Methylseleninic acid activates Keap1/Nrf2 pathway via up-regulating miR-200a in human oesophageal squamous cell carcinoma cells

Abstract

Oesophageal squamous cell carcinoma (ESCC) occurs at a very high rates in certain regions of China. There are increasing evidences demonstrating that selenium could act as a potential anti-oesophageal cancer agent, but the precise mechanisms involved are still not completely understood. Methylseleninic acid (MSA), as a potent second-generation selenium compound, is a promising chemopreventive agent. Previous studies demonstrated that the kelch-like ECH-associated protein 1 (Keap1)/nuclear factor E2-related factor 2 (Nrf2) system plays a critical role in cancer prevention, but little is known about its association with MSA in ESCC cells. In the present study, we observed that MSA treatment significantly down-regulated Keap1, induced nuclear accumulation of Nrf2 and enhance the antioxidant response element (ARE) promoter activity in ESCC cells. MSA could also significantly induce miR-200a expression and inhibit Keap1 directly. Antagomir-200a could attenuate MSA treatment-induced Keap1 down-regulation in ESCC cells. Moreover, MSA-induced miR-200a expression was dependent on the mediation of Krüpple-like factor 4 (KLF4). These results reaffirm the potential role of MSA as a chemopreventive agent via the regulation of KLF4/miR-200a/Keap1/Nrf2 axis in ESCC cells.

Keywords: Krüpple-like factor 4 (KLF4); kelch-like ECH-associated protein 1 (Keap1); methylseleninic acid (MSA); micro-ribonucleic acid-200a (miR-200a); nuclear factor E2-related factor 2 (Nrf2); oesophageal squamous cell carcinoma (ESCC).

Figure 1. MSA activated Keap1/Nrf2 pathway in ESCC cells

(A) KYSE180 and KYSE150 were treated with MSA (5 μM) for 24 h. Then, total cell lysates were prepared. Western blotting was performed to examine Keap1 and Nrf2. β-Actin was used as a loading control. (B) KYSE150 cells were treated with MSA (5 μM) for 24 h. Then, protein lysates were prepared. Keap1 and Nrf2 were detected in the cytoplasmic and nuclear extracts by western blot. β-Actin was used as a loading control of cytoplasmic protein and lamin B was shown as a loading control of nuclear protein. (C) KYSE150 cells were transfected with 100 ng of pGL3-ARE or pGL3-basic and 500 ng of pcDNA3-HA or pcDNA3-HA/Nrf2 plasmids with or without MSA treatment respectively. The pEGFP-C1 (20 ng) plasmid was co-transfected to normalize transfection efficiency. The luciferase activities were measured 48 h after transfection. Means ± S.D., n=3, *P<0.05.

Figure 2. MSA activates Keap1/Nrf2 pathway via up-regulating miR-200a

(A). KYSE150, KYSE 410, KYSE 180 and KYSE510 were treated with or without MSA (5 μM) for 24 h, then the expression level of miR-200a was detected by real-time PCR. U6 was used as an internal control. The level of miR-200a in each cell lines without MSA treatment was designated as unit 1 respectively. Means ± S.D., n=3, *P<0.05, **P<0.01. (B) Predicted duplex formation between human Keap1 3′-UTR and miR-200a. Luciferase activity of Keap1 3′-UTR wild-type (Keap1 3′-UTR-wt) or mutant (Keap1 3′-UTR-mut) reporter gene in KYSE150 cells with or without MSA treatment (5 μM, 24 h) were detected. Mean ± S.D. (n=3), *P<0.05. (C) KYSE180 and KYSE150 cells were transfected with either 30 nM Pre-miR miR-200a precursor or a Pre-miR negative control miRNA precursor. miRNA was extracted 24 h after transfection. Real-time PCR was performed to examine miR-200a level. The level of miR-200a in the corresponding cells that transfected with the negative control was designated as unit 1 respectively. Means ± S.D., n=3. (D) KYSE510 and KYSE150 cells were transfected with miR-200a precursor (30 nM) or pre-miR negative control (30 nM) respectively. Keap1 and Nrf2 protein levels were detected 24 h after transfection. β-Actin was used as a loading control. (E) miR-200a level in KYSE150 cells after the administration of MSA (5 μM) and/or antagomir-200a (20 μM) was measured by real-time PCR. U6 was used as the endogenous control. The level of miR-200a without MSA and antagomir-200a treatment was designated as unit 1. Means ± S.D., n=3. (F) The levels of Keap1 and Nrf2 in KYSE150 cells treated with MSA (5 μM) following antagomir-200a (20 μM) transfection were detected by western blot. β-Actin was used as a loading control.

Figure 3. KLF4 mediated miR-200a up-regulation induced by MSA in ESCC cells

KYSE180 cells were transfected with KLF4 siRNA or control siRNA for 48 h. Then, the cells were treated with or without MSA (5 μM) for additional 24 h. Total RNA was extracted. KLF4 mRNA (A) and miR-200a level (B) were determined by real-time PCR. The level of KLF4 or miR-200a in KYSE180 cells with control siRNA transfection and without MSA treatment was designated as unit 1 respectively. Means ± S.D., n=3. (C). The levels of Keap1 and Nrf2 in KYSE180 cells treated as indicated were detected by western blot. β-Actin was used as a loading control. (D). ChIP assay result showed the KLF4 that associated with miR-200a prompter. KYSE150 cells were treated with MSA (5 μM, 24 h). Then, the lysates were immunoprecipitated by KLF4 antibody and–1048—-804 region of miR-200a promoter was amplified with specific primers.