Reduced microRNA 375 in colorectal cancer upregulates metadherin-mediated signaling

Abstract

Background: The human microRNA 375 (MIR375) is significantly downregulated in human colorectal cancer (CRC) and we have previously shown that MIR375 is a CRC-associated miRNA. The metadherin (MTDH) is a candidate target gene of MIR375.

Aim: To investigate the interaction and function between MIR375 and MTDH in human CRC.

Methods: A luciferase reporter system was used to confirm the effect of MIR375 on MTDH expression. The expression levels of MIR375 and the target genes were evaluated by quantitative RT-PCR (qRT-PCR), western blotting, or immunohistochemistry.

Results: MTDH expression was found to be upregulated in human CRC tissues compared to that in healthy controls. We show that MIR375 regulates the expression of many genes involved in the MTDH-mediated signal transduction pathways [BRAF-MAPK and phosphatidylinositol-4,5-biphosphate-3-kinase catalytic subunit alpha (PIK3CA)-AKT] in CRC cells. Upregulated MTDH expression levels were found to inhibit NF-κB inhibitor alpha, which further upregulated NFKB1 and RELA expression in CRC cells.

Conclusion: Our findings suggest that suppressing MIR375 expression in CRC regulates cell proliferation and angiogenesis by increasing MTDH expression. Thus, MIR375 may be of therapeutic value in treating human CRC.

Keywords: Angiogenesis; Cell proliferation; Colorectal cancer; Metadherin; MicroRNA 375; Mitogen-activated protein kinase.

Figure 1

Metadherin is a direct target of microRNA 375. A: Sequence alignment of the wild type (WT) and mutated type (MT) microRNA 375 (MIR375) target site in the 3’-UTR of metadherin (MTDH); B: A luciferase reporter plasmid containing WT or MT MTDH 3’-UTR was co-transfected in HCT116 and Caco2 cells with pre-MIR1 as a negative control or pre-MIR375. Results are shown as relative firefly luciferase activity which is standardized to Renilla luciferase activity. Three independent experiments were conducted with duplicates; C: qRT-PCR analysis of MTDH mRNA expression in HCT116 and Caco2 cells. The data are presented as the fold change in MIR375 mimic transfected cells relative to non-transfected cells. Experiment was performed in duplicate and repeated 5 times; D: Cellular MTDH levels in MIR375 mimic-transfected and siMTDH-transfected HCT116, Caco2, and SW48 cells. Three independent experiments were conducted with duplicates. P values were calculated using Student’s t-test (^aP < 0.05, ^bP < 0.01, ^cP < 0.001). MTDH: Metadherin; MIR375: MicroRNA 375; ns: Not significant.

Figure 2

MicroRNA 375 regulates metadherin-mediated BRAF-MAPK signaling in colorectal cancer cell lines. A: Western blot analysis of KRAS, BRAF, mitogen-activated protein kinase 3/1 (MAPK3/1), pMAPK3/1 and β catenin (CTNNB1) expression levels in colorectal cancer (CRC) cells. Except for KRAS; BRAF, MAPK3/1, pMAPK3/1 and CTNNB1 expression levels were downregulated on microRNA 375 (MIR375) mimic and siMTDH transfection in HCT116 cells; B: Western blot analysis of KRAS, BRAF, MAPK3/1, pMAPK3/1 and CTNNB1 expression levels in CRC cells. Except for KRAS; BRAF, MAPK3/1, pMAPK3/1 and CTNNB1 expression levels were downregulated on MIR375 mimic and siMTDH transfection in Caco2 cells. Five independent experiments were performed with duplicates; C: Effects of MIR375 and siMTDH on cell viability in HCT116, Caco2, and SW480 cells. Cell viability was determined by MTT assay. Three independent experiments were performed with duplicates and P values were calculated using Student’s t-test (^aP < 0.05, ^bP < 0.01, ^cP < 0.001). ns: Not significant; MTDH: Metadherin; CTNNB1: β catenin; MAPK3/1: Mitogen-activated protein kinase 3/1; MIR375: microRNA 375; CRC: Colorectal cancer.

Figure 3

MicroRNA 375 regulates metadherin-mediated phosphatidylinositol-4,5-biphosphate-3-kinase catalytic subunit alpha-protein kinase B signaling in colorectal cancer cell lines. A: Western blot analysis of phosphatidylinositol-4,5-biphosphate-3-kinase catalytic subunit alpha (PIK3CA), protein kinase B (AKT), pAKT, and vascular endothelial growth factor A (VEGFA) expression levels in colorectal cancer (CRC) cells; B: Western blot analysis of PIK3CA, AKT, pAKT, and VEGFA expression levels in CRC cells; C: PIK3CA, AKT, pAKT, and VEGFA expression levels were downregulated on microRNA 375 (MIR375) mimic and siMTDH transfection in HCT116 cells; D: PIK3CA, AKT, pAKT, and VEGFA expression levels were downregulated on MIR375 mimic and siMTDH transfection in Caco2 cells. Five independent experiments were performed with duplicates and P values were calculated using Student’s t-test (^aP < 0.05, ^bP < 0.01, ^cP < 0.001). MTDH: Metadherin; PIK3CA: Phosphatidylinositol-4,5-biphosphate-3-kinase catalytic subunit alpha; AKT: Protein kinase B; VEGFA: Vascular endothelial growth factor A; MIR375: microRNA 375; CRC: Colorectal cancer.

Figure 4

MicroRNA 375 regulates metadherin-mediated NFKB1 signaling in colorectal cancer cell lines. Western blot analysis of NF-κB inhibitor alpha (NFKBIA), NFKB1 (p50), and RELA (p65) expression levels in colorectal cancer (CRC) cells. A: NFKBIA expression levels were upregulated on microRNA 375 (MIR375) mimic and siMTDH transfection in HCT116 cells; B: NFKBIA expression levels were upregulated on MIR375 mimic and siMTDH transfection in Caco2 cells; C: NFKB1 and RELA levels were downregulated on MIR375 mimic and siMTDH transfection in HCT116 cells; D: NFKB1 and RELA levels were downregulated on MIR375 mimic and siMTDH transfection in Caco2 cells. Four independent experiments were performed with duplicates and P values were calculated using Student’s t-test (^aP < 0.05, ^bP < 0.01). MTDH: Metadherin; MIR375: MicroRNA 375; NFKBIA: NF-κB inhibitor alpha; CRC: Colorectal cancer.

Figure 5

Endogenous metadherin expression in colorectal cancer tissues. A: metadherin (MTDH) expression levels were investigated in 15 colorectal cancer (CRC) tissue samples and matching healthy colorectal tissue samples. Results are shown for 8 colon cancer tissues in pairs. P1 to P8 indicate patients with colon cancer. Data are presented as fold change in the expression in tumor tissues relative to expression in matching healthy colon tissues. P values were calculated using Student’s t-test (^aP < 0.05); B: Relative endogenous NFKB1 (n = 5) and RELA (n = 4) expression levels in colon cancer tissue samples and matching healthy colon tissue samples. Data are presented as fold change in the expression in tumor tissues relative to expression in matching healthy colon tissues; C: Immunostaining of MTDH in human CRC and adjacent healthy colorectal samples (200 × magnification). Experiments were independently performed three times in duplicates. MTDH: Metadherin.

Figure 6

Diagrammatic representations of putative mechanisms of microRNA 375 in regulating metadherin-induced cell proliferation, cell migration, and angiogenesis in human colorectal cancer. Decreased microRNA 375 (MIR375) expression in colorectal cancer (CRC) cells leads to upregulation of cellular metadherin (MTDH) levels. Subsequently, upregulated expression of MTDH promotes inhibition of NFKBIA and thus, NFKB1 and RELA expression is upregulated in CRC tissues and CRC cells. Upregulated MTDH expression level stimulates the BRAF-MAPK and PIK3CA-AKT signaling pathway. However, KRAS expression is unaltered by upregulation of MTDH. Consequently, downregulated MIR375 expression levels in CRC leads to upregulation of cell proliferation, cell migration, and angiogenesis. This simple hypothetical mechanism of MIR375-mediated upregulation of angiogenesis is based on the results of previous studies and our current study. MTDH: Metadherin; MIR375: MicroRNA 375; CRC: Colorectal cancer.