Abstract Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor that is characterized by its high proliferative and migratory potential, leading to a high invasiveness of this tumor type. However, the underlying mechanism of GBM proliferation and migration has not been fully elucidated. In this study, at first, we used RNA-seq together with bioinformatics technology to screen for C-X-C motif ligand 1 (CXCL1) as a proliferation-related gene. And exogenous glial cell line-derived neurotrophic factor (GDNF) induced proliferation and up-regulated the level of CXCL1 in rat C6 glioma cells determined by sqPCR and ELISA. Then, we manipulated the CXCL1 expression by using a lentiviral vector (CXCL1-RNAi) approach. By this, the proliferation of C6 cells was decreased, suggesting that CXCL1 plays a key role in proliferation in these cells. We hypothesized that exogenous GDNF promoted NF-κB nuclear translocation and therefore, analyzed the interaction of CXCL1 with NF-κB by Western Blot and immunofluorescence. Additionally, we used BAY 11–7082, a phosphorylation inhibitor of NF-κB, to elucidate NF-κB mediated the effect of GDNF on CXCL1. These results demonstrated that GDNF enhanced the proliferation of rat C6 glioma cells through activating the NF-κB/CXCL1 signaling pathway. In summary, these studies not only revealed the mechanism of action of exogenous GDNF in promoting the proliferation of C6 glioma cells but may also provide a new biological target for the treatment of malignant glioma. Introduction Glioblastoma (GBM) is the most common primary malignant brain tumor, characterized by a high invasiveness [[34]1]. Like other tumors, GBM cells display an increased proliferation and migration potential that can eventually lead to the death of the patients [[35]2, [36]3]. However, the underlying mechanism of this cellular behavior has not been fully elucidated. Although the conventional treatment has some effect on glioma patients in early stages, the treatment effect of glioma patients in advanced stages is still very poor, mainly because glioma is prone to relapse and displays resistance to radio- and chemotherapy [[37]4, [38]5]. It has been proved that many oncogenes or tumor suppressors are dysregulated in glioma, which lead to a malignant progression of the disease [[39]4, [40]6, [41]7]. Therefore, studying the molecular mechanism of glioma development is conducive to exploring effective therapeutic strategies [[42]4]. GDNF is a member of the transforming growth factor beta (TGF-beta) superfamily, which was first isolated and purified in 1993 [[43]8]. The protein was previously considered to be an important differentiating factor with specific physiological roles in development and survival. Recent studies have shown that the tumorigenesis of glioma is associated with the abnormal expression of many cytokines, including GDNF [[44]9–[45]11]. Wiesenhofer and colleagues found an abnormal elevation of GDNF expression in primary glioma tissues and multiple glioma cell lines [[46]12]. It is noteworthy that this increase was positively correlated with the pathological grade of the tumors [[47]12]. Numerous studies suggest that exogenous GDNF plays an important role in proliferation and migration [[48]11, [49]13, [50]14]. Moreover, GDNF plays a regulatory role in glioma progression and is part of a complex multi-molecule network [[51]15]. However, up to now, it is unclear how GDNF influences this multi-molecular network to promote migration and proliferation of glioma cells. CXCL1, also known as GRO-α (growth related oncogene-α), is a proangiogenic and chemotactic factor, which mediates neutrophil recruitment by binding to CXCR2 receptors. Recent studies indicated that the proliferation and migration of cancer cells can be linked to some chemokines of the tumor microenvironment [[52]16, [53]17]. These molecules are secreted by tumor cells or other cell types present in the tumor microenvironment and that regulate the tumor immune response, angiogenesis, and proliferation [[54]18]. In addition, the production of CXCL1 has also been shown to be upregulated in glioma tissues where it mediates the proliferation of glial progenitor cells during neurodevelopment and contributes to glioma formation [[55]19]. However, the role of CXCL1 in the proliferation of glioma cells and its underlying mechanism remain unclear. In this study, we found CXCL1 was a proliferation-related gene by RNA-seq together with bioinformatics technology, and exogenous GDNF up-regulated the level of CXCL1 in rat C6 glioma cells determined by sqPCR and ELISA. Then, we suggested that CXCL1 plays a key role in proliferation in these cells by CXCL1-RNAi approach. Furthermore, we demonstrated exogenous GDNF promoted NF-κB nuclear translocation and the interaction of CXCL1 with NF-κB, and NF-κB mediated the effect of GDNF on CXCL1 by Western Blot and immunofluorescence. This study not only reveals the mechanism underlying the promotion of C6 cell proliferation after exogenous GDNF administration but also provides a new biological target for the treatment of malignant glioma. Materials and methods Cells and culture conditions The rat C6 glioma cell line was obtained from the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in F12/DMEM medium supplemented with 10% FBS for routine culture and with 0.1% FBS for hunger culture. The C6 cells were grown in 10 cm dishes and subjected to exogenous GDNF. Untreated C6 cells were used as a control. The AST cells were obtained from our laboratory at Xuzhou Medical University. Antibodies and reagents Antibodies were used at the following dilutions: Monoclonal mouse anti-β-catenin (BD610153; BD Biosciences, USA), 1:500; polyclonal rabbit anti-CXCL1 (GTX74108; GeneTex, USA), 1:2000; polyclonal rabbit anti-NF-κB (Cell Signaling Technology, USA), 1:2000; monoclonal mouse anti-β-actin (sc47778; Santa cruz, USA), 1:1000; polyclonal rabbit anti-Histone H3 (BS1660; Bioworld, USA), 1:500; IRDye 800CW goat (polyclonal) anti-rabbit IgG (926–32211; LI-COR, USA), 1:15000; IRDye 800CW goat (polyclonal) anti-mouse IgG (92632210; LI-COR, USA), 1:15000. BAY 11–7082 (S2913, Selleck, China). RNA-seq C6 cells were cultured and treated with GDNF for 0, 0.5, 1, and 24 h, respectively and total RNA was extracted from the four sample groups using Trizol reagent. All experiments were run in triplicate. After passing the electrophoretic quality inspection, performed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, US), total RNA was purified with a RNA clean XP kit and RNase free DNase set. Bidirectional RNA-Seq of the four samples were performed with an Illumina hiseq2500 high-throughput sequencer. Screening of differentially expressed genes DEGs The reads were converted into the number of reads per 1000 bases compared to exons in the reads per 1 million comparisons and the expression amount was standardized to calculate the gene’s fold change (FC). At the same time, the DEGs of different samples were analyzed by edger and the threshold value of P was determined by controlling the false discovery rate (FDR). The obtained P value was corrected by a multiple hypothesis test and the corrected P value was designated as Q value. Screening conditions for DEGs: Q value ≤ 0.05 and fold change ≥ 2 [[56]20]. Heat map and Venn map showed the distribution of DEGs In this study, we used the heatmap3 package of R language for data visualization [[57]21]. Additionally, Venny’s online software was used to draw a Venn graph. ([58]http://bioinfogp.cnb.csic.es/tools/venny/index.html) Enrichment analysis of Go terminology and KEGG pathway We used the David tool to perform GO and KEGG pathway enrichment analysis on the DEPs information. P < 0.05 was considered as being statistically significantly different [[59]22]. PPI network integration and module analysis In this study, we used the STRTING tool to construct a PPI network (medium confidence: 0.400) for DEPs, GDNF, and CXCL1 that were both rising in cytoplasm and nucleus, and thereafter used the Cytoscape software for further module analysis [[60]23]. Gene expression profiling interactive analysis (GEPIA) bioinformatics analysis For gene expression profiling and overall survival analysis, we conducted bioinformatics analysis on the GEPIA platform ([61]http://gepia.cancer-pku.cn/). GEPIA is an online analysis tool for processing high-throughput RNA sequencing expression data of bulk tumorous and normal samples based on the Cancer Genome Atlas (TCGA) ([62]https://portal.gdc.cancer.gov/) and the Genotype-Tissue Expression (GTEx, [63]https://www.gtexportal.org/) databases. We analyzed the correlation between CXCL1 expression and the overall survival (OS) of GBM patients and the expression of CXCL1 in GBM tissues. sqRT-PCR Total RNA was isolated from C6 glioma cells and its concentration and quality were determined with the help of a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and gel analysis. Then, the RNA samples were reverse transcribed into cDNA using a Transcriptor First Strand cDNA synthesis kit (Roche Applied Science, Penzberg, Germany). After this, semiquantitative real time PCR was performed to analyze the gene expression using the SYBR Green PCR master mix (Roche Applied Science, Mannheim, Germany). The 20 μL reaction mixture included 10 μL 2×SYBR® Premix Ex Taq (Takara, China), 0.5 μL each of 10 μM forward and reverse primers, 2 μL cDNA template and 7 μL RNase-free H[2]O. The PCR conditions included an initial denaturation step at 95°C for 5 min, followed by 45 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 15 s. The mRNA data were normalised to GAPDH. The used primer sequences were as follows: GAPDH F:CGGATTTGGCCGTATCGG R:TGAGGTCAATGAAGGGGTCG CXCL1 F:GCAGACAGTGGCAGGGATTC R:CGGTTTGGGTGCAGTGGG Cx3cl1 F:GAGGCAGGCAGTGGGGTTA R:TGGAGGCTCTGGTAGGCAAA Serpine1 F:CTGCCCCGCCTCCTCA R:CGCCACTGTGCCGCTCT Ddit3 F:CACCTCCCAAAGCCCTCG R:TGCTTGAGCCGCTCGTTCT Gadd45g F:CCGCTGGCGTCTACGAGTC R:GGCTATGTCGCCCTCATCTTC Mt1 F:CCAACTGCTCCTGCTCCACC R:GCACTTGTCCGAGGCACCTT Rap1gap F:CCAGCCACAGTGGGAGCTT R:CCCCGGCGTCCGAATC Klf10 F:AAGGAGGTTTGCTCGTTCCG R:GGCAGCTTCTTGGCTGATAGG Nfkbia F:TGACCTGGTCTCGCTCCTGT R:GACGCTGGCCTCCAAACAC Immunofluorescence For in situ staining, cells were washed with PBS, then fixed with 4% paraformaldehyde (PFA), washed 3× in PBS and placed in 10% normal goat serum with 0.3% Triton X-100 in PBS for 1 h. Afterwards, the cells were incubated in blocking solution with anti- NF-κB antibody overnight at 4°C. After that, the cells were washed 3× in PBS and incubated with goat anti-rabbit antibody in PBS for 1 h, washed 3× in PBS and incubated with DAPI (VIC112; VICMED, China). Finally, the cells were washed 3× with PBS and analyzed under a fluorescence microscope (IX-71; Olympus, Japan). Total protein, cytoplasmic and nuclear protein extraction For total protein extraction, cells of each group were washed with ice-cold phosphate-buffered saline and lysed in RIPA lysis buffer (P0013B; Beyotime, China) containing the protease inhibitor PMSF (1Mm; VPI003; VICMED, China). Cytoplasmic and nuclear protein extraction was subsequently performed. The samples were washed with ice-cold phosphate-buffered saline and treated with a nuclear protein extraction kit (P0028; Beyotime, China) according to the manufacturer’s protocol: The cell pellet was incubated in buffer A containing PMSF and thereafter incubated for 10 min. on ice. Then, buffer B was added and the samples were incubated for another 1 min. After centrifugation at 10,000 g for 5 min at 4°C, the cytoplasmic protein (the supernatant) could be isolated. The nuclei in the pellet were isolated by further centrifugation and resuspended in nuclear extraction buffer containing PMSF for 30 min at 4°C and vortexing every 5 minutes. After centrifugation at 10,000 g for 10 min at 4°C, the supernatant contained the nuclear protein. The protein concentration was determined with the help of a BCA assay kit (P0010, Beyotime, China) and was used according to the manufacturer’s protocol. The isolated proteins were stored at −80°C until use. Western blot analysis To detect the expression of specific proteins, 40 μg of total protein was separated on a SDS-polyacrylamide gel (KGP113K; KeyGEN, China) and wet transferred to a nitrocellulose transfer membrane (PALL66485F; VICMED, China). The membrane was probed with a primary and a secondary antibody. The protein bands were scanned and visualized using an Odyssey® Infrared Imaging System by LI-COR Biosciences (USA). Luciferase assay To monitor the expression of the GDNF-induced CXCL1, we generated a luciferase expression construct containing the CXCL1 promotor. Firstly, a Luciferase reporter gene vector was constructed by designing primers (F:CATGCTCGAGAAGAGCTGGAAGGCTTGCAGTCA; R:CATGAAGCTTGAAGTGAATCCCTGCCACTGTC) to amplify the CXCL1 promoter sequence. A Luciferase reporter gene vector (1 μg/well) and pRL-TK (50 ng/well), expressing Renilla luciferase as an internal control, were co-transfected in C6 cells (1×10^5) and incubated for various time points. Relative luciferase activity was assessed by the Promega dual-luciferase reporter assay system (Promega, Madison, WI). ELISA detection 50 μl of standard, 40 μl of sample diluent and 10 μl of sample were accurately added to the wells to be tested. After gentle shaking and mixing, the plate was closed and incubated at 37°C for 30 min. Then, 50 μL developer was added into each well, gently mixed and incubated for 10 min. at 37°C and protected from light for color development. After this, the wells were washed. Finally, 50 μL termination liquid were added into each well after which eventually the absorbance of each well was measured at 450 nm [[64]24]. Construction of the lentiviral vector The siRNA target was designed according to the transcripts of the r-CXCL1 gene. The siRNA sequence of the control virus vector was: TTCTCCGAACGTGCACGTAA. The interference sequence was: AGAACATCCAGTTGAAGGTGAT. The designed sequence was cloned into a pHB-U6-MCS-CMV-ZsGreen-PGK-PURO (Hanbio Biotechnology, Shanghai, China) to generate the lentiviral vector. Then, the expression vector together with the packaging vector were used to transfect 293T cells with lipofitertm (Hanbio Biotechnology, Shanghai, China). After 48 h, the supernatants containing HBLV-r-CXCL1 shRNA3-GFP-PURO and HBLV-GFP-PURO NC lentiviruses were collected. After that, the lentiviruses were purified by ultracentrifugation after which the virus titers were detected. ChIP assay A ChIP Chromatin Immunoprecipitation kit (Millipore, Bedford, MA, USA) was employed for the ChIP assay. Briefly, C6 cells were fixed with 1% formaldehyde for 10 min at 25°C and then washed with ice-cold phosphate-buffered saline buffer containing protease inhibitors. The nuclei were collected and resuspended in SDS lysis buffer containing protease inhibitor after which they were sonicated to generate crosslinked DNA fragments with 200–1000 bp length. The soluble supernatant was obtained after brief centrifugation whereupon it was incubated with Protein G Agarose. An aliquot of the supernatant was saved as a DNA input control and the remainder was incubated with anti-NF-κB antibody (Cell Signaling Technology, USA). A rabbit IgG antibody was used as a negative control. Immunoprecipitation was carried out overnight at 4°C and immune complexes were collected using Protein G Agarose beads. The immune complexes and the input were eluted and protein-DNA complexes were de-crosslinked overnight at 65°C. ChIP DNA was purified and subjected to PCR, which amplified the CXCL1 promoter sequence containing putative SRY-related protein-binding sites (site 1: position +951bp to +1116bp; site 2: position +356bp to +511bp; site 3: position +771 bp to +940bp). Specific ChIP primers used for PCR were as follows: CXCL1-1; F: TTGGGAGTGGAGCAAGGGG; R: TGGAGCTGGTTTAGGATCTGAGTC; CXCL1-2; F: CGTCCTCAGCCCAGAAAAAAC; R: CTCTTCTTTGCTTTTTGAACTCGG; CXCL1-3; F: TGGAGTCCTAGGTGGCGTGG; R: TTTTTGCTTTTTGCCCCAAAGTC. Cell viability assessment For cell viability assays, 1×10^3 cells were plated in clear bottom 96-well plates, processed according to the manufacturer’s instructions using a Cell Counting Kit-8 (CCK-8; CK04; Dojindo, Japan) and afterwards quantified on a microplate reader (Synergy2; BioTek, USA). Assessment of cell viability C6 cells (1×10^3 cells/well) were cultured for 12 h in 96-well plates before being treated with different concentrations of rapamycin BAY 11–7082 for 24 h. Cell viability was evaluated using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with absorbance measurement at 490 nm. Statistical analysis Statistical analysis was performed using SPSS 19.0 for Windows. The data are presented as the mean ± standard deviation obtained from three independent experiments. One-way ANOVA and Tukey’s post-hoc analysis were used to determine the differences between groups. P < 0.05 was considered to be statistically significantly different. Results 1 Bioinformatics-based screening of differentially expressed genes (DEGs) related to GDNF-induced proliferation of C6 cells It has shown that GDNF can significantly increase the proliferation and migration of glioma cells [[65]15]. In order to elucidate the molecular mechanism and target underlying the GDNF-induced C6 glioma cell proliferation, the C6 cells were incubated with 40 ng/ml GDNF [[66]15] for 0 h, 0.5 h, 1 h, and 24 h, respectively. The total RNA of the samples was extracted and sequenced by RNA-Seq. The results showed that the numerical regions of the four groups of samples were of high quality, with balanced distribution of bases ([67]S1A and S1B Fig). The percentage of tested genes increased with increasing sequencing depth, and the correlation between samples was high ([68]S1C and S1D Fig). Differentially expressed genes are displayed by a heat map drawn with the help of the R language software package. Compared to the 0 h group, 11, 37, and 87 genes were differentially expressed (DEG) in the 0.5 h, 1 h, and 24 h group, respectively, of which 5, 29, and 37 were up-regulated and 6, 8, and 50 were down-regulated ([69]Fig 1A). In order to find the intersection of DEGs, we used Venny’s online software to draw a Venn diagram. There were two common up-regulated DEGs (CXCL1 and [70]AABR07024908.1) and one common down-regulated DEG (LOC100910835) in the 0.5, 1, and 24 h group ([71]Fig 1B and 1C). Fig 1. The distribution of DEGs were showed by heat map and Venn diagram. [72]Fig 1 [73]Open in a new tab The heat map of DEGs. The abscissa indicated sample numbers, and NO0_1, NO1_1, NO2_1, NO3_1 represented the total RNA samples extracted from C6 glioma cells after exogenous GDNF treated 0, 0.5, 1 and 24 hours, respectively. The ordinate represented the gene name. The color key in the upper right corner indicated the color range, in which red indicated the up-regulated DEGs, and green indicated the down regulated DEGs. (B) Three sets of Venn plots showed the DEGs were up-regulated in C6 cells at 0.5h, 1 and 24h after GDNF treatment compared with 0 h. (C) Three sets of Venn plots showed the DEGs were down-regulated in C6 cells at 0.5h, 1 and 24h after GDNF treatment compared with 0 h. Furthermore, the DEGs were enriched for GO function and KEGG analysis. The number of GO terms mapped resulted in 18, 325, and 461 in the 0.5, 1, and 24 h group, respectively ([74]Fig 2). Then, the David software was used to enrich DEGs with KEGG. The results showed that 2 of the 5 DEGs were found to be located on the KEGG pathway. However, the enriched KEGG pathway resulted in 0 in the 0.5 h group (thresholds: count = 2, ease = 0.1) ([75]S2 Fig); However, 20 of the 29 DEGs were found to be located on the KEGG pathway and the enriched KEGG pathway resulted in 7 in the 1 h group (thresholds: count = 2, ease = 0.1) ([76]S3 Fig); 16 of the 37 DEGs genes were found to be located on the KEGG pathway with the enriched KEGG pathway result of 1 in the 24 h group (thresholds: count = 2, ease = 0.1) ([77]S4 Fig). Finally, STRING and Cytoscape software was used to analyze the protein-protein interaction (PPI) network of DEGs and further module analysis. The results showed that rich protein interactions only existed in the 1 h group and that there were mainly four functional modules: the TNF signaling pathway, the NF kappa B signaling pathway, the I-kappa B/NF-kappa B complex, and the beta interferon production pathway ([78]S5 Fig). In order to validate the RNA-seq results, gel electrophoresis was performed to prove the integrity of RNA at this time. [79]Fig 3A showed that the 18S and 28S bands of the target RNA were clear under agarose gel electrophoresis, indicating that the RNA is relatively complete and not degraded. In addition, for some genes a sqPCR analysis was carried out. As a result, the relative expression level of CXCL1 could be shown to be significantly increased in the 0.5, 1, and 24 h group ([80]Fig 3B). Because CXCL1 was up-regulated in the DEG analysis and is known to be involved in cellular proliferation in GO gene annotation (GO; 0008283), CXCL1 turned out to be the most noteworthy target gene. Fig 2. Go function enrichment scatter of DEGs. [81]Fig 2 [82]Open in a new tab DEGs were enriched for GO function and the scatter diagram of the top 30 was drawn after exogenous GDNF treated 0.5 (A), 1 (B) and 24 h (C) compared with 0 h respectively. Fig 3. The relative expression of genes was detected by sqPCR. [83]Fig 3 [84]Open in a new tab (A) RNA integrity was detected by gel electrophoresis. (B) The relative expression of genes was detected by sqPCR. 2 CXCL1 played a pivotal role in the proliferation of C6 cells and in GBM We used RNA-seq combined with bioinformatics technology to screen CXCL1 as a GDNF-induced proliferation-related gene which could not be confirmed by the experiments. Present studies show that CXCL1 acts as a carcinogen in glioma which promotes the proliferation and migration of glioma cells [[85]19]. Therefore, with the help of the GEPIA platform ([86]http://gepia2.cancer-pku.cn/#survival), we firstly analyzed the correlation between CXCL1 expression and the overall survival of GBM patients. We found that CXCL1 was negatively correlated to the OS of GBM patients ([87]Fig 4A). In addition, we found that the expression of CXCL1 was up-regulated in GBM tissues compared to normal tissues ([88]Fig 4B). To explore the expression and secretion of CXCL1, protein and supernatant from AST and C6 cells were extracted. AST cells were obtained by isolation and purification of rat primary cells with a positive GFAP rate in the cytoplasm of higher than 95% ([89]Fig 4C). Western blot and ELISA results showed that the CXCL1 protein expression level and the supernatant content in C6 cells were significantly higher than those in rat primary astrocytes, respectively ([90]Fig 4D–4F). These results demonstrated that CXCL1 was up-regulated in C6 cells and GBM. Fig 4. The expression level of CXCL1 in C6 cells and GBM. [91]Fig 4 [92]Open in a new tab (A) The correlation between CXCL1 and the overall survival of GBM patients on GEPIA. (B) The expression of CXCL1 in GBM tissues compared with related normal tissues. (C) Identification of AST. AST transmitted to the third generation were stained with anti-GFAP immunofluorescence. The cytoplasm of positive cells was stained red, and the nucleus of DAPI stained blue. Bar = 200 μm. (D) Western blot showed that CXCL1 protein expression level in C6 cells and AST. (E) The statistical results of (D). (F) ELISA showed that CXCL1 supernatant content in C6 cells and AST. (**P<0.01, ***P<0.001). To further validate the effect of CXCL1 on the proliferation of C6 glioma cells, we constructed a lentiviral vector with an interfering RNA (RNAi) targeting the rat CXCL1 gene and which was used to infect C6 cells. Western blot results showed that the expression level of CXCL1 was significantly down-regulated in the CXCL1 knockdown group (CXCL1-RNAi) compared to the control group (RNAi-vector) ([93]Fig 5A and 5B). The CCK-8 assay results showed that the OD450 value of the CXCL1-RNAi group was lower than that of RNAi-vector group ([94]Fig 5C). The above results suggest that knockdown of CXCL1 inhibits the proliferation of C6 cells. Fig 5. The effect of CXCL1 on proliferation of C6 cells. [95]Fig 5 [96]Open in a new tab (A) Western blot showed that the expression level of CXCL1 in the CXCL1 knockdown group (CXCL1-RNAi) and the control group (RNAi-vector). (B) The statistical results of (A). (C) CCK-8 showed that the OD450 value in CXCL1-RNAi and RNAi-vector group. (*P<0.05, **P<0.01). 3 CXCL1 mediated the proliferation of C6 cells induced by exogenous GDNF In order to detect the effect of exogenous GDNF on CXCL1, we used a double luciferase reporter gene activity assay to detect the effects of GDNF on the CXCL1 promoter region. The results showed that compared to the CXCL1 group, the CXCL1 promoter activity increased significantly after GDNF treatment ([97]Fig 6A). Fig 6. CXCL1 mediated the proliferation of C6 cells induced by exogenous GDNF. [98]Fig 6 [99]Open in a new tab (A) CXCL1 promoter activity increased significantly after GDNF treatment by double luciferase reporter gene activity. pGL 4.20 was an empty carrier. pRL-TK was internal reference. (B) The mRNA level of CXCL1 was analyzed by q-PCR. (C) Cell supernatants were collected and the secretion level of CXCL1 was detected by ELISA. (D) CCK-8 was used to detect the cell activity of each group. (*P<0.05, **P<0.01,***P<0.001). To validate the effect of GDNF on CXCL1, exogenous GDNF (40 ng/mL) was added to C6 cells and the CXCL1 mRNA level was analyzed by sqPCR. In addition, cell supernatants were collected and the secretion level of CXCL1 was detected by ELISA. The sqPCR results showed that GDNF could promote the expression of CXCL1 and ELISA results showed that GDNF could significantly promote the secretion of CXCL1 in C6 cells ([100]Fig 6B and 6C). In order to prove that CXCL1 is able to mediate the proliferation of C6 cells after GDNF-induction, C6 cells were infected with a CXCL1 knock-out lentiviral vector. After administration of exogenous GDNF (40 ng/mL) to the C6 cells, CCK-8 was used to detect the cell activity of each group. Compared to the control group, the activity of the C6 cells in the CXCL1-RNAi group was decreased. Moreover, compared to the GDNF treatment group, the C6 cell viability was decreased in the CXCL1-RNAi and the exogenous GDNF co-treatment group ([101]Fig 6D). These results demonstrate that CXCL1 mediates the proliferation of C6 cells after exogenous GDNF administration. 4 GDNF promoted NF-κB nuclear translocation and the interaction of CXCL1 with NF-κB In order to explore the possible mechanism of the GDNF promoted C6 cell proliferation via promotion of the CXCL1 gene transcription, we used a Qiagen online software to analyze the transcription factor (TF) that initiates the CXCL1 transcription at its corresponding binding sites (BS). The results showed that the most relevant transcription factors were NF-κB, NF-κB1, STAT1, STAT1 α, STAT1 β, C/EBP α, C/EBP β and P53, in which the TF-BS positions of NF kappa B on the CXCL1 gene promoter were NC_74723660, NC_74735035, and NC_74735036, respectively ([102]Fig 7A). In most tumors, NF-κB is abnormally activated and regulates various properties of cancer cells, including proliferation, invasion, inhibition of apoptosis, and angiogenesis [[103]25]. Accordingly, we designed and synthesized three primers able to bind to the CXCL1 promoter region and used the ChIP-qPCR assay to study the binding sites of transcription factor NF-κB in the CXCL1 promoter region in the control and GDNF treatment group. The results indicated that site 1 (promoter I + 951 BP to + 1116 bp), site 2 (promoter I + 356 BP to + 511 bp), and site 3 (promoter I + 771 BP to + 940 bp) were significantly correlated to the CXCL1 promoter regions after GDNF treatment with the highest binding affinity to site 3 ([104]Fig 7B). Fig 7. GDNF promoted NF-κB nuclear transformation and the interaction CXCEL with NF-κB. [105]Fig 7 [106]Open in a new tab (A) Transcription factors that regulate the gene CXCL1. Qiagen online software was used to analyze the TF that initiated CXCL1 transcription and its corresponding BS. (B) Exogenous GDNF promoted the interaction CXCEL with NF-κB by ChIP-qPCR assay. (C) Western blot showed exogenous GDNF promoted NF-κB nuclear transformation. The C6 cells were treated with exogenous GDNF for 48h, and nuclear protein were extracted to determine NF-κB (p65) levels by Western blot. Histone 3 was used as internal references. (D) The statistical results of (C). (E) NF-κB