Knockdown of miR-411-3p induces M2 macrophage polarization and promotes colorectal cancer progression by regulation of MMP7

Abstract

Colorectal cancer (CRC) is prone to metastasis, leading to a poor prognosis. miR-411-3p exhibits a tumor-suppressive function in CRC, but its exact mechanism is unclear. The malignant biological properties of CRC cells were detected by Carboxyfluorescein diacetate succinimidyl ester (CFSE) staining, scratch-wound and transwell assay. Levels of markers associated with macrophage polarization were evaluated by flow cytometry and ELISA kits. Bioinformatics analysis to screen whether the downstream target mRNA of miR-411-3p is matrix metalloproteinase 7 (MMP7), and Dual-Luciferase reporter assay verified the targeting relationship between the two. qRT-PCR tested miR-411-3p and MMP7 levels. MMP7 level was quantified by Western blot. Additionally, a nude mouse subcutaneous graft tumor model was constructed, Ki-67 expression was detected by immunohistochemistry, and the impact of miR-411-3p/MMP7 on the polarization of M2 macrophages was explored. miR-411-3p expression is downregulated in CRC. Knockdown of miR-411-3p elevated the amount of CFSE-positive, migrating, and invading cells, decreased apoptosis, and elevated the levels of M2 macrophage polarization markers. After overexpression of miR-411-3p, all of the above metrics were reversed in CRC cells. miR-411-3p targeted negative regulation of MMP7 expression, and MMP7 overexpression further enhanced the promotional effect of knockdown of miR-411-3p on the malignant progression of CRC and M2 macrophage polarization. Furthermore, knockdown of miR-411-3p upregulated the MMP7 level, elevated Ki-67-positive cells count, and induced M2 macrophage polarization in vivo. Knockdown of miR-411-3p upregulates MMP7 and induces M2 macrophage polarization, which in turn promotes malignant biological progression of CRC.

Figure 1.

miR-411-3p expression of in CRC. A) miR-411-3p level in adjacent tissues and CRC tissues (n=29) was detected through qRT-PCR. B) miR-411-3p level in cancer tissues of stage I+II (n=11) and stage III+IV (n=18) CRC patients. C) miR-411-3p level in normal colon epithelial cells (FHC) and CRC cell lines (SW480, LoVo, SW620 and HCT116), n=3. **p<0.01, ***p<0.001.

Figure 2.

Knockdown miR-411-3p promotes malignant progression in CRC cells. A) The mimics and inhibitors were transfected in CRC cells, and miR-411-3p level was assessed via qRT-PCR. B) CFSE probe was utilized to evaluate CRC cell proliferation after transfection. C,D) The number of invasion and migration of SW480 and HCT116 cells was determined by Transwell assay; magnification 20×; scale bar: 100 μm. E) Scratch-wound assay o detect cell migration rate; magnification 20×; scale bar: 100 μm. F) Flow cytometry was utilized to evaluate apoptosis rate. *p<0.05, **p<0.01, ***p<0.001.

Figure 3.

Knockdown miR-411-3p induces M2 macrophage polarization. A) M0 macrophage marker CD68 level was assessed by flow cytometry. B,C) M0 macrophages were induced into M1 macrophages or M2, and CRC cells were co-cultured with M1 macrophages for 48 h; utilizing qRT-PCR to evaluate M1 macrophage polarization markers levels, like CD86, iNOS, IL-1β and TNF-α. D) M1 macrophage marker CD86 level was determined by flow cytometry. E,F) M1 macrophage markers (iNOS, IL-1β, and TNF-α) levels were assessed by ELISA. G,H) CRC cells were co-cultured with M2 macrophages for 48 h, with CD206, IL-10, ARG-1, and CD163 (M2 macrophage polarization markers) levels were detected by qRT-PCR. I) M2 macrophage polarization markers CD206 and CD163 levels were assessed by flow cytometry. J-K) M2 macrophage markers IL-10 and ARG-1 levels were assessed by ELISA. *p<0.05, **p<0.01, ***p<0.001.

Figure 4.

miR-411-3p targets MMP7 and negatively regulates MMP7. A) The downstream target genes of miR-411-3p were predicted by GEPIA, miRDB and Home-miRWalk databases, and the MMP7 gene was obtained by taking the intersection of the three. B) The sequences of miR-411-3p and MMP7 combined were anticipated by TargetScan database. C,D) Dual-luciferase reporter assay verified miR-411-3p can target and regulate MMP7 expression. E) Examining MMP7 level in CRC cells through Western blot. F) The GEPIA database revealed an increased level of MMP7 in CRC. G,H) Examining MMP7 level in different tissues and cells through Western blot. I) miR-411-3p and MMP7 expression was analyzed by Pearson correlation analysis. *p<0.05, **p<0.01.

Figure 5.

Knockdown miR-411-3p induces M2 macrophage polarization via MMP7 and promotes malignant biological behaviour in CRC cells. A) OE-NC and OE-MMP7 were transfected in CRC cells, and the level of MMP7 mRNA was evaluated via qRT-PCR. B) After cocultured with SW480 and HCT116 cells for 48 h, CD206 and CD163 (M2 macrophage polarization markers) levels were detected through flow cytometry. C,D) IL-10 and ARG-1 (M2 macrophage markers) levels were evaluated by ELISA. E) After co-cultured with M2 macrophages, CFSE probe detected the CRC cell proliferation. F, G ) After co-cultured with M2 macrophages, the quantity of invasion and migration cells was quantified by Transwell assay; magnification 20×; scale bar: 100 μm. H) Scratch-wound assay detected cell migration rate; magnification 20×; scale bar: 100 μm. I) The apoptosis rate of CRC was evaluated via flow cytometry. *p<0.05, **p<0.01.

Figure 6.

Knockdown miR-411-3p upregulates MMP7 expression and promotes tumor growth through M2 macrophage polarization. A) miR-411-3p level in tumor tissues was assessed through qRT-PCR. B) Western blot detection of MMP7 level in CRC tissues; SW480 cells were mixed well with THP-1 cells after 24 h of PMA treatment and subcutaneously injected into mice, n=5. C) The size of the subcutaneous tumor was gauged using a vernier caliper on days 7, 14, 21, and 28; at d 28, mice were anesthetized and executed, followed by the removal and imaging of tumors D), and tumor weights were recorded (E). F) Immunohistochemical detection of Ki-67 expression in tumor tissues; magnification 40×, scale bar: 50 μm. G) Immunofluorescence detected CD206 and CD163 level in tumor tissues magnification 40×, scale bar: 50 μm. H,I) ELISA kit to determine IL-10 and ARG-1 levels in tumor tissues. *p<0.05, **p<0.01.