Abstract

Background

   The growth- and plasticity-associated protein-43 (GAP43) is biasedly
   expressed in indigestive system and nervous system. Recent study has
   shown that GAP43 is responsible for the development of neuronal growth
   and axonal regeneration in normal nervous tissue, while serves as a
   specific biomarker of relapsed or refractory neuroblastoma. However,
   its expression pattern and function in digestive system cancer remains
   to be clarified.

Methods

   In this study, we examined the GAP43 status with qRT-PCR and bisulfite
   genomic sequencing in colorectal cancer (CRC). We investigated the
   effect of overexpressed GAP43 in CRC cells with RNA-seq. The RNA-seq
   data was analyzed with DAVID and IPA.

Results

   GAP43 was downregulated in CRC compared to the adjacent tissues. DNA
   methylase inhibitor 5-Aza-CdR treatment could significantly induce
   GAP43, indicated that the silencing of GAP43 gene in CRC is closely
   related to DNA methylation. Bisulfite genomic sequencing confirmed the
   promoter methylation of GAP43 in CRC. To explore the transcriptional
   alterations by overexpressed GAP43 in CRC, we performed RNA-seq and
   found that upregulated genes were significantly enriched in the
   signaling pathways of ABC transporters and ECM-receptor interaction,
   while downregulated genes were significantly enriched in Ribosome
   signaling pathway. Further Ingenuity Pathway Analysis (IPA) showed that
   EIF2 signaling pathway was significantly repressed by overexpression of
   GAP43.

Conclusion

   Our findings provide a novel mechanistic insight of GAP43 in CRC.
   Transcriptome profiling of overexpressed GAP43 in CRC uncovered the
   functional roles of GAP43 in the development of human CRC.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12885-020-07728-x.

   Keywords: GAP43, Colorectal cancer, RNA-seq, DNA methylation,
   Transcriptome profiling

Background

   Colorectal cancer (CRC) is one of the most common type of the digestive
   tract cancers and it ranks the third in the world’s cancer incidence
   [[45]1]. CRC is mainly caused by genetic factors, chronic inflammation,
   unhealthy living habits, etc.; the geographical distribution of the
   disease is quite different, and the incidence of CRC is higher in
   developed areas [[46]2]. China is a low-incidence area for CRC, but the
   incidence rate is increasing [[47]3]. As of 2016, the number of new
   cases of CRC in China has exceeded 270,000 and more than 130,000 deaths
   [[48]4]. As the third malignancy in China, previous researches showed
   that CRCs are caused by various genetic and epigenetic changes,
   including WNT, PI3K-Akt, GPCR, FGFR, MAPK, TGFB, and TP53 signaling
   pathways [[49]5–[50]7]. To date, the exact cellular and molecular
   mechanisms leading to CRC have not been systematically evaluated.
   Further investigation of functional genes in CRC is critical for
   identifying novel potential targets for CRC detection and precision
   treatment.

   GAP43 was reported as a ‘growth’ or ‘plasticity’ protein, which was
   responsible for the development of neuronal growth and axonal
   regeneration [[51]8–[52]10]. Previous studies of GAP43 thus far were
   focused on the neuron biology. However, the role of GAP43 in human
   cancers, especially CRC, has not been explored to date. Previously,
   with the GEPIA database ([53]http://gepia.cancer-pku.cn/), we found
   that the expression level of GAP43 was significantly downregulated in
   colorectal cancer tissues (normal versus cancer tissues), including the
   colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ)
   (Supplementary Fig. S[54]1a).

   Here, we investigated the expression level of GAP43 in CRC cell lines.
   The mechanism of the silenced GAP43 gene expression in CRC tissues was
   further studied with the analysis of its promoter methylation.
   Furthermore, the transcriptome alteration of GAP43-overexpressed versus
   control CRC cell line was analyzed with RNA-seq. Taken together, we
   disclosed the potential functions of GAP43 in the development of human
   CRC.

Methods

Cell culture and transfection

   The CRC cell lines HCT116 (Resource number: 3131C0001000700099), SW620
   (Resource number: 3131C0001000700101), SW480 (Resource number:
   3131C0001000700086), HT-29 (Resource number: 3131C0001000700103), DLD-1
   (Resource number: 3131C0001000700134) were cultured in RPM1640 medium
   supplemented with 10% fetal calf serum and 2 mM L-glutamine. All CRC
   cell lines used in this study were originally purchased from the Type
   Culture Collection of the Chinese Academy of Sciences, Shanghai, China.
   Normal colorectal epithelial cell line NCM460 (Resource number:
   CVCL_0460, INCELL) was kindly provided by Dr. Ming Liu, Harbin Medical
   University, China. NCM460 was cultured in DMEM medium supplemented with
   10% fetal calf serum and 2 mM L-glutamine. All cell lines were
   authenticated by short tandem repeats (STR) analysis. All cell lines
   have been tested for mycoplasma contamination, and no contamination was
   found. SW620/GAP43 cell (SW620 cell overexpressing GAP43 protein) was
   generated by stabilizing a GAP43-expression plasmid that confers
   neomycin resistance in SW620 cell.

Patient samples

   The collection of three paired CRC samples used for quantitative
   Real-Time PCR, patient consent, and patient recruitment followed
   Institutional Review Board protocols from Hangzhou Cancer Hospital. The
   clinical information of these colorectal cancer tissues was listed in
   Supplementary Table S[55]6. The study has been approved by the Research
   Ethics Committee of Hangzhou Cancer Hospital (Hangzhou, P. R. China;
   approval number HZCH-2018-02). All written informed consents have been
   provided by the patients, and to be conducted in accordance with the
   Declaration of Helsinki.

Quantitative real-time PCR (qRT-PCR)

   Quantification of mRNA expression was performed with SYBR Green PCR
   Master Mix on an ABI 7500 real-time PCR system (Foster City, CA, USA).
   GAPDH was considered as an internal control. The qRT-PCR procedure was
   recommended according to the protocol. Primers for qRT-PCR are
   described in Supplementary Table S[56]7.

Bisulfite DNA sequencing

   Bisulfite DNA sequencing was performed as previously described
   [[57]11]. Briefly, the bisulfite modification of DNA was performed
   according to the protocol of the methylSEQr™ Bisulfite Conversion Kit
   (Applied Biosystems, Shanghai, CN). The bisulfite-treated DNA was
   amplified using nested PCR using the primers described in Table S[58]1.
   The amplified bisulfite-treated DNA was sequenced and compared to the
   original sequence of the GAP43 gene.

RNA-seq library preparation and sequencing

   Total RNA was isolated from 4X10^6 GAP43 overexpressed and control
   SW620 cells using RNeasy Mini Kit (Qiagen, Valencia, CA). Total RNA
   integrity was checked using an Agilent 2100 Bioanalyzer (Agilent
   Technologies, Inc., CA, USA) with an RNA integrity number (RIN) value
   greater than 8. The mRNA was purified and fragmented from total RNA
   (2 μg) using poly-T oligo attached magnetic beads with two rounds of
   purification. Then the cleaved RNA fragments were primed with random
   hexamers and were reverse transcribed into first-strand cDNA using
   reverse transcriptase and random primers. The RNA template was removed
   and synthesized a replacement strand to generate double-strand cDNA.
   End repair, A-tailing, adaptor ligation, cDNA template purification and
   enrichment were then performed. Constructed libraries were sequenced
   with 150 bp paired-end by an Illumina X10 sequencer, following
   manufacturer’s instructions.

Database for annotation, visualization and integrated discovery (DAVID) and
ingenuity pathway analysis (IPA)

   DAVID ([59]http://david.abcc.ncifcrf.gov), Functional Annotation
   Bioinformatics Microarray Analysis was used to identify significantly
   enriched gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes
   (KEGG) terms among the given list of genes that are differentially
   expressed in GAP43 overexpressed and control HCT116 cells.
   Statistically overrepresented GO and KEGG categories with p-value≤0.05
   were considered significant. For IPA, different expressional genes
   (DEGs) were submitted to IPA software (Qiagen bioinformatics, Ingenuity
   Pathway Analysis), the canonical pathways were considered significant
   with p-value≤0.05 and z-score ≥ 1 or z-score ≤ − 1.

Statistical analysis

   Statistical data were represented as mean ± standard deviation from at
   least three independent experiments. Genes expression were
   statistically analyzed by two-way repeated measures ANOVA. All graphs
   were plotted and analyzed with GraphPad Prism 7 software.

Results

GAP43 is expressed in human colon tissues, but not in colorectal cancer cells

   According to the RNA-seq data from normal tissues of 95 human
   individuals [[60]12], GAP43 gene is biasedly expressed in brain,
   adrenal, gall bladder, heart, placenta, prostate, salivary gland and a
   series of indigestive tissues, including duodenum, small intestine,
   colon and appendix (Fig. [61]1a). As previous observation of the
   downregulated GAP43 in CRC, we hypothesize that GAP43 may play role on
   the development of CRC. To investigate the expression level and
   mutations of GAP43 in CRC tissues, we analyzed the publicly available
   databases (cBioPortal, Oncomine). In cBioPortal database, we found that
   GAP43 was mutated in CRC cancer tissues with the 1–3% frequency
   (Supplementary Fig. S[62]1b), and in Oncomine, compared with normal
   colorectal tissues, we found that the expression of GAP43 was
   significantly repressed in CRC tissues (Supplementary Fig. S[63]1c).
   Further validations were performed with Quantitative real-time PCR
   (qRT-PCR) in 3 paired CRC tissues and the corresponding adjacent
   non-tumor tissues, 1 normal colorectal epithelial cell line NCM460 and
   5 CRC cell lines HCT116, SW620, SW480, HT29, DLD-1 (Fig. [64]1b). The
   results showed that GAP43 was significantly down-regulated in these 3
   CRC tissues and 5 CRC cell lines, compared to normal tissues and normal
   colon cell, respectively. Thus, these data indicated that GAP43 gene
   was expressed in human normal colon tissues, but significantly
   down-regulated in CRC.

Fig. 1.

   [65]Fig. 1
   [66]Open in a new tab

   GAP43 expression levels are detected in normal human tissues and
   colorectal cancer cell lines. a Relative mRNA levels are analyzed,
   according to the RNA-seq data from normal tissues of 95 human
   individuals. b qRT-PCR was performed in five CRC cell lines HCT116,
   SW620, SW480, HT29, DLD-1and 2 human adjacent non-cancer tissues. The
   relative mRNA levels are analyzed. The results represent at least three
   independent experiments and are reported as mean ± SEM. *p < 0.05

GAP43 level is repressed in CRC due to aberrant promoter methylation

   To investigate the mechanisms involved in the silencing of GAP43 gene
   in CRC, we investigated the DNA methylation and mRNA expression data of
   GAP43 from CRC tissue of the TCGA database. The results showed that the
   downregulated expression of GAP43 may due to the hyper DNA methylation
   of its promoter regions in CRC tissues (Supplementary Fig. S[67]2).

   To demonstrate that silencing of GAP43 expression in CRC cells is
   caused by the methylation of its promoter, we treated SW620 cells with
   the DNA demethylating agent 5-Aza-CdR. As shown in Fig. [68]2a, mRNA
   level of GAP43 was significantly increased after the treatment of SW620
   cells with high concentrations of 5-Aza-CdR for 72 h. When SW620 cells
   were treated with 10 μM 5-Aza-CdR, the mRNA level of GAP43 was
   significantly increased with the increasing treatment time (Fig.
   [69]2b). To further confirm the DNA methylation in the GAP43 promoter
   of CRC cells, we performed bisulfite genomic sequencing to analyze the
   methylation status of the GAP43 promoter in CRC cell lines SW620 and
   HCT116. The promoters of HAND1 M1 and HAND1 M2 have been known to be
   highly methylated [[70]13], and the promoters of ABCC1 and ABCG2 are
   rarely methylated [[71]14]. Thereby, we take them as positive or
   negative controls in bisulfite genomic sequencing analysis,
   respectively (Fig. S[72]3). The results showed that in the CRC,
   multiple CpG islands in the promoter region of the GAP43 gene were
   methylated (Fig. [73]2c). These results show that the downregulation or
   silencing of GAP43 in CRC is associated with its promoter methylation.

Fig. 2.

   [74]Fig. 2
   [75]Open in a new tab

   Silencing of GAP43 gene is due to aberrant promoter methylation. a
   SW620 cells were treated with the indicated concentration of DNA
   demethylating agent 5-Aza-CdRfor 72 h, or b SW620 cells were treated
   with 10 μM 5-Aza-CdR for indicated treatment time (0, 24, 48, 72 h),
   and then mRNA levels of GAP43 were analyzed by qRT-PCR. c Bisulfite
   genomic sequencing was performed to analyze the methylation status of
   the GAP43 promoter in CRC cell lines SW620 and HCT116. Multiple CpG
   islands in the promoter region of the GAP43 gene were analyzed. The
   results represent at least three independent experiments and are
   reported as mean ± SEM. *p < 0.05, **p < 0.001

Identification of transcriptome alterations in GAP43 overexpressed CRC cells
through RNA-seq

   As GAP43 expression is silenced in CRC cells, in order to investigate
   the transcriptome alterations, we performed RNA-seq on
   GAP43-overexpressed SW620 cells and control SW620 cells. For
   transcriptome analysis, we acquired valid RNA-seq data from stable
   transfected GAP43 (29,592,934 bp and 25,180,042 bp) and control
   (28,506,301 bp and 27,436,701 bp) SW620 cells with Q30 > 90%,
   respectively (Supplementary Table S[76]1). For more accurate results,
   the RNA-seq data was filtered with estimated FPKM values less than 1.0.
   Ultimately, with statistical analysis (Volcano plot), we found that the
   transcriptome of stable transfected GAP43 were significantly different
   from control SW620 cells (Fig. [77]3a). Totally 1056 differentially
   expressed genes (DEGs) were identified in stable transfected GAP43
   compared with control SW620 cells. Among these genes, 474 genes were
   upregulated and 582 genes were downregulated, including upregulated
   FILP1L (WNT suppressor) [[78]15], downregulated KRT6A (wound healing
   and epithelial migration) [[79]16] and S100A14 (cell proliferation and
   cell migration) [[80]17–[81]20], et al. (Supplementary Table S[82]2)
   (p-value< 0.05). The heatmap for some significantly altered genes by
   overexpressed GAP43 was shown in Fig. [83]3b. According to cellular
   functional categorization, significantly upregulated genes were
   classified as follows: transport (p-value = 2.60*E-04); glucose
   homeostasis (p-value = 5.51*E-04); retinoid metabolic process
   (p-value = 8.78*E-04); peripheral nervous system development
   (p-value = 8.93*E-04); extracellular matrix organization
   (p-value = 1.05*E-03). The significantly downregulated genes were
   classified as follows: translation (p-value = 8.02*E-56); SRP-dependent
   cotranslational protein, targeting to membrane (p-value = 2.98*E-54);
   nuclear-transcribed mRNA catabolic process, nonsense-mediated decay
   (p-value = 3.62*E-50); rRNA processing (p-value = 1.92*E-49);
   translational initiation (p-value = 7.24*E-49) (Supplementary Table
   S[84]3).

Fig. 3.

   [85]Fig. 3
   [86]Open in a new tab

   Heatmap and volcano plot. a The volcano plot was constructed by
   plotting the negative log of the log10 FDR value on the y-axis. These
   results in data points with low log10 FDR values (highly significant)
   appearing toward the top of the plot. The x-axis is the log (fold
   change) between GAP43-overexpressed and control SW620 cells. b The
   heatmap of up- and down-regulated genes in red and green, respectively

Transcriptome analyses of GAP43-overexpressed CRC cells through DAVID and IPA

   After bioinformatics analysis of RNA-seq data, the upregulated or
   downregulated genes in GAP43 stable transfected SW620 cells were
   summarized. We submitted these genes to DAVID (a web-based
   high-throughput functional genomics analysis tool) for systematically
   clustering these genes. In GOTERM_BP_FAT category, we identified
   significantly enriched GO terms that characterized GAP43 stable
   transfected SW620 cells. In total, 24 subcategories (p-value< 0.01)
   were found among upregulated genes and 38 subcategories (p-value< 0.01)
   among downregulated genes (Supplementary Table S[87]3). Among the
   up-regulated genes, we found that cell membrane proteins were
   significantly enriched, such as transport (p-value = 2.60*E-04) and
   extracellular matrix organization (p-value = 0.001); while among
   down-regulated genes, protein translation related genes were
   significantly enriched. In KEGG_PATHWAY category, we found that the
   upregulated genes were clustered into 6 subcategories (p-value< 0.05, 1
   subcategories with p-value< 0.01), including ABC transporters
   (p-value = 0.001) and ECM-receptor interaction (p-value = 0.019)
   (Fig. [88]4a). While the down-regulated genes were clustered into 12
   subcategories (p-value< 0.05, 10 subcategories with p-value< 0.01)
   (Fig. [89]4b), including ribosome (p-value = 2.98*E-62); biosynthesis
   of antibiotics (p-value = 6.87*E-08); biosynthesis of amino acids
   (p-value = 3.11*E-05); arginine and proline metabolism
   (p-value = 1.35*E-04); metabolic pathways (p-value = 1.61*E-04)
   (Supplementary Table S[90]4). These results showed that overexpressed
   GAP43 may influence the membrane transporters, and then repress the
   cancer cells’ metabolism and cause the repression of proliferation,
   invasion and migration effects in CRC cells.

Fig. 4.

   [91]Fig. 4
   [92]Open in a new tab

   Functional pathway enrichment analysis. a The upregulated DEGs or b
   down-regulated DEGs are involved in various KEGG biological pathways

   The expression levels of the most significant DEGs in RNA-seq data were
   confirmed by qRT-PCR in GAP43-overexpressed and control SW620 cells
   (Fig. [93]5). Furthermore, we applied these DEGs into IPA analysis, and
   we found that the most significant is the EIF2 signaling pathway
   (−log(p-value) = 25.5 and z-score = − 4.802) (Fig. [94]6a) [[95]21,
   [96]22]. In EIF2 signaling pathway, the translation initiation factor
   (eIF1) and transcriptional factor (ATF4 and ATF5) were significantly
   downregulated (Fig. [97]6b). These results suggested that except the
   function on membrane, GAP43 may also involve in the regulation of
   cellular translation through the EIF2 signaling pathway.

Fig. 5.

   Fig. 5
   [98]Open in a new tab

   The expression levels of the most significant DEGs in RNA-seq data were
   confirmed by qRT-PCR in GAP43-overexpressed and control SW620 cells

Fig. 6.

   [99]Fig. 6
   [100]Open in a new tab

   IPA analysis and EIF2 signaling pathway. a IPA analysis. b EIF2
   signaling pathway

Discussion

   In previous studies, the functional studies of GAP43 were focused on
   the neuronal development, and the protein was considered to play
   crucial roles in effective regenerative responses, and learning and
   memory processes of the nervous system [[101]23–[102]25]. GAP43 is
   highly expressed in nervous system, and also biasedly expressed in
   indigestive tissues, including duodenum, small intestine, colon and
   appendix. Recent study has shown that GAP43 serves as a specific
   biomarker of relapsed or refractory neuroblastoma [[103]26, [104]27].
   However, the relationship between GAP43 and CRC development has been
   uncovered. In this study, we confirmed that GAP43 is expressed in human
   colon tissues, but is downregulated in CRC cells and tissues due to the
   methylation of its promoter region.

   Aberrant epigenetic changes frequently occur in human colorectal
   cancer. The accumulation of numerous epigenetic alterations may drive
   the initiation and progression through the adenoma-carcinoma sequence
   [[105]28, [106]29]. We found the expression pattern of GAP43 in
   colorectal cancer cells was closely associated with cancer-linked DNA
   hypermethylation, and the methylation status of GAP43 promoter did not
   change in different colorectal cancer cell lines (Fig. [107]2c).
   Additionally, we found that the expression level of GAP43 in
   immortalized colorectal epithelial cell line NCM460 was significantly
   higher than those in CRC cell lines, but it was dramatically reduced
   compared to normal colorectal tissues (Fig. [108]1b). Thus, when
   comparing the GAP43 gene expression patterns in a series of CRC cell
   lines, immortalized colorectal epithelial cell line, tumor cells and
   normal cells, we deduced that the accumulation of aberrant methylation
   of GAP43 gene might drive the initiation of CRC.

   Further transcriptome analysis indicated multiple potential biological
   functions of GAP43 in CRC. The most significant different expression
   gene is upregulated FILIP1L, downregulated KRT6A and S100A14. While
   FILIP1L is a WNT pathway inhibitor [[109]15], and the reduction of
   FILIP1L in ovarian cancer is associated with poor survival, progression
   and chemoresistance [[110]30]. Previous studies showed that KRT6A and
   S100A14 were involved in cell migration and cell proliferation
   [[111]16, [112]17, [113]20]. These results remind us that the reduced
   level of GAP43 in CRC due to its transcriptional alterations might
   regulate some potential signaling pathways associated with
   tumorigenesis, such as WNT signaling.

   In KEGG_PATHWAY category, we found that the upregulated genes were
   clustered into 6 subcategories including ATP-binding cassette (ABC)
   transporters (p = 0.001). ABC transporters constitute a superfamily of
   membrane proteins that couple the hydrolysis of adenosine triphosphate
   (ATP) to vectorial transport of substrates across biological membranes
   [[114]31]. Previous studies have showed that ABC superfamily utilizes
   the free-energy from ATP hydrolysis to shuttle many different
   substrates across various biological membranes, and consequently, are
   involved in both normal and abnormal physiology [[115]32]. In this
   study, we found that with the overexpression of GAP43 in CRC cells, the
   expression of ABC transporters is also upregulated. However, the
   expression of bothGAP43 and ABC transporters is down-regulated in CRC
   tissues and cell lines, which indicates that ABC transporters might be
   related with the development of CRC.

   In addition, we analyzed the transcriptome of GAP43 overexpressed CRC
   cells through DAVID and IPA. The IPA analysis showed that EIF2
   signaling pathway is the most significant psychological phenomena
   (−log(p-value) =25.5 and z-score = − 4.802) (Fig. [116]6a,
   Supplementary Table S[117]5), and three important members (eIF1, ATF4,
   ATF5) of EIF2 signaling pathway were significantly downregulated (Fig.
   [118]6b). Incidentally, we also found that GAP43 is differentially
   expressed, elevated or decreased in gastric cancer. Survival analysis
   showed that there was no significant change in survival of gastric
   cancer patients with low or high expression of GAP43 (Fig. S[119]4).
   However, we found that patients with gastric cancer who were treated
   with the 5-FU anticancer drug had higher survival rates in patients
   with high GAP43 expression than those with low expression (Fig.
   S[120]4). This suggests that GAP43 is likely to be associated with drug
   resistance to cancer chemotherapy drugs. Considering that the tolerance
   of chemotherapy drugs is related with ABC transporters, and GAP43 level
   is associated with the expression of ABC transporters. Therefore, GAP43
   might affect the chemotherapy drug resistance in cancer patients
   through ABC transporter-mediated signaling pathway.

Conclusions

   GAP43 is expressed in human indigestive tissues, including colon
   tissues, but is downregulated in CRC due to the methylation of its
   promoter region. The RNA-Seq transcriptome data analysis showed the
   gene expression profile between GAP43-overexpressed and control CRC
   cells. The analysis of GAP43-overexpressed versus control CRC cell line
   data gives insight into potential genes and pathways that may uncover
   the functional roles of GAP43 in the development of human CRC and
   expand the knowledge of the CRC progression.

Supplementary Information

   [121]12885_2020_7728_MOESM1_ESM.pdf^ (1,006.3KB, pdf)

   Additional file 1: Supplementary Figure S1. Mutation and expression
   level analysis of GAP43 in CRC tissues with online database. a
   Expression of GAP43 in CRC (COAD and READ). b Mutation level of GAP43
   in CRC tissues with cBioPortal database. c Expression level of GAP43 in
   CRC tissues with Oncomine database.
   [122]12885_2020_7728_MOESM2_ESM.pdf^ (1.3MB, pdf)

   Additional file 2: Supplementary Figure S2. Correlation analysis of
   GAP43 expression level and its promoter methylation level in colon
   adenocarcinoma (a) and rectal adenocarcinoma (b).
   [123]12885_2020_7728_MOESM3_ESM.pdf^ (270.8KB, pdf)

   Additional file 3: Supplementary Figure S3. Bisulfite genomic
   sequencing was performed to analyze the methylation status of the ABCC1
   (a), ABCG2 (b), HAND1 M1 (c) and HAND1 M2 (d) promoters in CRC cell
   line HCT116. Multiple CpG islands in the promoter regions were
   analyzed.
   [124]12885_2020_7728_MOESM4_ESM.pdf^ (445.4KB, pdf)

   Additional file 4: Supplementary Figure S4. Kaplan-Meier Plotter
   survival analysis of GAP43 in gastric cancer with or without different
   treatments. a gastric cancer. b gastric cancer with surgery only. c
   gastric cancer with 5-FU based adjuvant.
   [125]12885_2020_7728_MOESM5_ESM.xlsx^ (11KB, xlsx)

   Additional file 5: Supplementary Table S1. Quality Control (QC) of our
   RNA-seq data.
   [126]12885_2020_7728_MOESM6_ESM.xlsx^ (210.7KB, xlsx)

   Additional file 6: Supplementary Table S2. GAP43 overexpression related
   genes revealed by RNA-seq. (p-value< 0.05).
   [127]12885_2020_7728_MOESM7_ESM.xlsx^ (26.2KB, xlsx)

   Additional file 7: Supplementary Table S3. DAVID analysis (GO_BP) of
   GAP43 overexpression related genes (p-value < 0.05).
   [128]12885_2020_7728_MOESM8_ESM.xlsx^ (12.4KB, xlsx)

   Additional file 8: Supplementary Table S4. DAVID analysis (KEGG) of
   GAP43 overexpression related genes (p-value < 0.05).
   [129]12885_2020_7728_MOESM9_ESM.xlsx^ (27.7KB, xlsx)

   Additional file 9: Supplementary Table S5. IPA Canonical Pathways of
   GAP43 overexpression related genes.
   [130]12885_2020_7728_MOESM10_ESM.xlsx^ (10.8KB, xlsx)

   Additional file 10: Supplementary Table S6. Clinical Information of
   three CRC samples used in expression analysis of GAP43.
   [131]12885_2020_7728_MOESM11_ESM.xlsx^ (11.2KB, xlsx)

   Additional file 11: Supplementary Table S7. Primers for qRT-PCR.

Acknowledgements