miR-195-5p/NOTCH2-mediated EMT modulates IL-4 secretion in colorectal cancer to affect M2-like TAM polarization

Abstract

Background: Tumor microenvironment (TME) is a complex environment containing tumor cells, tumor-associated macrophages (TAMs), interstitial cells, and non-cellular components. Epithelial-mesenchymal transition (EMT), as a major actor in cancer tumorigenicity and metastasis, was involved in the interaction between TAMs and tumor cells. However, the potential mechanisms of EMT and how EMT-programmed tumor cells affect M2-like TAMs still need further exploration.

Methods: An integrated analysis of nine CRC miRNA expression datasets was performed. Functional assays, including the EdU, clone formation, wound healing, and transwell assays, were used to determine the anticancer role of miR-195-5p in human CRC progression. Furthermore, RNA immunoprecipitation, RNA decay, and dual-luciferase reporter assays were used to determine the mechanism of miR-195-p CRC progression. Then co-culture, migration, and ELISA assays were applied to determine the role of miR-195-5p in macrophage recruitment and alternative polarization. Xenograft mouse models were used to determine the role of miR-195-5p in CRC tumorigenicity and TAM polarization in vivo.

Results: An integrated analysis confirmed that miR-195-5p was significantly downregulated in CRC tissues, and patients with a low level of miR-195-5p had significantly shortened overall survival as revealed by the TCGA-COAD dataset. Altered miR-195-5p in colon cancer cells led to distinct changes of proliferation, migration, invasion, and EMT. Mechanistically, miR-195-5p regulated NOTCH2 expression in a post-transcriptional manner by directly binding to 3'-UTR of the Notch2 mRNA. Subsequently, miR-195-5p/NOTCH2 suppressed GATA3-mediated IL-4 secretion in CRC cells and ultimately inhibited M2-like TAM polarization.

Conclusions: miR-195-5p may play a vital role in regulating NOTCH2-mediated tumor cell EMT, thereby affecting IL-4-related M2-like TAM polarization in CRC.

Keywords: CRC; EMT; IL-4; NOTCH2; TAMs; miR-195-5p.

Fig. 1

miR-195-5p was downregulated in CRC tumor samples with upregulation of NOTCH2. a miR-195-5p expressions in 6 pair CRC samples. b miR-195-5p is significantly decreased in primary human CRC tissues compared with ANT. Mean ± SD is shown. Statistical analysis was conducted using Student’s t test. c The levels NOTCH2 protein in 6 pairs of CRC samples. d NOTCH2 protein is significantly increased in primary human CRC tissues compared with ANT. Mean ± SD is shown. Statistical analysis was conducted using Student’s t test. e Scatter plots showing the negative correlation between miR-195-5p and NOTCH2 protein levels

Fig. 2

miR-195-5p inhibits HCT116 and DLD-1 cell proliferation, migration, and invasion in vitro. a, b Representative photomicrographs and quantifications of clone formation assay in DLD1 and HCT116 cells after transfection with miR-195-5p mimic, miR-195-5p mimic NC, miR-195-5p inhibitor, or miR-195-5p inhibitor NC for 48 h. c, d Representative photomicrographs and quantifications of EdU immunofluorescence staining assay in DLD1 and HCT116 cells. Bar = 50 μm. e, f Photomicrographs and quantifications of wound healing assay. Bar = 100 μm. g Transwell migration assays of DLD1 and HCT116 cells carrying different miRNAs. Bar = 100 μm. h Total number of cells in five fields was counted manually. i Transwell invasion assays of DLD1 and HCT116 cells. Bar = 100 μm. j Total number of cells in five fields was counted manually. Mean ± SD are shown. Statistical analysis was conducted using one-way ANOVA. *P < 0.05. **P < 0.01. ***P < 0.001

Fig. 3

miR-195-5p affects colon cancer cell EMT by modulating NOTCH2 expression in a post-transcriptional manner. Western blots of NOTCH2, Ad-NOTVH2, and EMT markers Vimentin and E-Cadherin in DLD1 and HCT116 cells transfected with different miRNAs (b) and quantifications of NOTCH2 protein (c). Assays were performed in triplicates. A schematic representation of the pmiR-RB-REPORTTM dual-luciferase reporter vector with 3′-UTR of Notch2 mRNA harbors two miR-195-5p cognate sites (d). Relative luciferase activity of reporter plasmids carrying wild-type or mutant Notch2 3′-UTR in DLD1 and HCT116 cells co-transfected with miR-195-5p mimic or mimic negative control (mimic NC) (e). Notch2 mRNA decay curves of HCT116 cells carrying miR-195-5p mimic or mimic NC (f). The decay model was fit in a one-phase exponential decay model. AGO2-RNA immunoprecipitation assay (RIP) showing that miR-195-5p interacted with NOTCH2 in HCT116 cells (g). (**P < 0.01, ***P < 0.001)

Fig. 4

Silenced NOTCH2 abrogates the role of the miR-195-5p inhibitor in CRC cell clone formation, proliferation, migration, and invasion abilities. Representative photomicrographs (a) and quantifications (b) of clone formation assay in HCT116 cells after transfection with the si-NOTCH2, miR-195-5p inhibitor, or si-NOTCH2 together with the miR-195-5p inhibitor for 48 h. Representative photomicrographs (c) and quantifications (d) of EdU immunofluorescence staining assay. Bar = 50 μm. Transwell migration assays of transfected HCT116 cells (e). Bar = 100 μm. Total number of cells in five fields was counted manually (f). Transwell invasion assays of HCT116 cells (g). Bar = 100 μm. Total number of cells in five fields was counted manually (h). Mean ± SD are shown. Statistical analysis was conducted using one-way ANOVA. *P < 0.05. **P < 0.01. ***P < 0.001.

Fig. 5

miR-195-5p inhibits M2-like TAM activation and recruitment. a Fluorescent probe in situ hybridization (FISH) experiment to demonstrate the distribution of miR-195-5p, NOTCH2, and CD163. b Western blots of NOTCH2, Ad-NOTCH2, GATA3, and IL-4 of CRC tissues, miR-195-5p-transfected HCT116 (c), and si-NOTCH2 or IL-4 inhibitor-treated HCT116 (d). e ELISA detected the IL-4 levels in cell supernatants. f–i RT-qPCR tested the macrophage-associated markers after being co-cultured with transfected HCT116. Assays were performed in triplicates. Mean ± SD is shown. Statistical analysis was conducted using one-way ANOVA. *P < 0.05. **P < 0.01. ***P < 0.001

Fig. 6

miR-195-5p suppresses tumor growth and M2-likeTAM infiltration in vivo. a Immunohistochemistry of tumor tissues treated with or without miR-195-5p. b Western blots of E-cadherin, Vimentin, NOTCH2, Ad-NOTCH2, GATA3 and IL-4 in the tumors. GAPDH was used as a loading control. c Representative photomicrographs and quantifications (d) of CTCs captured from blood of mice. Assays were performed in triplicates. Mean ± SD is shown. Statistical analysis was conducted using Student’s t test. f Interaction between CRC EMT and M2-like TAMs in TME