope (magnification, ×200; http://www.jnoec.com). The percentage of the area that was positive for CHL1 staining on each whole tissue point was evaluated using ImageTool (IT) software [version 3.0; Department of Dental Diagnostic Science at the University of Texas Health Science Center (UTHSCSA); magnification, ×200], which indicated the expression levels of CHL1 in the CAN tissue and glioma tissues graded from I to IV.

The human glioblastoma U-87 MG cell line (cat. no. CL-0238; unknown origin) and the human glioma U251 cell line (cat. no. CL-0237) were purchased from Procell Life Science and Technology Co., Ltd. The human glioma cell line SHG-44 (cat. no. SHG44) was purchased from Guangzhou Jennio Bioech Co., Ltd; http://www.jennio-bio.com.). All cell lines were authenticated using short tandem repeat analysis and tested for mycoplasma contamination. Cell lines were maintained in Dulbecco's modified Eagle's medium (HyClone; Thermo Fisher Scientific, Inc.) supplemented with 50 U/ml penicillin/streptomycin mixture (Beijing Solarbio Science and Technology Co., Ltd.) and 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.). The cells were routinely cultured in 75 cm² cell culture plates (Corning Inc.) at 37°C in a humidified incubator with 5% CO₂.

One control siRNA and siRNA against CHL1 (Table I), and one control siRNA and three different siRNAs against NRG1 (Table II), as well as the transfection reagent siRNA-Mate were purchased from Shanghai GenePharma Co., Ltd.. To test the effect of NRG1 on CHL1 expression, cells (5×10⁴ cells/well) in normal culture medium were allowed to adhere overnight in 48-well plates. When 80% confluence was achieved, the media were aspirated and replaced with fresh medium containing RNA/siRNA-Mate complexes (10 nM of either the random control siRNA or the targeting siRNAs/well) and the cells were further cultured at 37°C in a humidified incubator with 5% CO₂ for 48 h before western blot analysis.

To determine the effects of CHL1 on the level of proliferation cell nuclear antigen (PCNA), cells (2×10⁴/well) in normal culture medium were allowed to adhere overnight in an 8-well Lab-Tek Chamber Slide™ (cat. no. 154941PK; Nalge Nunc International). The media were then aspirated and replaced with fresh medium containing RNA/siRNA-Mate complexes (10 nM of either the random control siRNA or the siRNA against CHL1/well) and the cells were further cultured for 48 h. Cells were fixed with 4% paraformaldehyde in PBS (pH 7.3) at 4^ºC for 15 min, and were then subjected to double immunofluorescence staining of both CHL1 and PCNA.

The effects of CHL1 on cell senescence was assessed with the application of CHL1 siRNA in human glioblastoma U-87 MG cells. Cells (1×10⁵ cells/well) in culture medium were allowed to adhere to 24-well plates overnight. When 80% confluence was achieved, the medium was aspirated and replaced with fresh medium containing RNA-siRNA-Mate complexes (10 nM of either the control or the CHL1 siRNA/well). The cells were further cultured at 37°C in a humidified incubator with 5% CO₂ for 48 h. After 48 h of treatment, the cells were fixed for 20 min at 4°C with 4% paraformaldehyde in PBS. A β-galactosidase/X-Gal complex was added to each well and incubated overnight at 37°C according to the manufacturer's protocol (Beyotime Institute of Biotechnology). Cell senescence was determined by the development of a deep blue color in the cytoplasm, which reflected the activation of β-galactosidase.

To investigate the effects of NRG1α and β on CHL1 expression levels and NRG1-induced activation of ERK1/2 in vitro, U-87 MG, U251 and SHG-44 cells (1×10⁵ cells/well) were allowed to adhere to 24-well plates (Costar; Corning Inc.). After an overnight incubation, the medium was aspirated and replaced with serum-free culture medium containing NRG1α or NRG1β at concentrations of 0, 0.5, 1.0, 1.5, 2.5 and 5.0 nM for 48 h. Cells were lysed using RIPA lysis buffer mixture supplemented with PMSF (1:200; both Beijing Solarbio Science and Technology Co., Ltd.). The cell lysates were centrifuged at 14,000 × g for 15 min at 4°C, and the supernatants were collected for western blot analysis.

For immunofluorescence staining, 2×10⁴ cells/well were seeded overnight on an 8-well Lab-Tek Chamber Slide™ (Nunc). After the overnight incubation, the medium was aspirated and replaced with serum-free culture medium containing NRG1α or NRG1β at concentrations of 2.5 nM for 48 h. The cells were then subjected to fixation with 4% paraformaldehyde in PBS (pH 7.3) at 4°C for 15 min, which was followed by immunofluorescence staining for both CHL1 and p-Neu, as aforementioned.

Cells were then lysed with RIPA buffer (Solarbio Life Sciences). The cell lysate was then subjected to concentration determination with a BCA kit (Solarbio Life Sciences). A total of 20 µg of protein from each cell lysate were heated at 95°C in 20% sample loading buffer (0.125 M Tris-HCl, pH 6.8, 20% glycerol, 10% SDS, 0.1% bromophenol blue and 5% β-mercaptoethanol), then resolved using SDS-PAGE (with an 8% gel) and transferred onto polyvinylidene difluoride membranes (EMD Millipore). Non-specific protein binding sites were blocked with 5% skimmed milk diluted in Tris-buffered saline (pH 7.4) buffer containing 0.05% Tween-20 (TBST). The membranes were incubated overnight at 4°C with a mouse monoclonal anti-NRG1 antibody, a rat anti-human CHL1 antibody, a mouse monoclonal anti-ERK1/2 antibody and a mouse monoclonal anti-p-ERK1/2 antibody. To control protein loading, a mouse monoclonal anti-GAPDH antibody was used to blot the membrane overnight at 4°C. After 3 washes of 5 min each with TBST at room temperature, horseradish peroxidase-conjugated goat anti-mouse and rabbit anti-rat secondary antibodies diluted in TBST were incubated with the membranes for 1 h at room temperature, which was followed by 3 washes with TBST for 5 min each at room temperature. The antigens were visualized using enhanced chemiluminescence (Beyotime Institute of Biotechnology). The signal intensity was quantified using IT software as the average densitometry multiplied by the area (measured as the number of pixels) (22).

Deparaffinized human glioma tissue microarray sections (4-µm thick and deparaffinized) were rehydrated through incubation with a graded ethanol series (from 100, 90, 80, 70, 60 to 50%) that culminated with PBS. Antigen retrieval was performed using 10 mM citrate buffer (pH 6.0) at 100°C for 1 h. The samples were blocked with 10% normal donkey serum (Beijing Solarbio Science and Technology Co., Ltd.) in PBS at room temperature for 60 min. The sections were incubated with the following primary antibodies: Rat anti-human CHL1 (1:200) and rabbit anti-PCNA antibodies (1:200). The cultured cells were incubated with rat anti-human CHL1 (1:200) antibody together with either mouse anti-PCNA antibody (1:200) or rabbit anti-p-Neu antibody (1:100). After being washed in PBS 3 times for 5 min, the cells were incubated at room temperature for 90 min with a donkey anti-rat secondary antibody conjugated to Dylight™ 488 (1:500) and a donkey anti-rabbit secondary antibody conjugated to Dylight™ 594 (1:1,000). After being washed in PBS 3 times for 5 min, the tissue samples were incubated at room temperature for 90 min with a donkey anti-mouse secondary antibody conjugated to Dylight™ 488 (1:500) and a donkey anti-rat secondary antibody conjugated to Dylight™ 594 (1:1,000). The tissues and cells were co-stained with DAPI at room temperature for 20 min and mounted using an anti-fade mounting solution (Beyotime Institute of Biotechnology). Confocal images were captured using an Olympus confocal system (FV-1000; Olympus Corporation). DAPI, Dylight™ 488 and Dylight™ 594 were excited at 405, 488 and 594 nm, respectively.

CHL1 and PCNA immunofluorescence images for each sample were rearranged onto one panel based on the tumor grade. The protein level (measured as the integrated immunofluorescence intensity) was evaluated on the basis of gray scale ranging from 0 to 255, which was obtained using Image Tool II software. The correlation between CHL1 and PCNA was evaluated via Pearson's correlation analysis, using SPSS software (version 17.0; SPSS Inc.).

In vitro experiments were repeated at least 3 times using independent culture preparations. All numerical data are presented as group mean ± standard error of the mean. Statistical analyses were performed using SPSS version 10.0 (SPSS, Inc.). The analysis of CHL1 levels in glioma samples was undertaken using one-way ANOVA followed by Tukey's post hoc test. The effects of NRG1s on CHL1 were analyzed using one-way ANOVA followed by Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Human tissues from a commercial glioma tissue microarray were subjected to H&E staining and immunohistochemical staining of CHL1. Representative staining from the CAN tissue and tissues of different grades is shown in Fig. 1A. H&E staining showed regularly arranged cells in the CAN tissue, whereas abnormally proliferating cells were observed in the structurally disordered glioma tissues graded from I to IV (Fig. 1A), CHL1 was weakly stained in the CAN tissue, whereas it was strongly expressed in glioma tissue graded from I to IV, with the highest staining intensity detected in one grade II glioma tissue (Fig. 1A). The percentage of CHL1-positive area in each tissue point was measured to evaluate the expression levels of CHL1. As shown in Fig. 1B, the percentages of CHL1 positivity in CAN, and grade I, I–II, II, III and IV tumor tissues, were 32.33±5.00, 52.61±2.91, 66.67±4.43, 64.58±2.85, 46.67±5.92 and 42.90±5.33%, respectively. As was indexed by the percentage of CHL1-positive area in each tissue point, CHL1 expression levels in I–II and II grade gliomas were significantly higher compared with that in CAN tissue samples (P<0.05 for grade I–II and P<0.001 for grade II). By contrast, CHL1 expression levels in grade IV tissues were significantly lower than those in grade I–II samples (P<0.05). In addition, the CHL1 expression levels in grade III and IV tissues were significantly lower than those in grade II samples (P<0.05 for grade III and P<0.001 for grade IV). It was thus concluded that CHL1 expression levels is associated with the malignancy of glioma.

To identify the differential expression of CHL1 between low-grade and high-grade gliomas, the distribution of CHL1 expression between gliomas graded II and IV were further compared. Fig. 1C-a and -e showed one grade IV and grade II tissue containing both low-cell-density and high-cell-density areas, and Fig. 1C-i showed one grade II tissue only with a low-cell-density area, as revealed by H&E staining. The limited distribution of CHL1 in grade IV and II glioma tissues and the scattered distribution of CHL1 in grade II are presented in Fig. 1C-b, -f and -j, respectively. Fig. 1C-b and -d showed a restricted CHL1 expression in one grade IV glioblastoma, and that CHL1 expression level was associated with cell density within the glioma tissue. Generally, CHL1 was distributed in low-cell-density areas (Fig. 1C-d), while it was not notably expressed in high-cell-density areas (Fig. 1C-c). Similarly, CHL1 was largely expressed in the cell sparse areas in grade II glioma tissues (Fig. 1C-f, -h and -h′), with limited distribution found in the cell dense area (Fig. 1C-f, -g and -g′). In grade II gliomas with a scattered cell distribution, CHL1 was also dispersedly localized (Fig. 1C-j). Taken together, these results indicated that CHL1 was predominately expressed in low-cell-density area relative to that in the high-cell-density area within the glioma tissue.

First, the colocalization of CHL1 and PCNA in human glioma tissues was analyzed. Representative staining of CHL1 and PCNA are shown, in CAN and grade I–II and II human glioma tissues, in which the expression levels of CHL1 is highly expressed relative to that in the other grades of gliomas (Fig. 2A). In CAN tissue, both PCNA and CHL1 were weakly colocalized in the nucleus. In one grade I–II sample, CHL1 was solely localized on the cell membrane, while PCNA was weakly detected in the nucleus. However, in one grade II sample, CHL1 was not only detected on the cell membrane but it was also detected in the nucleus, where intensive colocalization with PCNA was observed. These data led to the hypothesis that CHL1 expression levels may be associated with the proliferation level of glioma cells. The correlation between CHL1 and PCNA on human gliomas of each grade was then evaluated.

Correlations between CHL1 and PCNA expression levels in the microarray data of human glioma tissues (n=23, 18, 85, 27 and 11 for grades I, I–II, II, III and IV, respectively) were then analyzed using Pearson's correlation (Fig. 2B). R values derived from Pearson correlation assay were 0.919, 0.872, 0.911, 0.882, and 0.857 in I, I–II, II, III and IV-grade glioma tissues, respectively, suggesting that CHL1 expression levels were highly positively correlated with the expression of PCNA and that CHL1 may modulate the proliferation of glioma cells, as indicated by PCNA. Immunofluorescence staining of CHL1 and PCNA was undertaken in U-87 MG cells treated with either control siRNA or siRNA targeting CHL1. The results demonstrated that downregulation of CHL1 resulted in the reduction of PCNA expression levels, suggesting that CHL1 regulates glioma cell proliferation (Fig. 2C).

To investigate whether downregulation of CHL1 can lead to cell senescence, senescence staining was conducted using a β-galactosidase activity assay. Enhanced senescence was observed in U-87 MG cells treated with an siRNA targeting CHL1. Compared with the negative control siRNA, cells treated with an siRNA targeting CHL1 demonstrated greater staining intensity by 48 h as shown in the dotted circle, which is indicative of a higher β-galactosidase activity and suggests that glioblastoma cells with less CHL1 activity are more prone to senescence (Fig. 2D).

Different concentrations of NRG1α and β were added to SHG-44 and U251 human glioma and U-87 MG human glioblastoma cells, and western blotting was used to investigate the effects of the different NRG1 isoforms on CHL1 protein expression levels. A total of 48 h after the application, NRG1α treatment resulted in a dose-dependent increase in the protein expression levels of CHL1 in both SHG-44 and U251 cells, with the highest levels observed at 2.5 and 5 nM, respectively. In U-87 MG cells, CHL1 levels were significantly increased in cells treated with 1.0–2.5 nM NRG1α, with the highest level observed following 2.5 nM treatment (Fig. 3A). Statistical significances were found in SHG44 an U-87 MG cells treated with 2.5 nM of NRG1αand in U251 cells treated with 5.0 nM of NRG1α (P<0.05; Fig. 3A).

A total of 48 h after application, NRG1β treatment resulted in a dose-dependent increase in the expression levels of CHL1 in both SHG-44 and U-87 MG cells, with the highest level observed at 5.0 nM (Fig. 3B). In contrast, NRG1β showed no significant effects on CHL1 expression levels at any of the concentrations (Fig. 3B).

Statistical significances were found in SHG44 cells treated with both 2.5 and 5.0 nM of NRG1βand in U-87 MG cells treated with 5.0 nM of NRG1β (P<0.05; Fig. 3B).

Subsequently U-87 MG glioblastoma cells, whose malignancy is relatively high, were treated with a control siRNA and 3 individual NRG1 siRNAs for 48 h. The siRNA results demonstrated that all siRNAs targeting NRG1 markedly reduced the protein level of NRG1, which was accompanied by a reduction in CHL1 (Fig. 3C). Since all the three siRNAs worked in reducing the targeted protein NRG1, no statistical analysis was applied.

Among the ErbB receptors for NRG1, NEU is a commonly shared receptor that can form heterodimers with either ErbB3 or ErbB4 (14). The colocalization of CHL1 and p-Neu was further investigated as a result of NRG1 activation in both U251 human glioma cells and U-87 MG human glioblastoma cells. The cell lines were treated with both NRG1s at 2.5 nM, a concentration at which NRG1s can sufficiently work in both cell lines. The immunofluorescence staining results demonstrate that NRG1α increased the protein level of CHL1 in the cytoplasm and at the plasma membrane of U251 cells. Consistent with the western blot results, NRG1β failed to increase the expression levels of CHL1 at 2.5 nM, although increased activation of p-Neu was detected in U251 cells (Fig. 4A). The immunofluorescence staining results demonstrate that both NRG1α and NRG1β increased CHL1 expression levels in the cytoplasm and at the plasma membrane of U-87 MG cells. However, the fluorescence signaling of p-Neu was not increased following treatment with NRG1β at 2.5 nM (Fig. 4B).

Accumulating evidence has indicated that the Ras/MAPK/ERK signaling pathways contribute to cell growth, proliferation and survival (1). Thus, western blot analysis was performed to assess p-ERK1/2 expression levels in U251, SHG-44 and U-87 MG cells in response to treatment with NRG1s. It was demonstrated that p-ERK1/2 protein expression levels were upregulated in U251 and U-87 MG human glioma/glioblastoma cells in response to 48 h of rNRG1α treatment, and that the response was dose-dependent; however, rNRG1α showed no effect on the protein expression levels of p-ERK1/2 in the SHG-44 human glioma cell line (Fig. 5A). Similarly, rNRG1β increased p-ERK1/2 expression levels in a dose-dependent manner in SHG-44, U251 and U-87 MG cells. The p-ERK1/2 expression levels peaked when the rNRG1β concentration was 1.5 nM and then p-ERK1/2 expression levels gradually returned to the baseline level (Fig. 5B).