Silenced lncRNA H19 and up-regulated microRNA-129 accelerates viability and restrains apoptosis of PC12 cells induced by Aβ25-35 in a cellular model of Alzheimer's disease

Abstract

Accumulating data manifest that long non-coding RNA (lncRNAs) are involved in all kinds of neurodegenerative disorders, consisting of the onset and progression of Alzheimer's disease (AD). The study was for the research of the mechanism of lncRNA H19 (H19) in viability and apoptosis of PC12 cells induced by Aβ_25-35 in a cellular model of AD with the regulation of microRNA (miR)-129 and high mobility group box-1 protein (HMGB1). An AD cellular model of PC12 cells was established using Aβ_25-35. The Aβ_25-35-induced PC12 cells were transfected with si-H19 or miR-129 mimic to figure their roles in cell viability,apoptosis, mitochondrial membrane potential dysfunction and oxidative stress in AD. Luciferase reporter assay and RNA-pull down assay were employed for verification of the binding relationship between H19 and miR-129 and the targeting relationship between miR-129 and HMGB1. An AD mouse model was induced and brain tissues were collected. H19, miR-129 and HMGB1 were detected in Aβ_25-35-treated cells and brain tissues of AD mice. Elevated H19, HMGB1 and decreased miR-129 were found in Aβ_25-35-treated PC12 cells as well as in brain tissues of AD mice. Silenced H19 or elevated miR-129 promoted viability, inhibited apoptosis, prevented mitochondrial membrane potential dysfunction and decreased oxidative stress in Aβ_25-35-treated PC12 cells. H19 could specifically bind to miR-129. MiR-129 specifically suppressed HMGB1 expression. This study suggests that silenced H19 and up-regulated miR-129 accelerates viability and represses apoptosis of PC12 cells stimulated by Aβ_25-35 in AD, which is beneficial for AD treatment.

Keywords: Alzheimer’s disease; Aβ25-35; MicroRNA-129; PC12 cells; apoptosis; high mobility group box-1 protein; long non-coding RNA H19; viability.

Figure 1.

Observation of the morphology of PC12 cells induced by Aβ_25-35. A. Observation of the morphology of PC12 cells induced by Aβ_25-35 under a microscope; B. The viability of PC12 cells induced by Aβ_25-35 tested by MTT assay, N = 3. Data were expressed as mean ± standard deviation. One way ANOVA was functioned for comparison among multiple groups, followed by Tukey's post hoc test

Figure 2.

Elevation of H19, HMGB1 and reduction of miR-129 exhibit in PC12 cells after induction of Aβ_25-35 A. H19, miR-129, HMGB1 expression in PC12 cells treated with different concentrations of Aβ_25-35 detected via RT-qPCR; B. HMGB1 protein bands in PC12 cells treated with different concentrations of Aβ_25-35; C. HMGB1 protein expression in PC12 cells treated with different concentrations of Aβ_25-35 detected via western blot analysis; D. H19, miR-129, HMGB1 expression in Aβ_25-35-treated PC12 cells after transfection detected via RT-qPCR; E. HMGB1 protein bands in Aβ_25-35-treated PC12 cells after transfection detected via western blot analysis; F. HMGB1 protein expression in Aβ_25-35-treated PC12 cells after transfection detected via western blot analysis. The data in the figure were all measurement data, in the form of mean ± standard deviation. Student’s t test was used to evaluate significant differences between two groups of data. One-way ANOVA was functioned for comparison among multiple groups. The pairwise comparison after ANOVA analysis used Tukey’s post hoc test, N = 3, In the A and C, + vs 0 μmol/L, P < 0.05; In the D and F, * vs the si-NC group, P < 0.05; # vs the mimic NC group; & vs the OE-H19 + mimic NC group, P < 0.05

Figure 3.

Silence of H19 or elevation of miR-129 facilitate viability and colony formation ability of PC12 cells induced by Aβ_25-35. A. The viability of cells detected by MTT assay; B. The cell colony formation ability tested by colony formation assay; C. Quantification results of panel B; D. Ki-67 and P53 mRNA expression in PC12 cells tested by RT-qPCR; The data in the figure were all measurement data, in the form of mean ± standard deviation. One-way ANOVA was functioned for comparison among multiple groups. Tukey’s post hoc test was employed in pairwise comparison after ANOVA analysis. N = 3, * vs the si-NC group, P < 0.05; # vs the mimic NC group, P < 0.05; & vs the OE-H19 + mimic NC group, P < 0.05

Figure 4.

Decreased H19 or elevated miR-129 accelerate cell cycle entry and restrain apoptosis of PC12 cells induced by Aβ_25-35. A. Aβ_25-35-treated PC12 cell cycle distribution tested by flow cytometry; B. Quantification results of panel A; C. The apoptosis of Aβ_25-35-treated PC12 cells tested by flow cytometry; D. Quantification results of panel C; E. The apoptosis of Aβ_25-35-treated PC12 cells detected via Hoechst 33,258 staining; F. Bax and Bcl-2 mRNA expression in Aβ_25-35-treated PC12 cells tested by RT-qPCR; The data in the figure were all measurement data, in the form of mean ± standard deviation. One-way ANOVA was functioned for comparison among multiple groups. Tukey’s post hoc test was employed in pairwise comparison after ANOVA analysis. N = 3, * vs the si-NC group, P < 0.05; # vs the mimic NC group, P < 0.05; & vs the OE-H19 + mimic NC group, P < 0.05

Figure 5.

Reduction of H19 or elevation of miR-129 decrease ROS and elevate MMP expression in PC12 cells induced by Aβ_25-35. A. ROS in Aβ_25-35-treated PC12 cells after transfection; B. MMP in Aβ_25-35-treated PC12 cells after transfection. The data in the figure were all measurement data, in the form of mean ± standard deviation. One-way ANOVA was functioned for comparison among multiple groups. Tukey’s post hoc test was employed in pairwise comparison after ANOVA analysis. N = 3, * vs the si-NC group, P < 0.05; # vs the mimic NC group, P < 0.05; & vs the OE-H19 + mimic NC group, P < 0.05

Figure 6.

Inhibited H19 or up-regulated miR-129 elevate SOD and CAT and reduce MDA in Aβ_25-35 induced PC12 cells. A. MDA in Aβ_25-35-treated PC12 cells after transfection; B. SOD activity in Aβ_25-35-treated PC12 cells after transfection; C. CAT expression in Aβ_25-35-treated PC12 cells after transfection. The data in the figure were all measurement data, in the form of mean ± standard deviation. One-way ANOVA was functioned for comparison among multiple groups. Tukey’s post hoc test was employed in pairwise comparison after ANOVA analysis. N = 3, * vs the si-NC group, P < 0.05; # vs the mimic NC group, P < 0.05; & vs the OE-H19 + mimic NC group, P < 0.05

Figure 7.

H19 competitively regulates miR-129. A. H19 subcellular localization predicted via online analysis site; B. H19 subcellular localization verified by FISH assay; C. The binding site of H19 to miR-129 predicted via RNA22 site; D. The binding of H19 to miR-129 verified via luciferase activity assay; E. The enrichment of miR-129 on H19 tested via RNA pull-down assay; The data in the figure were all measurement data, in the form of mean ± standard deviation. Student’s t test was used to evaluate significant differences between two groups of data. One-way ANOVA was functioned for comparison among multiple groups, followed by Tukey’s post hoc test. N = 3

Figure 8.

The targeting relationship between HMGB1 and miR-129. A. The target site for binding of HMGB1 and the corresponding miR-129 predicted via Target Scan; B. Detection results of dual-luciferase reporter gene assay. The data represented the mean ± standard deviation of three independent experiments. Student’s t test was used to evaluate significant differences between two groups of data. N = 3.* vs the mimic NC group, P < 0.05

Figure 9.

H19 and HMGB1 are elevated and miR-129 is suppressed in AD mice. A. H19, miR-129 and HMGB1 expression in brain tissues of AD mice via RT-qPCR; B. HMGB1 protein expression in brain tissues of AD mice via Western blot analysis. The two-group comparison was performed by independent sample t test for statistical analysis. n = 10. * vs the sham group, P < 0.05