The let-7 microRNA enhances heme oxygenase-1 by suppressing Bach1 and attenuates oxidant injury in human hepatocytes

Abstract

The let-7 microRNA (miRNA) plays important roles in human liver development and diseases such as hepatocellular carcinoma, liver fibrosis and hepatitis wherein oxidative stress accelerates the progression of these diseases. To date, the role of the let-7 miRNA family in modulation of heme oxygenase 1 (HMOX1), a key cytoprotective enzyme, remains unknown. Our aims were to determine whether let-7 miRNA directly regulates Bach1, a transcriptional repressor of the HMOX1 gene, and whether indirect up-regulation of HMOX1 by let-7 miRNA attenuates oxidant injury in human hepatocytes. The effects of let-7 miRNA on Bach1 and HMOX1 gene expression in Huh-7 and HepG2 cells were determined by real-time qRT-PCR, Western blot, and luciferase reporter assays. Dual luciferase reporter assays revealed that let-7b, let-7c, or miR-98 significantly decreased Bach1 3'-untranslated region (3'-UTR)-dependent luciferase activity but not mutant Bach1 3'-UTR-dependent luciferase activity, whereas mutant let-7 miRNA containing base complementarity with mutant Bach1 3'-UTR restored its effect on mutant reporter activity. let-7b, let-7c, or miR-98 down-regulated Bach1 protein levels by 50-70%, and subsequently up-regulated HMOX1 gene expression by 3-4 fold, compared with non-specific controls. Furthermore, Huh-7 cells transfected with let-7b, let-7c or miR-98 mimic showed increased resistance against oxidant injury induced by tert-butyl-hydroperoxide (tBuOOH), whereas the protection was abrogated by over-expression of Bach1. In conclusion, let-7 miRNA directly acts on the 3'-UTR of Bach1 and negatively regulates expression of this protein, and thereby up-regulates HMOX1 gene expression. Over-expression of the let-7 miRNA family members may represent a novel approach to protecting human hepatocytes from oxidant injury.

Figure 1. let-7 miRNA mimics repress wild type but not mutant reporter activities

(A) The firefly luciferase reporter construct pLSV40-GL3/Bach1 containing two seed match sites for let-7 miRNA is schematically illustrated; (B) Four nucleotides in two seed match sites of the Bach1 3’-UTR (highlighted) were mutated, leading to a partial seed match at positions 2–8 of let-7b, let-7c and miR-98 that failed to meet the criteria for miRNA binding. Effects of let-7 miRNA on WT and mutant reporter activities were illustrated. Huh-7 cells were co-transfected with 0.4 µg/mL of pLSV40-GL3/Bach1-WT or pLSV40-GL3/Bach1-Mut, 0.4 µg/mL of pRL-TK (renilla) and with 50 nM let-7 mimic, or MMNC as negative control by Lipofectamine 2000 as indicated. 48 h after transfection, the luciferase reporter activities were measured using Dual Luciferase Assay System from Promega. Firefly luciferase activities were normalized to renilla luciferase activities and total protein, as indicated in Materials and Methods. Values for cells with 50 nM of MMNC transfection were set equal to 1. Data are presented as means ± SE, n=3; (C) let-7b, let-7c and miR-98 significantly inhibit WT but not mutant reporter activities (*differs from MMNC only, p=0.012, let-7b; p=0.016, let-7c; and p=0.012, miR-98).

Figure 1. let-7 miRNA mimics repress wild type but not mutant reporter activities

(A) The firefly luciferase reporter construct pLSV40-GL3/Bach1 containing two seed match sites for let-7 miRNA is schematically illustrated; (B) Four nucleotides in two seed match sites of the Bach1 3’-UTR (highlighted) were mutated, leading to a partial seed match at positions 2–8 of let-7b, let-7c and miR-98 that failed to meet the criteria for miRNA binding. Effects of let-7 miRNA on WT and mutant reporter activities were illustrated. Huh-7 cells were co-transfected with 0.4 µg/mL of pLSV40-GL3/Bach1-WT or pLSV40-GL3/Bach1-Mut, 0.4 µg/mL of pRL-TK (renilla) and with 50 nM let-7 mimic, or MMNC as negative control by Lipofectamine 2000 as indicated. 48 h after transfection, the luciferase reporter activities were measured using Dual Luciferase Assay System from Promega. Firefly luciferase activities were normalized to renilla luciferase activities and total protein, as indicated in Materials and Methods. Values for cells with 50 nM of MMNC transfection were set equal to 1. Data are presented as means ± SE, n=3; (C) let-7b, let-7c and miR-98 significantly inhibit WT but not mutant reporter activities (*differs from MMNC only, p=0.012, let-7b; p=0.016, let-7c; and p=0.012, miR-98).

Figure 2. Mutant let-7b mimic inhibits the luciferase activity of mutant Bach1 3’-UTR reporter

(A) Four nucleotides in the seed match sites of let-7b were mutated, resulting in formation of perfect seed region matches between mutant let-7b and mutant Bach1 3’-UTR; (B) Effect of wild type or mutant let-7b mimic on mutant Bach1 3’-UTR reporter activity. Huh-7 cells were co-transfected with 0.4 µg/mL of pLSV40-GL3/Bach1, 0.4 µg/mL of pRL-TK and with 0, 10, 20 or 50 nM wild type let-7b or mutant let-7b mimic for 48 h, firefly and renilla luciferase activities were measured using Dual Luciferase Assay System from Promega. Firefly luciferase activities were normalized to renilla luciferase activities and total protein, as indicated in Materials and Methods. Values for cells without miRNA transfection were set equal to 1. Data are presented as means ± SE, n=3. *differs from 0, p=0.017, 10 nM; p=0.004, 20 nM; and p=0.003, 50 nM).

Figure 3. let-7 miRNA mimics down-regulate Bach1 protein levels in human Huh-7 cells

Human Huh-7 cells were transfected with 0–50 nM of let-7 miRNA mimics, or MMNC as control by Lipofectamine 2000 for indicated times. Cells were harvested, and total RNA and proteins were extracted. The levels of Bach1 mRNA and protein were assessed by real-time qRT-PCR and WB, respectively, as described in Materials and Methods. The amounts of Bach1 mRNA and protein were normalized to GAPDH, which did not vary with transfection. Values for cells at time 0 or with a MMNC transfection were set equal to 1. Data are presented as means ± SE, n=3. * differs from time 0 or negative control, P<0.05. (A) WB analysis of time-course effects of let-7b mimic on Bach1 protein levels; (B) Time-course effects of let-7b mimic on Bach1 mRNA levels; (C) WB analysis of effects of let-7b, let-7c and miR-98 mimics on Bach1 protein levels; (D) Effects of let-7b, let-7c and miR-98 mimics on Bach1 mRNA levels.

Figure 3. let-7 miRNA mimics down-regulate Bach1 protein levels in human Huh-7 cells

Human Huh-7 cells were transfected with 0–50 nM of let-7 miRNA mimics, or MMNC as control by Lipofectamine 2000 for indicated times. Cells were harvested, and total RNA and proteins were extracted. The levels of Bach1 mRNA and protein were assessed by real-time qRT-PCR and WB, respectively, as described in Materials and Methods. The amounts of Bach1 mRNA and protein were normalized to GAPDH, which did not vary with transfection. Values for cells at time 0 or with a MMNC transfection were set equal to 1. Data are presented as means ± SE, n=3. * differs from time 0 or negative control, P<0.05. (A) WB analysis of time-course effects of let-7b mimic on Bach1 protein levels; (B) Time-course effects of let-7b mimic on Bach1 mRNA levels; (C) WB analysis of effects of let-7b, let-7c and miR-98 mimics on Bach1 protein levels; (D) Effects of let-7b, let-7c and miR-98 mimics on Bach1 mRNA levels.

Figure 4. let-7 miRNA inhibitors up-regulate Bach1 protein levels in human Huh-7 cells

Huh-7 cells were transfected with let-7 miRNA inhibitors which contain a combination let-7a, let-7c and let-7e inhibitors, or MINC as control by Lipofectamine 2000. 48 h after transfection, cells were harvested and total RNA and proteins were extracted. Bach1 mRNA and protein levels were assayed by real-time qRT-PCR and WB, respectively. The amounts of Bach1 mRNA and protein were normalized to GAPDH, which did not vary with transfection. Values for cells with a mock transfection (0) were set equal to 1. Data are presented as means ± SE, n=3. (A) Up-regulation of Bach1 protein levels by let-7 miRNA inhibitors; (B) Densimetric analysis of Bach1 and GAPDH band intensities (*p=0.009, let-7 inhibitors vs MINC); (C) Lack of effect of let-7 miRNA inhibitors on Bach1 mRNA levels.

Figure 5. Effects of let-7 miRNA mimics and inhibitors on expression of the HMOX1 gene in human Huh-7 cells

Huh-7 cells were transfected with let-7 miRNA mimics, MMNC, let-7 miRNA inhibitors (a combination of let-7a, let-7c and let-7e inhibitors) or MINC by Lipofectamine 2000 for 48 h. Cells were harvested, and total RNA was then extracted. The levels of HMOX1 mRNA were assessed by real-time qRT-PCR. The amounts of HMOX1 mRNA were normalized to GAPDH which did not vary with transfection. Values for cells with mimic or inhibitor negative control transfection were set equal to 1. Data are presented as means ± SE, n=3. (A) let-7 miRNA mimics up-regulate HMOX1 mRNA levels (*differs from MMNC only, p=0.004, let-7b; p=0.002, let-7c; and p=0.001, miR-98); (B) let-7 miRNA inhibitors down-regulate HMOX1 mRNA levels (*p=0.005, let-7 inhibitors vs MINC).

Figure 6. Over-expression of let-7b protects cells from oxidative injury in Huh-7 cells

Huh-7 cells were transfected for 48 h with 50 nM of let-7b or MMNC by Lipofectamine 2000. Cells were then incubated with the indicated concentrations of tBuOOH for 4 or 6 h. CellTiter-Glo® Reagent was added for CellTiter-Glo luminescent cell viability assay on a Synergy HT microplate reader with integration time set for 0.25 to 1 second. Decreases in luminescence were taken as an index of cellular cytotoxicity. Data represent means ± SE of triplicate determinations. Data are presented as means ± SE, n=3. * differs from MMNC only, P<0.05. Susceptibility of Huh-7 cells transfected with MMNC and let-7b to tBuOOH induced oxidative injury was compared. (A) 4 h after tBuOOH treatment (*differs from MMNC, p= 0.03); (B) 6 h after tBuOOH treatment (*differs from MMNC, p=0.03); (C) WB analysis of over-expression of Bach1 by pCMV-Bach1; (D) The protection of cells by let-7b against oxidative injury was ablated following over-expression of Bach1. Huh-7 cells were transfected with 0.4 µg/mL of pCMV-Bach1 or pCMV-XL5, a control vector without Bach1 cDNA insertion for 24 h, and then transfected with 50 nM of let-7b or MMNC for 48 h. Cells were then incubated with 400 µM of tBuOOH for 6 h. Cell viability assays were performed as described above. Data are presented as means ± SE, n=3. ^#p=0.006 and *p=0.001.

Figure 6. Over-expression of let-7b protects cells from oxidative injury in Huh-7 cells

Huh-7 cells were transfected for 48 h with 50 nM of let-7b or MMNC by Lipofectamine 2000. Cells were then incubated with the indicated concentrations of tBuOOH for 4 or 6 h. CellTiter-Glo® Reagent was added for CellTiter-Glo luminescent cell viability assay on a Synergy HT microplate reader with integration time set for 0.25 to 1 second. Decreases in luminescence were taken as an index of cellular cytotoxicity. Data represent means ± SE of triplicate determinations. Data are presented as means ± SE, n=3. * differs from MMNC only, P<0.05. Susceptibility of Huh-7 cells transfected with MMNC and let-7b to tBuOOH induced oxidative injury was compared. (A) 4 h after tBuOOH treatment (*differs from MMNC, p= 0.03); (B) 6 h after tBuOOH treatment (*differs from MMNC, p=0.03); (C) WB analysis of over-expression of Bach1 by pCMV-Bach1; (D) The protection of cells by let-7b against oxidative injury was ablated following over-expression of Bach1. Huh-7 cells were transfected with 0.4 µg/mL of pCMV-Bach1 or pCMV-XL5, a control vector without Bach1 cDNA insertion for 24 h, and then transfected with 50 nM of let-7b or MMNC for 48 h. Cells were then incubated with 400 µM of tBuOOH for 6 h. Cell viability assays were performed as described above. Data are presented as means ± SE, n=3. ^#p=0.006 and *p=0.001.