miR-455/GREM1 axis promotes colorectal cancer progression and liver metastasis by affecting PI3K/AKT pathway and inducing M2 macrophage polarization

Abstract

Background: Colorectal cancer is among the most common malignant tumors affecting the gastrointestinal tract. Liver metastases, a complication present in approximately 50% of colorectal cancer patients, are a considerable concern. Recently, studies have revealed the crucial role of miR-455 in tumor pathogenesis. However, the effect of miR-455 on the progression of liver metastases in colorectal cancer remains controversial. As an antagonist of bone morphogenetic protein(BMP), Gremlin 1 (GREM1) may impact organogenesis, body patterning, and tissue differentiation. Nevertheless, the role of miR-455 in regulating GREM1 in colorectal cancer liver metastases and how miR-455/GREM1 axis influences tumour immune microenvironment is unclear.

Methods: Bioinformatics analysis shows that miR-455/GREM1 axis plays crucial role in liver metastasis of intestinal cancer and predicts its possible mechanism. To investigate the impact of miR-455/GREM1 axis on the proliferation, invasion, and migration of colorectal cancer cells, colony formation assay, wound healing and transwell assay were examined in vitro. The Dual-Luciferase reporter gene assay and RNA pull-down assay confirmed a possible regulatory effect between miR-455 and GREM1. In vivo, colorectal cancer liver metastasis(CRLM) model mice was established to inquiry the effect of miR-455/GREM1 axis on tumor growth and macrophage polarization. The marker of macrophage polarization was tested using immunofluorescence(IF) and quantitative real-time polymerase chain reaction(qRT-PCR). By enzyme-linked immunosorbent assay (ELISA), cytokines were detected in culture medium supernatants.

Results: We found that miR-455 and BMP6 expression was increased and GREM1 expression was decreased in liver metastase compared with primary tumor. miR-455/GREM1 axis promotes colorectal cancer cells proliferation, migration, invasion via affected PI3K/AKT pathway. Moreover, downregulating GREM1 augmented BMP6 expression in MC38 cell lines, inducing M2 polarization of macrophages, and promoting liver metastasis growth in CRLM model mice.

Conclusion: These data suggest that miR-455/GREM1 axis promotes colorectal cancer progression and liver metastasis by affecting PI3K/AKT pathway and inducing M2 macrophage polarization. These results offer valuable insights and direction for future research and treatment of CRLM.

Keywords: AKT; Colorectal cancer; GREM1; Liver metastasis; Macrophage polarization; PI3K; miR-455.

Fig. 1

Differential expression of mRNAs and miRNAs between LM and primary tumors (PT) of CRC. (A) Volcano plots for DEGs from three mRNA datasets (GSE41258, GSE49355, GSE81558). (B) The Venn diagram for consistent total DEGs. (C) Heatmaps for DEGs of three mRNAs datasets. (D) Volcano plots for differential expression of miRNAs from GSE81582. (E) The Venn diagram of the predicted mRNAs and above differential mRNAs. (F) The KM survival analysis of seven key genes. (G) GO (Biological process, Cellular component, and Molecular function) and KEGG pathway enrichment analysis of consistent DEGs

Fig. 2

Prediction of GREM1 downstream pathway and its effect on tumor immune microenvironment. (A) Gene sets enrichment analyses of three mRNAs datasets. (B) immune infiltration analysis of GSE49355 dataset. (C) Differential Expression of GREM1 affect Macrophages Polarization according to TIME database

Fig. 3

GREM1 expression is downregulated in LM of CRC.(A-E) The expression level of GREM1, miR-455, BMP6, BMP4, BMP2 in the liver metastases, primary tumors of CRC patients, and the adjacent normal tissues detected by qRT-PCR. (F) The expression level of GREM1, BMP6, in the liver metastases, primary tumors of CRC patients, and the adjacent normal tissues detected by WB

Fig. 4

The effect of miR-455 on the human colorectal cancer cells by targeting GREM1. (A) qRT-PCR analysis of miR-455 or GREM1 expression in human colorectal cancer cells overexpressing miR-455 or control. (B) Analysis of human colorectal cancer cells overexpressing miR-455 or control migration by wound-healing assays at 0, 24, and 48 h. (C) The colony formation assay was performed to evaluate the proliferation of human colorectal cancer cells overexpressing miR-455 or control. (D) The migration of miR-455 overexpressing or control human colorectal cancer cells were assessed using Transwell migration assays. (E) qRT-PCR analysis of GREM1 expression in human colorectal cancer cells overexpressing GREM1 or control. (F) Analysis of human colorectal cancer cells overexpressing GREM1 or control migration by wound-healing assays at 0, 24, and 48 h. (G) The colony formation assay was performed to evaluate the proliferation of human colorectal cancer cells overexpressing GREM1 or control. (H) The migration of GREM1 overexpressing or control human colorectal cancer cells were assessed using Transwell migration assays. (I) Analysis of human colorectal cancer cells of control group, miR-455 mimic group, pc-GREM1 group and miR-455 mimic + pc-GREM1 group migration by wound-healing assays at 0, 24, and 48 h. (J) The colony formation assay was performed to evaluate the proliferation of human colorectal cancer cells of control group, miR-455 mimic group, pc-GREM1 group and miR-455 mimic + pc-GREM1 group. (K) The migration of control group, miR-455 mimic group, pc-GREM1 group and miR-455 mimic + pc-GREM1 group was assessed using Transwell migration assays. (L) TargetScan predicted that GREM1 was a potential target of miR-455. (M) miR-455 mimic reduced luciferase ability of GREM1 wild-type mRNA 3 ‘-UTR, while it did not change GREM1 mutant 3’-UTR. (N) GREM1 protein expression level of control group, miR-455 mimic group, pc-GREM1 group and miR-455 mimic + pc-GREM1 group migration were detected by Western blot. (O) The expression level of GREM1 in RNA pull-down test was detected by Western blot assay and qRT-PCR analysis

Fig. 5

Deletion of GREM1 promoted liver metastasis of colorectal cancer and induced the M2 polarization of macrophages. (A) qRT-PCR analysis of GREM1 expression in MC38 cells of sh-NC and sh-GREM1 group. (B) Western blot assay was performed to detect the level of GREM1, BMP6, p-PI3K, PI3K, p-AKT, AKT in MC38 cells of different groups. (C) The representative images of liver metastases from the sh-NC or sh-GREM1 injected mice treated with CLD or not. Statistical results of the detectable tumor numbers of liver metastasis and liver weight. (D) Immunofluorescence staining for CD206 and iNOS from metastatic lesions. Statistical results of immunofluorescence staining for CD206. (E) The expression levels of IL-10,TGF-β in metastatic nodules of the liver were measured by ELISA. (F) The expression levels of IL-1β, TNF-a in metastatic nodules of the liver were measured by ELISA

Fig. 6

Knocking down GREM1 promotes M2 polarization of macrophages via increasing BMP6 expression. (A-F) The levels of GREM1, BMP6, IL-10, TGF-β, IL-1β, TNF-a in the supernatant of sh-NC, sh-GREM1, sh-NC + BMDM, sh-GREM1 + BMDM, BMP6 + BMDM group measured by ELISA. (G) Immunofluorescence staining for CD206 and iNOS from BMDMs co-cultured with MC38/sh-GREM1(sn), MC38/sh-NC(sn), MC38/sh-GREM1(sn) + anti-BMP6 neutralizing antibodies, MC38/sh-NC(sn) + anti-BMP6 neutralizing antibodies. (H) Representative histograms of the relative mRNA levels of ARG1, MRC1 for BMDMs treated with MC38/sh-GREM1(sn), MC38/sh-NC (sn), MC38/sh-GREM1(sn) + anti-BMP6 neutralizing antibodies, MC38/sh-NC(sn) + anti-BMP6 neutralizing antibodies. (I) Representative histograms of the relative mRNA levels of CD86, NOS2 for BMDMs treated with MC38/sh-GREM1(sn), MC38/sh-NC (sn), MC38/sh-GREM1(sn) + anti-BMP6 neutralizing antibodies, MC38/sh-NC (sn) + anti-BMP6 neutralizing antibodies

Fig. 7

BMP6 promotes M2 polarization by inducing IL-10 release from macrophages. (A) The levels of IL-10 in the supernatant of BMDMs after co-cultured with MC38/sh-GREM1(sn), MC38/sh-NC (sn), MC38/sh-GREM1(sn) + anti-BMP6 neutralizing antibodies, MC38/sh-NC (sn) + anti-BMP6 neutralizing antibodies measured by ELISA. (B) Representative histograms of the concentration of IL-10 in liver metastases from CRLM model mice treated with AS101 or not. (C)The detectable tumor numbers in liver metastasis from the sh-NC or sh-GREM1-injected mice treated with AS101 or not