Silencing of Long Non-Coding RNA (lncRNA) Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) Protects PC-12 Cells from LPS-Induced Injury via Targeting miR-29a

Abstract

BACKGROUND Spinal cord injury (SCI) is a debilitating neuropathological condition that significantly affects the quality of life. The present study is basic research examining the underlying mechanisms of NEAT1 and miR-29a in regulating LPS-induced PC-12 cell injury. MATERIAL AND METHODS The model of cell injury was induced by the treatment of PC-12 cells with LPS. The expressions of NEAT1, miR-29a, and inflammatory cytokines were measured by real-time quantitative polymerase chain reactions (RT-qPCR). Cell proliferation and apoptosis were evaluated by CCK-8 and flow cytometry, respectively. Finally, the target between miR-29a and NEAT1 as well as miR-29a and BCL2L11 was investigated by luciferase and RNA pull-down assays. RESULTS Knockdown of NEAT1 can inhibit inflammatory cytokine expression and PC-12 cell apoptosis and promote PC-12 cell proliferation by targeting miR-29a. However, the variation caused by NEAT1 knockdown can be reversed by the silencing of miR-29a and the overexpression of BCL2L11, which is the direct target gene of miR-29a. CONCLUSIONS High NEAT1 levels can increase LPS-induced injury in PC-12 cells through the miR-29a/BCL2L11 pathway. lncRNA NEAT1 may, therefore, be a promising target for SCI treatment.

Figure 1

The effect of LPS treatment on PC-12 cells. (A) PC-12 cell viability after LPS treatment was detected by CCK-8. (B) PC-12 cell apoptosis after LPS treatment was detected by flow cytometry. (C, D) Expression levels of inflammatory cytokines after LPS treatment were detected by ELISA. (E) Relative expression of lncRNA NEAT1 after LPS treatment was assessed by qPCR. * P<0.05 vs. 0 μg/ml, ** P<0.01 vs. 0 μg/ml.

Figure 2

The effect of NEAT1 knockdown on PC-12 cells after LPS treatment. (A) The relative expression of LncRNA NEAT1 after Ad-si-NEAT1 infection. (B) PC-12 cell viability after LPS treatment and Ad-si-NEAT1 infection was detected by CCK-8. (C) Expression levels of inflammatory cytokines after LPS treatment were evaluated by ELISA. (D, E) PC-12 cell apoptosis after LPS treatment and Ad-si-NEAT1 infection was detected by flow cytometry and TUNEL staining. * P<0.05 vs. control,** P<0.01 vs. control, ^# P<0.05 vs. LPS+si-nc.

Figure 3

Reciprocal inhibition between lncRNA NEAT1 and miR-29a in HEK-293 cells. (A) The sequences of wild-type and mutant-type vectors and putative binding sites of miR-29a within lncRNA NEAT1 mRNA. (B) The relative luciferase activities were detected by dual-luciferase reporter assay in HEK-293 cells co-transfected with wild-type and mutant-type vectors together with miR-29a mimics and miR-nc. (C) The expression level of lncRNA NEAT1 mRNA was measured by RT-qPCR in the sample pulled down by biotinylated miR-29a. (D) The relative expression of miR-29a in PC-12 cells after infection with Ad-si-NEAT1 or si-nc. * P<0.05 vs. miR-nc, ** P<0.01 vs. biotin-nc or si-nc.

Figure 4

Upregulation of miR-29a counteracted the changes induced by Ad-si-NEAT1 in PC-12 cells after LPS treatment. (A) The relative expression of miR-29a in different groups was evaluated by qPCR. (B) PC-12 cell viability after LPS treatment or transduction of Ad-si-NEAT1 or si-miR-29a was assessed by CCK-8. (C–E) PC-12 cell apoptosis was evaluated by flow cytometry and TUNEL staining. (F) Expression levels of inflammatory cytokines was detected by ELISA assay. * P<0.05 vs. control, ** P<0.01 vs. control, ^# P<0.05 vs. LPS+si-nc, ^& P<0.05 vs. LPS+si-NEAT1+si-nc.

Figure 5

miR-29a targeted the BCL2L11 gene in HEK-293 cells. (A) The sequences of wild-type and mutant-type vectors and putative binding sites of miR-29a within BCL2L11 mRNA. (B) The relative luciferase activities were detected in HEK-293 cells co-transfected with vectors containing the wild- or mutant-type of BCL2L11 together with miR-29a mimics or miR-nc. (C, D) The relative mRNA and protein expression of BCL2L11 after transfection of miR-29a mimic or si-miR-29a. (E) The expression level of BCL2L11 mRNA was measured by RT-qPCR in the sample pulled down by biotinylated miR-29a. (F) The correlation between miR-29a and BCL2L11 was analyzed by Pearson analysis. * P<0.05 vs. miR-nc or biotin-nc, ** P<0.01 vs. miR-nc, ^# P<0.01 vs. si-nc.

Figure 6

NEAT1 modulated the BCL2L11 and the apoptotic signal pathway. (A) Pearson analysis was performed to evaluate the correlation between NEAT1 and BCL2L11. (B) The expression of BCL2L11, bax, pro-caspase3, caspase3, and bcl-2 was evaluated by Western blot.