HIF-1α promotes the migration and invasion of cancer-associated fibroblasts by miR-210

Abstract

Metastasis is the major cause of death in colorectal cancer (CRC) patients. Inhibition of metastasis will prolong the survival of patients with CRC. Cancer cells bring their own soil, cancer-associated fibroblasts (CAFs), to metastasize together, promoting the survival and colonization of circulating cancer cells. However, the mechanism by which CAFs metastasize remains unclear. In this study, CAFs were derived from adipose mesenchymal stem cells (MSCs) after co-culture with CRC cell lines. Transwell assays showed that CAFs have stronger migration and invasion abilities than MSCs. In a nude mouse subcutaneous xenograft model, CAFs metastasized from the primary tumour to the lung and promoted the formation of CRC metastases. The expression of HIF-1α was upregulated when MSCs differentiated into CAFs. Inhibition of HIF-1α expression inhibited the migration and invasion of CAFs. Western blot and ChIP assays were used to identify the genes regulated by HIF-1α. HIF-1α regulated the migration and invasion of CAFs by upregulating miR-210 transcription. Bioinformatics analysis and luciferase reporter assays revealed that miR-210 specifically targeted the 3'UTR of VMP1 and regulated its expression. Downregulation of VMP1 enhanced the migration and invasion of CAFs. In vivo, inhibition of miR-210 expression in CAFs reduced the metastasis of CAFs and tumour cells. Therefore, the HIF-1α/miR-210/VMP1 pathway might regulate the migration and invasion of CAFs in CRC. Inhibition of CAF metastasis might reduce CRC metastasis.

Keywords: HIF-1α; cancer-associated fibroblasts; colorectal cancer; invasion; migration.

Figure 1.

MSCs were induced to CAFs by colorectal cancer cells. (A) Western blot showing that the expression of the CAF characteristic proteins α-SMA and FAPA was upregulated after MSCs were co-cultured with LOVO, HCT-116, and HCT-8 cells. (B) Comparison of adipogenic (Oil Red O staining) and osteogenic (alizarin red and alkaline phosphatase staining) differentiation abilities between MSCs and CAFs. Oil Red O staining and alizarin red staining were performed after 12 days of culture. Alkaline phosphatase staining was performed after 5 days of culture. (C) Effect of combined MSC transplantation on primary tumour volume in nude mice. HCT-116 group: HCT-116 5 × 10⁶ cells; MSC + HCT-116 group: HCT-116 5 × 10⁶ cells + MSC 1 × 10⁶ cells. (D) In vivo fluorescence image showing the effect of combined MSC transplantation on tumour metastasis. Two groups of mice were transplanted with HCT-116 5 × 10⁶ cells and HCT-116 5 × 10⁶ cells + MSC 1 × 10⁶ cells. The HCT-116 cell lines carried GFP.

Figure 2.

CAFs had stronger migration and invasion abilities than MSCs. (A) Transwell assays (n = 4) showed that CAFs had stronger migration and invasion abilities than MSCs. * P < 0.05. Scale bar: 200μm. (B) In vivo fluorescence image showing CAFs expressing luciferase in parts other than the primary foci in the HCT-116 + MSC group of the nude mouse subcutaneous transplantation model. Lentivirus vectors carrying mCherry-luciferase were transfected into MSCs. HCT-116 5 × 10⁶ cells and HCT-116 5 × 10⁶ + MSC 1 × 10⁶ cells were transplanted into the two groups. (C) Multiple immunofluorescence staining showing colorectal cancer cells and CAFs originating from the primary tumour in the lung metastases in mice of the HCT-116 + MSC group. DAPI: nucleus (blue), CDX-2: colorectal cancer cells (green), α-SMA: CAFs (red), mCherry: exogenous CAFs (violet). White arrows: CDX-2-positive cells (CRC cells), yellow arrows: both α-SMA- and mCherry-positive cells (exogenous CAFs), red arrow: only α-SMA-positive cells (endogenous CAFs). Scale bar: 20μm.

Figure 3.

HIF-1α regulated the migration and invasion of CAFs. (A) Western blot assay of HIF-1α protein levels during the differentiation of MSCs into CAFs induced by HCT-116 cells. (B) Western blot assay of HIF-1α protein levels in the nucleus during the differentiation of mesenchymal stem cells (MSCs) into CAFs induced by HCT-116 cells. (C) Transwell assays (n = 4) showing that the migration and invasion capacities of CAFs were significantly decreased after treatment with the HIF-1α inhibitor KC7F2. * P < 0.05. Scale bar: 200 μm.

Figure 4.

HIF-1α regulated the migration and invasion of CAFs by upregulating the expression of miR-210. (A) Binding sites of HIF-1α in the miR-210 promoter region. (B) ChIP assay revealed that HIF-1α could bind to the miR-210 promoter region. (C) qRT-PCR showing that the expression of miR-210 was upregulated during the differentiation of MSCs into CAFs induced by HCT-116 cells. * P < 0.05. (D) Transwell assays (n = 4) revealed that the migration and invasion of CAFs decreased significantly after interference with miR-210 in CAFs. * P < 0.05. Scale bar: 200μm. (E) Transwell assays (n = 4) revealed that the migration and invasion of CAFs increased significantly after miR-210 overexpression in MSCs. * P < 0.05. Scale bar: 200μm.

Figure 5.

miR-210 regulated the migration and invasion of CAFs by regulating VMP1. (A) Bioinformatics analysis showing that miR-210 could directly bind to the 3’UTR of VMP1. (B) Western blot showing the downregulated expression of the VMP1 protein during the differentiation of MSCs into CAFs. (C) Dual-luciferase reporter assay suggested that miR-210 could bind to the 3’UTR of VMP1 (VMP1 WT: VMP1 3’UTR Wild type; VMP1 MT: VMP1 3’UTR mutation type; mimic: microRNA control). * P < 0.05. (D) Western blot analysis showing the upregulated and downregulated expression of the VMP1 protein after interference with and overexpression of miR-210, respectively. (E) Transwell assays (n = 4) showing that the migration and invasion of CAFs significantly increased after interference with VMP1. * P < 0.05. Scale bar: 200μm.

Figure 6.

Validation of the effect of the HIF-1α/MIR-210 pathway on the metastasis of cancer cells and CAFs. The metastasis of cancer cells and CAFs was detected in a subcutaneous xenograft model of colorectal cancer. Lung metastases of colorectal cancer cells (arrows in A, CDX-2 IHC, scale bar: 2.5 mm.) and exogenous CAFs (tagged violet mCherry in B, scale bar: 5000 μm.) were reduced after interference with the expression of miR-210 in the subcutaneously transplanted MSCs.