The role of the miR-99b-5p/mTOR signaling pathway in neuroregeneration in mice following spinal cord injury

Abstract

The present study aimed to investigate the role of microRNA (miR)‑99b‑5p in spinal cord injury (SCI). Reverse transcription‑quantitative polymerase chain reaction demonstrated that, compared with control mice, the expression levels of miR‑99b‑5p were upregulated in the mouse spinal cord following SCI. Mechanistic target of rapamycin (mTOR) was predicted to be the possible target of miR‑99b‑5p according to TargetScan and microrna databases. Dual‑luciferase reporter assay verified that miR‑99b‑5p was able to target mTOR. Furthermore, the results of an apoptosis analysis demonstrated that there were few apoptotic neurons in the control group, whereas SCI induced a significant increase in the number of apoptotic cells. Conversely, apoptosis was inhibited following transfection with a miR‑99b‑5p inhibitor. The effects of miR‑99b‑5p on neurite growth were also evaluated. The results of an immunofluorescence analysis indicated that neurite growth was normal in the control group, whereas SCI induced a reduction in neurite growth, which was rescued following transfection with a miR‑99b‑5p inhibitor. The protein expression levels of mTOR were detected in the three groups by western blotting. The results demonstrated that, compared with the control group, the protein expression levels of mTOR were significantly reduced in SCI neurons, whereas transfection with a miR‑99b‑5p inhibitor suppressed the SCI‑induced reduction of mTOR. In conclusion, treatment with a miR‑99b‑5p inhibitor may attenuate SCI‑induced harmful alterations in spinal cord neurons via the regulation of mTOR expression.

Figure 1.

SCI upregulates the expression levels of miR-99b-5p in the mouse spinal cord. The expression levels of miR-99b-5p were significantly upregulated in SCI mice compared with in Con mice. *P<0.05. Con, control; miR, microRNA; SCI, spinal cord injury.

Figure 2.

miR-99b-5p targets mTOR. (A) mTOR was predicted as a potential target gene of miR-99b-5p. (B) Interaction between mTOR and miR-99b-5p was analyzed using a LUC reporter assay. LUC activity was lower in cells co-transfected with the mTOR-WT plasmid and miR-99b-59 compared with the control group; no obvious difference was detected in fluorescence between the groups transfected with mTOR-MUT. *P<0.05. C, control; LUC, luciferase; miR, microRNA; mTOR, mechanistic target of rapamycin; MUT, mutant; UTR, untranslated region; WT, wild type.

Figure 3.

miR-99b-5p inhibitor suppresses SCI-induced neuronal apoptosis. (A) Compared with the Con group, the number of apoptotic cells was increased in the SCI group; however, miR-99b-5p inhibitor transfection suppressed SCI-induced apoptosis. (B) Statistical analysis of apoptosis assay. *P<0.05 SCI group vs. control group; inhibitor group vs. SCI group. Con, control; FITC, fluorescein isothiocyanate; miR, microRNA; PI, propidium iodide; SCI, spinal cord injury.

Figure 4.

miR-99b-5p inhibitor rescues SCI-induced neurite growth inhibition. (A) Neurite growth was normal in the Con group; however, it was markedly reduced by SCI and rescued by miR-99b-5p inhibitor transfection; magnification, ×200. (B) Statistical analysis of immunofluorescence. *P<0.05 SCI group vs. control group; inhibitor group vs. SCI group. Con, control; miR, microRNA; SCI, spinal cord injury.

Figure 5.

miR-99b-5p inhibitor rescues SCI-induced reduced mTOR expression. Compared with the Con group, significantly reduced expression levels of mTOR were detected in SCI neurons, whereas transfection with a miR-99b-5p inhibitor reversed SCI-induced reduced mTOR expression. (A) Protein expression levels of mTOR; (B) mRNA expression levels of mTOR. *P<0.05 SCI group vs. control group; inhibitor group vs. SCI group. Con, control; miR, microRNA; mTOR, mechanistic target of rapamycin; SCI, spinal cord injury.