RHOJ Induces Epithelial-to-Mesenchymal Transition by IL-6/STAT3 to Promote Invasion and Metastasis in Gastric Cancer

Abstract

Background: Recently, the molecular classification of gastric cancer (GC) promotes the advances of GC patients' precision therapy and prognosis prediction. According to the Asian Cancer Research Group (ACRG), GC is classified as microsatellite instable (MSI) subtype GC, microsatellite stable/epithelial-to-mesenchymal transition (MSS/EMT) subtype GC, MSS/TP53- subtype GC, and MSS/TP53+ subtype GC. Due to the easy metastasis of EMT-subtype GC, it has the worst prognosis, the highest recurrence rate, and the tendency to occur at a younger age. Therefore, it is curious and crucial for us to understand the molecular basis of EMT-subtype GC. Methods: The expression of RHOJ was detected by quantitative real-time PCR (qPCR) and immunohistochemistry (IHC) in GC cells and tissues. Western blotting and immunofluorescence (IF) were conducted to examine the effects of RHOJ on the EMT markers' expression of GC cells. The GC cells' migration and invasion were investigated by transwell assay. The tumor growth and metastasis were demonstrated correspondingly in different xenograft models. Results: Firstly, it was noticed that RHOJ was significantly upregulated in EMT-subtype GC and RHOJ has close relationships with the EMT process of GC, based on the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) databases. Next, transwell assay and tail vein metastasis models were conducted to verify that RHOJ mediates the EMT to regulate the invasion and metastasis of GC in vitro and in vivo. In addition, weakened tumor angiogenesis was observed after RHOJ knockdown by the angiogenesis assay of HUVEC. RNA-seq and further study unveiled that RHOJ aggravates the malignant progression of GC by inducing EMT through IL-6/STAT3 to promote invasion and metastasis. Finally, blocking the IL-6/STAT3 signaling overcame RHOJ-mediated GC cells' growth and migration. Conclusions: These results indicate that the upregulation of RHOJ contributes to EMT-subtype GC invasion and metastasis via IL-6/STAT3 signaling, and RHOJ is expected to become a promising biomarker and therapeutic target for EMT-subtype GC patients.

Keywords: EMT; Gastric Cancer; IL-6/STAT3 signaling; Metastasis; RHOJ.

Figure 1

Upregulated in EMT-subtype GC, RHOJ is correlated with poor GC prognosis. (A) Kaplan-Meier analysis showed the patients' overall survival (OS) and disease-free survival (DFS) in the four GC subtypes of ACRG, according to the GSE62254 dataset. (B) Heatmap of different expression patterns of EMT-subtype GC-related genes in the four GC subtypes of ACRG. (C) In the GSE62254 dataset, RHOJ (labeled by seven independent molecular probes) expression levels in the four GC subtypes of ACRG. (D) Kaplan-Meier analysis showed the OS and DFS in RHOJ high expression group and low expression group GC patients, according to the GSE62254 dataset. (E) IHC staining tested RHOJ expression levels of the EMT-subtype GC and Non-EMT-subtype GC tissue specimens, scale bar, 50 µm. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data were expressed as Mean±SD.

Figure 2

RHOJ mediates EMT to regulate the migration and invasion of GC cells. (A) Pearson correlation analysis assessed the links between the expression levels of the EMT-related genes (CDH1, VIM, ZEB1, ZEB2, and FN1) with RHOJ in the GSE62254 dataset. (B) RHOJ relative expression levels of GC cells line were assessed by qPCR. (C) Protein levels of RHOJ, ZEB1, SNAI2, N-cadherin, Vimentin, and E-cadherin were detected by western blotting in RHOJ knockdown cells (SGC7901, SNU-1) and RHOJ overexpression cells (SNU-1, MKN-45). (D) IF staining representative images and quantified charts of E-cadherin (Red), Vimentin (Green), and DAPI (Blue) in SNU-1 cells captured by a confocal microscope, magnification, 200× and 800×, scale bar, 40 µm. (E) Representative images and statistical charts of transwell migration or invasion assay in RHOJ knockdown cells (SGC7901, SNU-1), scale bar, 100 µm. (F) Representative images and statistical charts of transwell migration or invasion assay in RHOJ overexpression cells (SNU-1, MKN-45), scale bar, 100 µm. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data were expressed as Mean±SD.

Figure 3

RHOJ promotes the metastasis of GC through EMT in vivo. (A) Representative photographs of mice lung metastatic foci (SGC7901, SNU-1) in the tail vein metastasis model. (B) Statistics on the number of mice lung surface metastatic foci from (A). (C) Representative images of HE staining from (A), scale bar, 200 µm. (D) Representative photographs of mice liver metastatic foci (SGC7901) in the tail vein metastasis model. (E) Statistics on the number of mice liver surface metastatic foci from (D). (F) Mice liver metastasis rate of GC cells (SGC7901) in the tail vein metastasis model. (G) Representative images of HE staining from (D), tumor tissues and normal tissues were noted as T and N, respectively, scale bar, 200 µm. (H) IHC staining representative images of E-cadherin and Vimentin in mice lung metastatic foci, scale bar, 50 µm. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data were expressed as Mean±SD.

Figure 4

RHOJ enhances the angiogenesis of GC. (A) Pathway enrichment analysis of the genes highly related to RHOJ expression, based on the GSE62254 dataset. (B) Pathway enrichment analysis of the genes highly related to RHOJ expression, based on the TCGA database. (C) Pearson correlation analysis evaluated the links between the expression levels of PECAM1 (CD31) with RHOJ in the GSE62254 dataset. (D) Pearson correlation analysis evaluated the links between the expression levels of PECAM1 (CD31) with RHOJ in the TCGA database. (E) Representative images and statistical charts of the formed tubes in HUVEC angiogenesis assay, scale bar, 200 μm. (F) Protein levels of RHOJ and VEGFA were detected by western blotting in RHOJ knockdown cells (SGC7901, SNU-1). (G) IHC staining representative images of CD31 in mice lung metastatic foci, scale bar, 50 μm. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data were expressed as Mean±SD.

Figure 5

RHOJ facilitates GC cells' proliferation and tumor growth. (A) Colony-forming assay measured the proliferation ability of RHOJ knockdown cells (SGC7901, SNU-1). (B) CCK-8 assay assessed the viability of RHOJ knockdown cells (SGC7901, SNU-1) at 0 h, 72 h, 96 h, and 120 h, respectively. (C) Representative images and statistical charts of EdU proliferation assay in RHOJ knockdown cells (SGC7901, SNU-1), scale bar, 100 µm. (D) Colony-forming assay measured the proliferation ability of RHOJ overexpression cells (SNU-1, MKN-45). (E) CCK-8 assay assessed the viability of RHOJ overexpression cells (SNU-1, MKN-45) at 0 h, 48 h, 72 h, and 96 h, respectively. (F) Cell counting assay recorded the growth of RHOJ overexpression cells (SNU-1, MKN-45). (G) Representative photographs of mice back tumors (SGC7901, SNU-1) in the subcutaneous xenograft model. (H) The volumes of mice back tumors were recorded weekly with Vernier calipers. (I) Tumors were weighed and counted in each group from (G). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data were expressed as Mean±SD.

Figure 6

RHOJ induces S and G2/M phase transition and inhibits GC cells' apoptosis. (A) Flow cytometry analyzed the cell cycles of RHOJ knockdown cells (SGC7901 and SNU-1) and displayed the percentage in the G1, S, and G2/M phases of each group cell with statistical charts. (B) Annexin V-FITC/PI staining assay analyzed the percentage of apoptosis in RHOJ knockdown cells (SGC7901 and SNU-1) and displayed it with statistical charts. (C) Representative images and statistical charts of TUNEL assay in RHOJ knockdown cells (SGC7901, SNU-1), scale bar, 100 µm. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data were expressed as Mean±SD.

Figure 7

RHOJ regulates the EMT of GC via IL-6/STAT3 signaling. (A) Heatmap of the top 50 differentially expressed genes in RHOJ overexpression cells (SNU-1) identified by RNA-seq. (B) GO enrichment analysis of the upregulated genes in RNA-seq data from (A). (C) Relative expression levels of RHOJ, TNF-α, IL-1β, and IL-6 were assessed by qPCR in RHOJ overexpression cells (SNU-1) and RHOJ knockdown cells (SNU-1). (D) IL-6 levels were tested by ELISA in RHOJ overexpression cells (SNU-1) and RHOJ knockdown cells (SNU-1). (E) Protein levels of RHOJ, STAT3, p-STAT3 (Y705), and p-STAT3 (S727) were detected by western blotting in RHOJ knockdown cells (SGC7901, SNU-1) and RHOJ overexpression cells (SNU-1, MKN-45). (F) Treated with different concentrations of Stattic for 96 h, the CCK-8 assay assessed the viability of SNU-1 cells. (G) RHOJ overexpression and control cells (SNU-1) were treated with or without Stattic (5 µM) for 1.5 h, protein levels of RHOJ, STAT3, p-STAT3 (Y705), and p-STAT3 (S727) were detected by western blotting. (H) Representative images and statistical charts of transwell migration assay in RHOJ overexpression and control cells (SNU-1) that were treated with or without Stattic (5 µM), scale bar, 100 µm. (I) Treated with or without Stattic (5 µM) for 96 h, the CCK-8 assay assessed the viability of RHOJ overexpression and control cells (SNU-1). (J) RHOJ overexpression and control cells (SNU-1) were transfected with siRHOJ, protein levels of RHOJ, STAT3, p-STAT3 (Y705), p-STAT3 (S727), Vimentin, and E-cadherin were detected by western blotting. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data were expressed as Mean±SD.

Figure 8

Schematic illustration of RHOJ induces EMT by IL-6/STAT3 to promote invasion and metastasis in GC. RHOJ activates the IL-6 production firstly and enhances the phosphorylation level of STAT3 via IL-6/STAT3 signaling, which induces the EMT process of GC cells and angiogenesis, further promoting GC invasion, metastasis, and tumor growth.