MIR4435-2HG in exosomes promotes gastric carcinogenesis by inducing M2 polarization in macrophages

Abstract

Gastric cancer (GC) is a cancer with a high mortality rate. lncRNAs play a role in regulating GC tumorigenesis. In this paper, we analyzed differentially expressed lncRNAs between GC and adjacent normal tissues using multiple bioinformatics tools to identify new potential targets in GC. Cell viability and migration ability were detected using the Cell Counting Kit-8 (CCK-8) and transwell assays, MIR4435-2HG was negatively correlated with the survival rate of GC patients, and by inhibiting the activity of MIR4435-2HG, the viability and migration ability of GC cells could be reduced. In addition, RT- qPCR and western blot to detect gene and protein level expression, transmission electron microscopy and nanoparticle tracking analysis (NTA) to study the efficiency of exosome isolation, and flow cytometry to observe cell differentiation were employed, delivery of MIR4435-2HG shRNA via MKN45 cell-derived exosomes significantly reversed the MKN45 exosome-induced M2 polarization in macrophages. Furthermore, the low expression of MIR4435-2HG in MKN45 cell-derived exosomes inhibited the Jagged1/Notch and JAK1/STAT3 pathways in macrophages; MIR4435-2HG downregulated exosomes were found to significantly inhibit GC tumor growth in vivo by establishing a mouse model. In short, MKN45 cell-derived exosomes deliver lncRNA MIR4435-2HG, which promotes gastric carcinogenesis by inducing macrophage M2 polarization.

Keywords: GC; JAK1/STAT3; Jagged1/Notch; MIR4435-2HG; MKN45.

Figure 1

Macrophage M2 polarization pathway mapped based on our findings.

Figure 2

Differential analysis of TCGA dataset for gastric cancer. (A) Differential expression results of the TCGA dataset; yellow indicates upregulated genes, blue indicates downregulated genes. (B). Analysis of MIR4435-2HG expression in gastric cancer tissues and normal tissues adjacent to cancer, blue indicates tumor, and red indicates normal tissue. *** indicates P<0.001.

Figure 3

MIR4435-2HG expression results. (A) Expression of MIR4435-2HG in GES-1, MKN45, AGS and SNU-5 cells by RT-Qpcr. (B) MKN45 or AGS cells were transfected with MIR4435-2HG OE. Then, the expression of MIR4435-2HG in GC cells was detected by RT-qPCR. (C) MKN45 or AGS cells were transfected with MIR4435-2HG shRNA1 or shRNA2. Then, the expression of MIR4435-2HG in GC cells was detected by RT-qPCR. (D, E) The viability of gastric cancer cells was tested by CCK8 assay. **P < 0.01 compared with control (OE-NC; shRNA-NC).

Figure 4

Overexpression of MIR4435-2HG increased the migration of gastric cancer cells. (A, B) The migration of gastric cancer cells was measured by the transwell assay. (C) The protein levels of E-cadherin, N-cadherin, and vimentin in GC cells were detected by western blot. **P < 0.01 compared with the control group.

Figure 5

Results of exosome extraction and identification. (A) The separation efficiency of exosomes was examined by TEM. The particle sizes of exosomes were measured by NTA. (B) The expressions of TSG101 and CD63 in GES-1, MKN45 cells or exosomes derived from GES-1 or MKN45 cells were detected by WB. (C) The levels of MIR4435-2HG in exosomes derived from GES-1 or MKN45 cells were measured by RT-qPCR. (D) The expression of TSG101 in exosomes derived from MKN45 cells was detected by western blot. (E) The expression of MIR4435-2HG in MKN45 or exosomes derived from MKN45 cells was detected by RT-qPCR. (F) THP-1 cells were treated with 100 ng/ml of PMA and co-cultured with MKN45 cell-derived exosomes, and the location of exosomes was observed by immunofluorescence staining. (G) Macrophages were co-cultured with MKN45-Exo-OE NC, MKN45-Exo-MIR4435-2HG OE, MKN45-Exo-shRNA NC, or MKN45-Exo-MIR4435-2HG shRNA2. Then, the expression of MIR4435-2HG in macrophages was detected by RT-qPCR. **P < 0.01 compared with the control group.

Figure 6

Exosome MIR4435-2HG promotes M2 polarization in macrophages. (A, B) Macrophages were co-cultured with MKN45-Exo-NC, MKN45-Exo-MIR4435-2HG OE, or MKN45-Exo-MIR4435-2HG shRNA2 for 24 h, and then the distribution rate of CD86 or CD206 in macrophages was detected by flow cytometry. (C) The levels of Arginase-1 and iNOS in macrophages were tested by RT-qPCR. (D) The levels of TGF-β and IL-10 in supernatants of macrophages were detected by ELISA. **P < 0.01.

Figure 7

Exosome-induced M2 macrophages promote the migration of gastric cancer cells by inhibiting the EMT process. (A) MKN45 cells were co-cultured with macrophages, ExoNC-treated macrophages, Exo-MIR4435-2HGOE-treated macrophages, and Exo-MIR4435-2HGshRNA2-treated macrophages for 24 h, and the migration of MKN45 cells was detected by the transwell method. (B) The morphology of MKN45 cells was observed under a microscope. (C) The E-cadherin, N-cadherin, and vimentin protein levels in macrophages were detected by western blot. **P < 0.01.

Figure 8

Exosomes from MKN45 cells promote M2 polarization in macrophages by regulating the Jagged1/Notch and JAK1/STAT3 axes. (A) The Jagged1, Notch1, Notch2, Hes1, and Hes5 protein levels in macrophages were detected by WB. (B) WB detection of p-JAK1, JAK1, p-STAT3, and STAT3 protein levels in macrophages.

Figure 9

M2 m acrophages induced by exosomes significantly promotes the tumor growth of gastric cancer. (A) Tumor volumes of nude mice were tested. (B) Tumor tissues of nude mice were pictured. (C) Tumor weights of nude mice were measured. (D) The expression of E-cadherin, Ncadherin, and vimentin in tissues of nude mice were detected by WB. **P < 0.01 compared with MKN45.