miR-497 and miR-302b regulate ethanol-induced neuronal cell death through BCL2 protein and cyclin D2 - PubMed (original) (raw)
miR-497 and miR-302b regulate ethanol-induced neuronal cell death through BCL2 protein and cyclin D2
Sanjay Yadav et al. J Biol Chem. 2011.
Abstract
In chronic alcoholism, brain shrinkage and cognitive defects because of neuronal death are well established, although the sequence of molecular events has not been fully explored yet. We explored the role of microRNAs (miRNAs) in ethanol-induced apoptosis of neuronal cells. Ethanol-sensitive miRNAs in SH-SY5Y, a human neuroblastoma cell line, were identified using real-time PCR-based TaqMan low-density arrays. Long-term exposure to ethanol (0.5% v/v for 72 h) produced a maximum increase in expression of miR-497 (474-fold) and miR-302b (322-fold). Similar to SH-SY5Y, long-term exposure to ethanol induced miR-497 and miR-302b in IMR-32, another human neuroblastoma cell line. Using in silico approaches, BCL2 and cyclin D2 (CCND2) were identified as probable target genes of these miRNAs. Cotransfection studies with 3'-UTR of these genes and miRNA mimics have demonstrated that BCL2 is a direct target of miR-497 and that CCND2 is regulated negatively by either miR-302b or miR-497. Overexpression of either miR-497 or miR-302b reduced expression of their identified target genes and increased caspase 3-mediated apoptosis of SH-SY5Y cells. However, overexpression of only miR-497 increased reactive oxygen species formation, disrupted mitochondrial membrane potential, and induced cytochrome c release (mitochondria-related events of apoptosis). Moreover, ethanol induced changes in miRNAs, and their target genes were substantially prevented by pre-exposure to GSK-3B inhibitors. In conclusion, our studies have shown that ethanol-induced neuronal apoptosis follows both the mitochondria-mediated (miR-497- and BCL2-mediated) and non-mitochondria-mediated (miR-302b- and CCND2-mediated) pathway.
Figures
FIGURE 1.
Effect of either short-term (4 h, A and B) or long-term (72 h, C and D) ethanol exposure on viability of SH-SY5Y cells transfected with NTC siRNA or dicer siRNA. Cell viability is measured by MTT (A and C) and NRU (B and D) assays. The viability of cells transfected with NTC siRNA and exposed to different concentration of ethanol (either short-term or long-term) is compared with the viability of cells transfected with NTC siRNA and not exposed to ethanol (control = 100%). Similarly, the viability of cells transfected with dicer siRNA and exposed to ethanol (either short-term or long-term) is compared with the viability of cells transfected with dicer siRNA and not exposed to ethanol. All values are mean of four different experiments, and the error bars represent + S.E. of the mean. Statistical significance between control and different ethanol exposed groups was calculated by one-way analysis of variance followed by a post hoc Tukey test. Values showing p < 0.05 in the Tukey test are considered statistically significant and are marked a, b, c, d, e, and f. (Dicer KD: dicer knockdown (KD)).
FIGURE 2.
Global expression profiling of miRNAs by real-time PCR using TLDA arrays in SH-SY5Y cells. A, volcano plot of miRNA expression in SH-SY5Y cells after either short-term exposure (A and B) or long-term exposure (C and D) to ethanol. Fig. 1, A and C, represents the set of miRNAs present in pool A of the TLDA plates. Fig. 1, B and D, represents the set of miRNAs present in pool B of the TLDA plates. For short-term exposure, cells are exposed to 2.0% v/v ethanol for 4 h, and for long-term exposure, cells were exposed to 0.5% v/v ethanol for 72 h. The volcano plot is plotted between log2 of the fold changes versus its p value obtained from the Student's t test. The black line with the arrowhead in the center indicates a fold change in the gene expression of 1. Parallel to the central line, two vertical lines, one on each side, indicate a + 3-fold change in gene expression. The single horizontal line inside the boxes indicates the threshold for the p value of the t test, set as 0.05. E, the top five miRNAs altered after short-term exposure of ethanol. F, the top five miRNAs altered after long-term exposure to ethanol. Cells not exposed to ethanol are considered as control and are used for calculation of the fold change with the -ΔΔ cycle threshold method (control = 1). #, MiR-369–3p had not amplified in control sample, so for calculations, their Ct is taken as 35 (final number of the cycle). In the bar diagram, fold change is represented by multiplying it from 104. All values are the mean of three individual experiments. Significant changes are calculated by Student's t test. *, p < 0.05.
FIGURE 3.
Induction of miR-497 and miR-302b by ethanol exposure in IMR-32 cells (A) and effect of ectopic expression of these miRNAs on apoptosis of SH-SY5Y (B and C) and IMR-32 (D and E) cells. A, real-time PCR of miR-497 and miR-302b in control and ethanol-exposed (long-term exposure, 0.5% v/v × 72 h) IMR-32 cells. B, dot plots of cell death analysis by FITC annexin V staining followed by flow cytometry analysis in SH-SY5Y cells transfected with either NTC, Syn-miR-497, or Syn-mir-302b. C, bar diagram analysis of total cell death in SH-SY5Y cells as observed by flow cytometry analysis. D, dot plots of cell death analysis by FITC annexin V staining followed by flow cytometry analysis in IMR-32 cells transfected with either NTC, Syn-miR-497, or Syn-mir-302b. E, bar diagram analysis of total cell death of IMR-32 cells as observed by flow cytometry analysis. Dot plots are plotted between FITC-A and PE-Texas Red. Q1, quadrant I (necrotic cells); Q2, quadrant 2 (late apoptotic cells); Q3, quadrant 3 (living cells); Q4, quadrant 4 (early apoptotic cells). For calculating fold change in total cell death, fold change in early apoptosis, late apoptosis, and necrosis is calculated individually and added. Cells transfected with NTC are considered as control and used for calculating fold change in early apoptosis, late apoptosis, or necrosis of cells transfected with Syn-miR-497 or Syn-miR-302b. All values are the mean of three individual experiments. Significant changes are calculated by Student's t test. *, p < 0.05. RQ, relative quantification.
FIGURE 4.
Ectopic expression of miR-497 or miR-302b induced caspase 3 activity (A) and down-regulated expression of BCL2 and/or CCND2 (B_–_D) by targeting 3′-UTR of these genes (E-I and E-II). A, caspase 3 activity measured in lysates of cells transfected with NTC (control = 100%), Syn-miR-497, or Syn-miR-302b using the fluorogenic substrate Ac-DEVD-AMC. Fluorescence was measured using a multiple-well plate reader at an excitation wavelength of 380 nm and an emission wavelength of 450 nm. B, effect of ectopic expression of miR-497, miR-302b, or NTC (control = 1) on transcript levels of BCL2 or CCND2 as measured by real-time PCR. Expression of BCL2 and CCND2 in cells transfected with Syn-miR-497 or Syn-miR-302b is compared with their expression in cells transfected with NTC (control = 1). C, effect of ectopic expression of miR-497, miR-302b, or NTC on protein levels of BCL2 or CCND2 measured by immunoblotting in cell lysates of SH-SY5Y cells. Lane 1, NTC; lane 2, Syn-miR-497; lane 3, Syn-miR-302b. D, densitometry of immunoblots of BCL2 and CCND2 shown in B. E, luciferase assays in SH-SY5Y cells cotransfected with the pEZX-MT01 vector with 3′-UTR of BCL2 (I) or CCND2 (II) and miRNA mimics or NTC (control = 100%). When cells were transfected with NTC siRNAs, the empty pEZX-MT01 vector was used for cotransfection. Both firefly luciferase and Renilla luciferase activities were measured as described under “Experimental Procedures,” and firefly luciferase activity was normalized with Renilla luciferase activities in the same well. All the values are the mean of three individual experiments. Significant changes are calculated by Student's t test. *, p < 0.05.
FIGURE 5.
Effect of ectopic expression of miR-497, miR-302b, or NTC (control) on ROS formation (A and B) and MMP loss (C–E) in SH-SY5Y cells. A, ROS formation was measured by flow cytometry in cells transfected with Syn-miR-497, Syn-miR-302b, or NTC (control) as described under “Experimental Procedures. P2, basal fluorescence; P3, increased bright fluorescence. B, % change in ROS formation as measured by flow cytometry in A. C, flow cytometry analysis of MMP loss using JC-1, a dual-emission potential-sensitive probe in SH-SY5Y cells transfected with Syn-miR-497, Syn-miR-302b, or NTC (control). D, bar diagram for representing percent of MMP loss observed after flow cytometry in C. E, fluorescence imaging of cells incubated with JC-1 for validating MMP loss induced by ectopic expression of miRNAs. 1-3, cells transfected with NTC. 4-6, cells transfected with Syn-miR-497. 7-9, cells transfected with Syn-miR-302b. All values are the mean of three individual experiments. Significant changes are calculated by Student's t test. *, p < 0.05.
FIGURE 6.
Effect of ectopic expression of miR-497, miR-302b, or NTC (control) on cytochrome c release in SH-SY5Y cells. A, levels of VDAC-1 (mitochondrial loading control) and β-actin (cytosolic loading control) in mitochondrial fraction (Mito-F) and cytosolic fraction (Cyto-F) of cells transfected with NTC, Syn-miR-497, or Syn-miR-302b. B, immunoblotting of cytochrome c in Mito-F and Cyto-F of cells transfected with NTC, Syn-miR-497, or Syn-miR-302b. C, percent distribution of cytochrome c between mitochondrial and cytosolic fraction calculated by densitometry of cytochrome c immunoblots. After densitometry, the amount of cytochrome c detected in the mitochondrial and cytosolic fraction of NTC-, Syn-miR-497-, or Syn-miR-302b-transfected cells is added separately and considered as 100%. All values are the mean of three individual experiments. Significant changes are calculated by Student's t test. *, p < 0.05.
FIGURE 7.
Effect of pre-exposure of GSK-3B inhibitors on ethanol-induced death of SH-SY5Y cells. A, cells were exposed to ethanol (E, 0.5% × 72 h), lithium (L, 20 m
m
) + E, TDZD-8 (T, 10 μ
m
) + E, or vehicle control (VC). The cell death assay was carried out by FITC annexin V staining followed by flow cytometry analysis. Data are presented as dot plots (FITC-A plotted against PE-Texas Red). Q1, quadrant I (necrotic cells); Q2, quadrant 2 (early apoptotic cells); Q3, quadrant 2 (living cells); Q4, quadrant 4 (late apoptotic cells). B, bar diagram analysis of total cell death as observed by flow cytometry analysis in A. For calculating fold change in total cell death, the fold change in early apoptosis, late apoptosis, and necrosis is calculated individually and added. The fold change in early apoptosis, late apoptosis, and necrosis of E-, L+E-, or T+E-exposed groups is calculated against the vehicle control. Significant changes are calculated by one-way analysis of variance followed by a pairwise Tukey test. Values showing p < 0.05 in the Tukey test are considered statistically significant. a, p < 0.05 when compared with vehicle control; b, p < 0.05 when compared with ethanol-exposed cells).
FIGURE 8.
Effect of pre-exposure of GSK-3B inhibitors (L, lithium or T, TDZD-8) on ethanol-induced alterations in expression of miR-497, miR-302b, BCL2, or CCND2. A–D, expression of miR497 (A), miR-302b (B), BCL2 (C), and CCND2 (D) measured by real-time PCR in SH-SY5Y cells exposed with GSK-3B inhibitors and ethanol. E, immunoblotting of BCL2, CCND2, and β-actin in total cell lysates of SH-SY5Y cells exposed with GSK-3B inhibitors and ethanol. F, densitometry of immunoblots. The concentrations used for exposure are ethanol (E), 0.5% × 72 h; lithium (L), 20 m
m
; and TDZD-8 (T), 10 μ
m
. The expression of miR-497 and miR-302b was measured by using TaqMan assays, whereas expression of BCL2 and CCND2 was measured using SYBR Green chemistry as mentioned under “Experimental Procedures.” The fold change in expression of miR-497 and miR-302b was calculated by considering their levels as 1-fold in cells exposed to vehicle control. Significant changes are calculated by one-way analysis of variance followed by a pairwise Tukey test. a, p < 0.05 versus vehicle control; b, p < 0.05 versus ethanol.
FIGURE 9.
Pictorial summary of results. The arrows with ● at the bottom indicate a connection between two events. ↑ indicate an increase in activity or expression, and ↓ indicate decreased expression.
References
- Li X., Jin P. (2010) Nat. Rev. Neurosci. 11, 329–338 - PubMed
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