GREM2 maintains stem cell-like phenotypes in gastric cancer cells by regulating the JNK signaling pathway

Abstract

Gastric cancer (GC) is one of the major malignancies worldwide. This study was conducted to explore the mechanism by which GREM2 maintains biological properties of GC stem cells (GCSCs), and proved that GREM2 could potentially regulate the proliferation, apoptosis, invasion, migration and tumorigenic ability of GCSCs through the regulation of the JNK signaling pathway. In silico analysis was utilized to retrieve expression microarray related to GC, and differential analysis was conducted. The cell line with the highest GREM2 expression was overexpressed with GREM2 mimic, silencing GREM2 by siRNA, or treated with activator or inhibitor of the JNK signaling pathway. Subsequently, expression of GREM2, JNK signaling pathway-, apoptosis- or migration and invasion-associated factors were determined. Proliferation, migration, invasion, apoptosis of GCSCs in vitro and tumorigenic ability and lymph node metastasis of GCSCs in vivo were determined. Based on the in silico analysis of GSE49051, GREM2 was determined to be overexpressed in GC and its expression was the highest in the MKN-45 cell line, which was selected for the subsequent experiments. Silencing of GREM2 or inhibition of the JNK signaling pathway suppressed the proliferation, migration and invasion, while promoting apoptosis of GCSCs in vitro as well as inhibiting tumorigenesis and lymph node metastasis in vivo. In conclusion, the aforementioned findings suggest that the silencing of GREM2 suppresses the activation of the JNK signaling pathway, thereby inhibiting tumor progression. Therefore, GREM2-mediated JNK signaling pathway was expected to be a new therapeutic strategy for GC.

Keywords: GREM2; JNK signaling pathway; biological properties; gastric cancer stem cells.

Figure 1.

GREM2 was overexpressed in GC. A total of 2535 differentially expressed genes were screened out from GC microarray GSE49051, including 856 highly expressed genes and 1679 poorly expressed genes, and 50 genes with greater fold changes were selected. Furthermore, GO functional enrichment analysis was performed on the 2535 DEGs obtained from the analysis. A, Differential analysis of GC expression microarray. The horizontal coordinate represents the samples, and the vertical coordinate represents the genes. The upper dendrogram represents sample type clustering. The left dendrogram represents the clustering results among genes; each square indicates the expression of a gene in a sample. The upper right histogram color indicates the color gradation. B: GO enrichment analysis. The three bar charts represent the enrichment results of BP, CC and MF, respectively. The horizontal coordinate represents the name of the GO item and the vertical coordinate represents the number of genes. GREM, gremlin; GO, gene ontology; BP, biological process; CC, cellular component; MF, molecular function.

Figure 2.

The MKN-45 cell line had the highest expression of GREM2. A, mRNA expression of GREM2 in human gastric mucosa epithelial cell line GES-1 and human GC cell lines AGS, SGC-7901, MKN-28, MKN-45 and MKN-74 detected by RT-qPCR. B and C, protein expression of GREM2 in human gastric mucosa epithelial cell line GES-1 and human GC cell lines AGS, SGC-7901, MKN-28, MKN-45 and MKN-74 detected by western blot analysis. *, p < 0.05 vs. the GES-1 cells; #, p < 0.05 vs. the MKN-45 cells. The data of RT-qPCR were the measurement data, expressed as mean value of standard error; multiple groups were compared by one-way analysis of variance; the experiment was repeated 3 times; GREM, gremlin; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Figure 3.

SP cells were increased and positive rate of CD44⁺ was upregulated in GCSCs. A, flow cytometry analysis of SP cells; B, histogram analysis of SP cells; C, immunofluorescence figure of immunomagnetic bead CD44⁺ sorting (400 ×); D, histogram of CD44⁺ positive rate of cells; *, p < 0.05 vs. the MKN-45 cells. The number of negative or weak positive staining of Hoechst33342 and the rate of CD44⁺ positive cells were taken as the measurement data, which were expressed as the mean value of standard error; multiple groups were compared by one-way analysis of variance; the experiment was repeated 3 times; SP, side population.

Figure 4.

The proliferation of GCSCs was diminished by si-GREM2 or JNK inhibitor. a, EdU chromatogram of each group (400 ×); b, proliferation of GCSCs in response to the treatment of GREM2, siRNA-GREM2, SP600125, or Juglanin; c, cell cycle distribution of cells in each group; d, histogram of cell cycle in each group; *, p< 0.05 vs. the blank group; the transfection values and cell cycle percentage of cells in each group were taken as the measurement data, and expressed as mean value of standard error; multiple groups were compared by one-way analysis of variance; the experiment was repeated 3 times; GREM, Gremlin; EdU, 5-ethynyl-2ʹ-deoxyuridine; JNK, c-Jun NH2-terminal kinase.

Figure 5.

Depletion of GREM2 or the JNK signaling pathway could accelerate the apoptosis of GCSCs. (a), Hoechst33528 staining of cells in each group (400 ×); (b), apoptosis rate analysis for GCSCs in response to the treatment of GREM2, siRNA-GREM2, SP600125, or Juglanin; (c), the mRNA levels of Bcl-2 and Bax of GCSCs in response to the treatment of GREM2, siRNA-GREM2, SP600125, or Juglanin determined by RT-qPCR; (d), the grey value and protein expression of Bcl-2 and Bax of GCSCs in response to the treatment of GREM2, siRNA-GREM2, SP600125, or Juglanin determined by western blot analysis; *, p< 0.05 vs. the blank group; the number of apoptotic cells and values of the RT-qPCR as well as western blot analysis after transfection in each group were taken as the measurement data, expressed as mean value of standard error; multiple groups were compared by one-way analysis of variance; the experiment was repeated three times; GREM, gremlin; JNK, c-Jun NH2-terminal kinase; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Figure 6.

Si-GREM2 or the JNK signaling pathway reduction inhibits the sphere-forming ability of GCSCs. (a), the number of sphere cells in GCSCs in response to the treatment of GREM2 overexpression, siRNA-GREM2, SP600125, or Juglanin; (b), cell colony formation of GCSCs in response to the treatment of GREM2, siRNA-GREM2, SP600125, or Juglanin; (c), histogram of cell colony number of GCSCs in response to the treatment of GREM2, siRNA-GREM2, SP600125, or Juglanin; (d), the mRNA expression of genes (Sox-2, Nanog, and Oct-4) related to stemness of GCSCs detected by RT-qPCR; (E) and (F), the protein expression of genes (Sox-2, Nanog, and Oct-4) related to stemness of GCSCs detected by western blot analysis. *, p< 0.05 vs. the blank group; the sphere numbers and cell colonies after transfection were taken as the measurement data, represented by mean value of standard error; multiple groups were compared by one-way analysis of variance; the experiment was repeated three times; GREM, gremlin; JNK, c-Jun NH2-terminal kinase; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Figure 7.

The migration and invasion of GCSCs could be suppressed via si-GREM2 or inhibition of the JNK signaling pathway. (a), the scratch width of GCSCs in response to the treatment of GREM2, siRNA-GREM2, SP600125, or Juglanin; (b), statistical analysis of cell migration of GCSCs in response to the treatment of GREM2, siRNA-GREM2, SP600125, or Juglanin; (c), representative figures of invading cells by Transwell assay (200 ×); (d), average invasion cells from three independent experiments; (e), the mRNA levels of MMP-2 and MMP-9 of GCSCs in response to the treatment of GREM2, siRNA-GREM2, SP600125, or Juglanin determined by RT-qPCR; (f), the grey value of MMP-2 and MMP-9 protein bands of GCSCs in response to the treatment of GREM2, siRNA-GREM2, SP600125, or Juglanin; (g), the protein levels of MMP-2 and MMP-9 of GCSCs in response to the treatment of GREM2, siRNA-GREM2, SP600125, or Juglanin determined by western blot analysis; *, p < 0.05 vs. the blank group; measurement data were represented by mean value of standard error; multiple groups were compared by one-way analysis of variance; the experiment was repeated three times; GREM, gremlin; JNK, c-Jun NH2-terminal kinase; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Figure 8.

Si-GREM2 served as an inhibitor of activation of the JNK signaling pathway. (a), the mRNA levels of GREM2, JNK and c-jun of GCSCs in response to the treatment of GREM2, siRNA-GREM2, SP600125, or Juglanin determined by RT-qPCR; (b), the grey value of GREM2, JNK, c-jun, p-JNK and p-c-jun protein bands of GCSCs in response to the treatment of GREM2, siRNA-GREM2, SP600125, or Juglanin; (c), the protein levels of GREM2, JNK, c-jun and the extent of JNK and c-jun phosphorylation of GCSCs in response to the treatment of GREM2, siRNA-GREM2, SP600125, or Juglanin determined by western blot analysis; *, p < 0.05 vs. the blank group; the results of RT-qPCR and western blot analysis were taken as the measurement data, and were expressed as mean value of standard error; multiple groups were compared by one-way analysis of variance; the experiment was repeated three times; GREM, gremlin; JNK, c-Jun NH2-terminal kinase; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Figure 9.

Tumorigenic ability of GCSCs in nude mice was inhibited by si-GREM2 or inhibited JNK signaling pathway. (a), subcutaneous transplanted tumor of MKN-45 cells in nude mice; (b), tumor weight of nude mice implanted subcutaneously with MKN-45 cells in each group; (c), tumor volume of nude mice implanted subcutaneously with mkn-45 cells in each group *, p < 0.05 vs. the blank group. The tumor size and weight of nude mice with subcutaneous implantation were measurement data, expressed as mean value of standard error. Data comparison was conducted with one-way analysis of variance (n = 5). GREM, gremlin; JNK, c-Jun NH2-terminal kinase.

Figure 10.

Si-GREM2 or the JNK signaling pathway inhibitor prohibited the metastasis of lymph node in GCSCs. (a), HE staining results of xenograft tumor in nude mice implanted subcutaneously with MKN-45 cells in each group (100 ×); (b), statistical chart of positive lymph node metastasis of MKN-45 cells in each group; *, p < 0.05 vs. the blank group. The value of lymph node metastasis of xenograft tumor in each group was taken as the measurement data, represented by mean value of standard error, and one-way analysis of variance was used for data analysis; n = 5; HE, hematoxylin and eosin; GREM, gremlin; JNK, c-Jun NH2-terminal kinase.

Figure 11.

Map of molecular mechanisms involved in GREM2 regulation in migration, invasion and apoptosis of GCSCs. In GC, the expression of GREM2 was significantly increased, which therefore accelerated the activation of the JNK signaling pathway, and increased expression of JNK, c-jun phosphorylation to promote the expression of MMP-2, MMP-9, Bcl-2 and inhibit the expression of Bax. Ultimately, the migration and invasion of GC would be promoted and the apoptosis of GCSCs would be inhibited.