miR-526b-3p functions as a tumor suppressor in colon cancer by regulating HIF-1α

Abstract

HIF-1α is an important transcriptional factor, which plays roles in cancer development and progression. But its regulation by miRNAs is not clear. Here, to investigate the regulation of HIF-1α by miRNAs, miRNAs were predicted and miR-526b-3p was verified as a regulator of HIF-1α in colon cancer cells. Using TaqMan RT-PCR analysis, we analyzed the expression of miR-526b-3p in tumor tissues and cell lines and found that miR-526b-3p was consistently under-expressed in cancer tissues and cell lines compared with their normal controls. When miR-526b-3p was induced into the colon cancer cells, cell proliferation, metastasis and glycolysis of colon cancer cells were suppressed. We also found that miR-526b-3p was down-regulated in metastatic colon cancer tissues and negatively with HIF-1α mRNA in colon cancer tissues. In a summary, miR-526b-3p plays as a tumor suppressor by down-regulation of HIF-1α expression in colon cancer and may be a new diagnosis or therapeutic target.

Keywords: HIF-1α; colon cancer; glycolysis; miR-526b-3p.

Figure 1

miR-526b-3p is identified as a potential regulator of HIF-1α in colon cancer cells. A. The expression miRNAs in HT-29 colon cancer cells that regulates HIF-1α. miRNA expression was examined by real time RT-PCR. B. miR-526b-3p was down-regulated in various colon cancer cells. *p < 0.05, **p < 0.01 vs control.

Figure 2

HIF-1α is a direct target of miR-526b-3p in colon cancer cells. A. Wild typed and mutant target sites for miR-526b-3p sequences in 3’UTR of HIF-1α were shown. B. Luciferase reporter assays were performed to verify the binding of miR-526b-3p in 3’-UTR of HIF-1α. WT: wild type; Mut: mutation. C. qRT-PCR assay was performed to detect the mRNA level of HIF-1α in HT29 cells treated with miR-526b-3p mimics. miR-526b-3p was measured by miRNA real-time RT-PCR in HT29, SW480 and HCT116 cells after miR-526b-3p mimic transfection. D. Western blotting analysis was used to measure HIF-1α protein in colon cancer cells exposing in hypoxia (1% O₂) treated with miR-526b-3p mimics. *p < 0.05, **p < 0.01 vs control.

Figure 3

Low expression of miR-526b-3p is positively correlated with clinic features of colon cancer. A. miR-526b-3p was down-regulated in various colon cancer tissues. B. miR-526b-3p was down-regulated in metastatic colon cancer tissues. C. miR-526b-3p was related to colon cancer features in clinic. D. HIF-1α mRNA expression was negatively with miR-526b-3p expression in colon cancer tissues. *p < 0.05, **p < 0.01 vs control.

Figure 4

miR-526b-3p suppresses colon cancer cell proliferation and invasion by down-regulation of HIF-1α. A. Cell grow was assayed in HT-29 cells with miR-526b-3p or HIF-1α transfection. B. Cell grow was assayed in SW480 cells with miR-526b-3p or HIF-1α transfection. C. Cell migration was assayed in HT-29 and SW480 cells with miR-526b-3p or HIF-1α transfection. D. Cell invasion was assayed in HT-29 and SW480 cells with miR-526b-3p or HIF-1α transfection. *p < 0.05, **p < 0.01 vs control.

Figure 5

miR-526b-3p regulates colon cancer cell glycolysis through HIF-1α. A. Glycolysis rate in HT-29 cells co-transfected miR-526b-3p or HIF-1α/ΔODD. B. Glycolysis rate in SW480 cells co-transfected miR-526b-3p and HIF-1α/ΔODD. C. Lactate production in HT-29 cells co-transfected miR-526b-3p and HIF-1α/ΔODD. D. Lactate production in SW480 cells co-transfected miR-526b-3p and HIF-1α/ΔODD. E. The changes of glycolysis associated protein in cells transfected miR-526b-3p or HIF-1α/ΔODD. *p < 0.05, **p < 0.01 vs control.