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, 16 (3), 2522-2528

MicroRNA‑21 Promotes Neurite Outgrowth by Regulating PDCD4 in a Rat Model of Spinal Cord Injury

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MicroRNA‑21 Promotes Neurite Outgrowth by Regulating PDCD4 in a Rat Model of Spinal Cord Injury

Yuqing Jiang et al. Mol Med Rep.

Abstract

Altered expression levels of microRNA‑21 (miRNA‑21) have been observed in a series of pathological processes, including cancer and central nervous system injury; however, the involvement of miRNA‑21 in the molecular pathophysiology of spinal cord injury (SCI) has not been well documented. The present study examined the expression levels of miRNA‑21 and its predicted target genes, programmed cell death 4 (PDCD4) and phosphatase and tensin homolog (PTEN), in rats using quantitative polymerase chain reaction and western blotting to further understand the role of miRNA‑21 and the mechanisms underlying repair following SCI. The present study demonstrated that compared with uninjured spinal cords, miRNA‑21 expression levels were significantly downregulated in injured spinal cords 4 and 8 h, and 1 day post‑SCI, and were significantly upregulated after 3 and 7 days. Conversely, expression levels of PDCD4 and PTEN were significantly decreased at days 3 and 7 post‑SCI compared with the control group. miRNA‑21 overexpression in monolayer‑cultured postnatal rat spinal cord neurons promoted neurite outgrowth and downregulated protein expression levels of PDCD4; however, PTEN protein expression levels were unaltered. To confirm that miRNA‑21 directly targets PDCD4, a pRL‑CMV luciferase reporter construct was used to detect miRNA‑21 interactions with the PDCD4 3'‑untranslated region. The results demonstrated that miRNA‑21 decreased luciferase activity compared with a rat PDCD4 control reporter. The results of the present study suggested that increased miRNA‑21 expression levels following SCI may promote the repair of injured spinal cords by inhibiting the expression of its target gene PDCD4.

Figures

Figure 1.
Figure 1.
Altered expression of miR-21, PDCD4 and PTEN following SCI. (A) miR-21 expression levels were significantly reduced 4 and 8 h, and 1 day post-SCI compared with the control group, whereas miR-21 expression levels were significantly increased at days 3 and 7 post-SCI compared with the control group. (B) Representative western blot images and quantification of (C) PDCD4 and (D) PTEN protein expression levels. The expression levels of PDCD4 and PTEN were decreased significantly at days 3 and 7 post-SCI compared with the control group. Data are expressed as the mean ± standard deviation (n=4/group). *P<0.05 vs. control group. miR-21, microRNA-21; SCI, spinal cord injury; PDCD4, programmed cell death 4; PTEN, phosphatase and tensin homolog; con, control.
Figure 2.
Figure 2.
miR-21 promotes neurite outgrowth in rat spinal cord neurons. (A) Neurite outgrowth (detected by β-III tubulin in red) observed in control or miR-21-transfected spinal cord neurons. Scale bar, 100 µm. (B) miR-21 increased the mean length of the longest neurite (per neuron) compared with the control group (n=5/group). (C) Representative western blot images and (D) quantification of PDCD4 protein expression levels following transfection with pri-miR-21 or in-miR-21. β-actin served as an internal control. miR-21 significantly downregulated the expression levels of PDCD4. Data are expressed as the mean ± standard deviation (n=4/group). *P<0.05 vs. control group. miR-21, microRNA-21; PDCD4, programmed cell death 4; PTEN, phosphatase and tensin homolog; pri, primary; in, inhibitor; nc, negative control.
Figure 3.
Figure 3.
miR-21 targets the PDCD4 gene. (A) Alignment of the 3′-UTR sequence of PDCD4 in three species with miR-21, highlighting the fully conserved sequence in bold. (B) Relative luciferase expression in rat PDCD4 luciferase reporters co-transfected with miR-21-expressing constructs in HEK293 cells. miR-21 decreased luciferase activity in the miR-21-transfected group by 45±5.1% compared with the control and negative control groups. Data are expressed as the mean ± standard deviation (n=5/group). **P<0.01. miR-21, microRNA-21; PDCD4, programmed cell death 4; neg, negative control.

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