MiR-19a suppresses ferroptosis of colorectal cancer cells by targeting IREB2

Abstract

Colorectal cancer (CRC) is the most common malignant tumor occurred in digestive system. However, the prognosis of CRC patients is poor. Therefore, it is urgent to illuminate the mechanism suppressing CRC and explore novel targets or therapies for CRC treatment. MicroRNAs (miRNAs) are a class of non-coding RNAs with a length of 20-23 nucleotides encoded by endogenous genes, which are associated with the development of a variety of cancers, including CRC. Studies have shown that miR-19a is identified as oncogenic miRNA and promotes the proliferation, migration and invasion of CRC cells. However, the relationship between miR-19a and ferroptosis in CRC remains unknown. Here, we reported that iron-responsive element-binding protein 2 (IREB2), as an inducer of ferroptosis, was negatively regulated by miR-19a. IREB2 is a direct target of miR-19a. In addition, ferroptosis was suppressed by miR-19a through inhibiting IREB2. Thus, we proposed a novel mechanism of ferroptosis mediated by miR-19a in CRC cells, which could give rise to a new strategy for the therapy of CRC.

Keywords: cell proliferation; colorectal cancer; ferroptosis; iron-responsive element-binding protein 2; miR-19a; miRNA.

Graphical abstract

Figure 1.

miR-19a inhibits the expression of IREB2 at the translation level. (a) Bioinformatical analysis shows that IREB2 is a target of miR-19a. (B&C) HT29 cells were treated with NC inhibitor or miR-19a inhibitor before harvest, and the mRNA levels of both miR-19a (b) and IREB2 (c) were determined by RT-qPCR. (d) The expression of IREB2 was determined by western blotting in miR-19a inhibitor treatment HT29 cells.

Figure 2.

Identification of miR-19a has an interaction with IREB2. (a) RNA-RNA pull down analysis the relationship of IREB2 and miR-19a, and the expression of IREB2 mRNA in different treatment assays were determined by RT-qPCR. (b) Agarose gel electrophoresis analysis in HT29 cells shows the interaction between miR-19a and IREB2.

Figure 3.

miR-19a negatively regulates IREB2. (a-d) HT29 cells were transfected with indicated plasmids and were treated with miR-19a or miR-19a inhibitor, IREB2 promoter activity was determined by luciferase values. IREB2: wild-type 3’UTR; mut-IREB2-1: 347–353 binding sites mutant of IREB2; mut-IREB2-2: 1413–1420 binding sites mutant of IREB2; mut-IREB2-3: both of 347–353 and 1413–1420 binding sites mutant of IREB2. Error bars represent data from three independent experiments (mean ± SD). * P < 0.05, ** P < 0.01.

Figure 4.

miR-19a attenuates ferroptosis of colorectal cancer cells by suppression of IREB2. (A&B) The cell proliferation in HT29 cells expressing the indicated proteins or treat with indicated inhibitor was tested by the CCK-8 assay. Results presented represent the means of triplicate experiments ± SEM. *P < 0.05, **P < 0.01. (c) Inhibition rates of miR-19a inhibitor and IREB2 gene sensitivity in HT-29 cells, Inhibition rate = (1- mean OD value of the experimental group/mean OD value of the control group) ×100% (same time as cell proliferation). (D&E) HT29 cells were treated as in (a) and LDH overflow detection and related inhibition rate were determined by CCK-8 assay. Results presented represent the means of triplicate experiments ± SEM. * P < 0.05, ** P < 0.01.