miR-98 and its host gene Huwe1 target Caspase-3 in Silica nanoparticles-treated male germ cells

Abstract

Silica nanoparticles (NP) is one of the most commonly used nanomaterials with potential health hazards. However, the effects of Silica NP on germ cells and the underlying mechanisms are still unclear. In this study, GC-2 and TM-4, which are two different types of male germ cells were exposed to Silica NP for 24h, and then general cytotoxicity and multi-parameter cytotoxicity were evaluated. Our results showed that Silica NP could induce apoptosis in GC-2 cells. Transmission electron microscopy (TEM) results showed that Silica NP was localized in the lysosomes of GC-2 cells. High content screening (HCS) showed that Silica NP exposure could increased cell permeabilization and decreased mitochondrial membrane potential in GC-2 cells. The mRNA and protein levels of apoptosis markers (Bax, Caspase-3, Caspase-9) in GC-2 cells were significantly increased, while Bcl-2 was decreased. Accordingly, the expression level of miR-98, which can regulate Caspase-3, was significantly decreased. Huwe1, the host gene of miR-98, was positively associated with miR-98 expression after Silica NP exposure. Dual luciferase reporter assay suggested that miR-98 directly targets Caspase-3. These results suggest that Silica NP induces apoptosis via loss of mitochondrial membrane potential and Caspase-3 activation, while miR-98 plays key role in modulating this effect.

Figure 1. The hydrodynamic diameter and TEM images of cells.

(A) The hydrodynamic diameter of Silica NP in serum medium. (B) The TEM image of Silica NP. (C) Randomly selected three ultrathin slices were observed under TEM. Randomly selected cells from ten fields of each ultrathin slice were surveyed. Representative images of GC-2 cells exposed to Silica NP and control for 24 h.

Figure 2. Effects of Silica NP on cell viability in GC-2 and TM-4 cells.

(A and B) Cell viability was determined by MTT assay after exposure to various concentrations of Silica NP for 24 h and 48 h. Values of the experiment were represented as means ± S.E. from five separate experiments in which treatments were performed in quadruplicate.

Figure 3. Effects of Silica NP on cell cycle/apoptosis in GC-2 and TM-4 cells.

Cells were cultured with various concentrations of Silica NP (0.1, 1, 10, 100 μg/ml) or control medium for 24 h. (A and B) Results quantitated in cell cycle were shown in A and B. (C and D) The percentage of apoptotic cells in GC-2 and TM-4 cells were also separately presented in histogram. LL indicated that they were live cells. LR indicated cells were in the early stages of apoptosis. UR indicated cells were in late stages of apoptosis. UL indicated that they were dead cells. Each data point was represented as the means ± S.E. from three separate experiments in which treatments were performed in triplicate. *indicates significant difference when the values were compared to that of the control (p < 0.05).

Figure 4. Representative images from the high-content screen (HCS) after Silica NP exposure to GC-2 cells.

(A) Staining for nucleus (blue), cell membrane permeability (green), cytochrome c (yellow) and mitochondrial membrane potential (red) in GC-2 cells. Images were acquired with the ArrayScan HCS Reader with a 20 objective. (B) The relative expression of nuclear size, permeability, cytochrome c and mitochondrial membrane potential in GC-2 cells. *indicates significant difference when the values were compared to that of the control (p < 0.05).

Figure 5. The expression of apoptosis factors and miR-98 in GC-2 cells.

Cells were cultured with various concentrations of Silica NP (0.1, 1, 10, 100 μg/ml) or control medium for 24 h. (A) Caspase-3/Caspase-9/Bcl-2/Bax mRNA levels were determined by quantitative RT-PCR using a housekeeping gene GAPDH as an internal control. (B) Caspase-3/Caspase-9/Bcl-2/ Bax protein levels were determined by western blot. The blots in panel band were performed on the same blot membranes and shown as cropped images. (C and D) The expression levels of miR-98 and Huwe1 were determined by quantitative RT-PCR. (E) Correlation between the levels of miR-98 and Caspase-3 by Pearson correlation analysis (R² = 0.7809, p < 0.001). (F) Correlation between the levels of miR-98 and Huwe1 by Pearson correlation analysis (R² = 0.3838, p < 0.001). Each data point was normalized to the control and represented the means ± S.E. from three independent experiments. *indicates significant difference when the values were compared to that of the control (p < 0.05).

Figure 6. Over-expression of miR-98 reduced Caspase-3 expression.

Cells were transfected with 50 nM miR-98 mimics or control for 24 h. (A) qRT-PCR was performed to evaluate the expression level of miR-98. (B) The relative mRNA expression levels of Caspase-3 after transfection. (C and D) The protein levels of Caspase-3 after transfection. The blots in panel band were performed on the same blot membranes and shown as cropped images. (E) Sequence alignment of miR-98 with 3′ UTR of Caspase-3. Bottom: mutations in the 3′UTR of Caspase-3 in order to create the mutant luciferase reporter constructs. (F) Cells were co-transfected with miR-98 mimics or control, renilla luciferase vector pRL-SV40 and Caspase-3 3′UTR luciferase reporters for 24 h. Both firefly and Renilla luciferase activities were measured in the same sample. Firefly luciferase signals were normalized with Renilla luciferase signals. Reporter activity was significantly decreased after miR-98 overexpression compared to control. *indicates significant difference compared with that of control cells (p < 0.05). All tests were performed in triplicate and presented as means ± SE.