circ_014260/miR-384/THBS1 aggravates spinal cord injury in rats by promoting neuronal apoptosis and endoplasmic reticulum stress.

Objective: To explore the mechanism of circ_014260 regulating neuronal apoptosis, oxidative stress, and endoplasmic reticulum stress in rats with spinal cord injury (SCI) miR-384/THBS1 axis.

Methods: T9-L10 spinal cord segments of Sprague Dawley rats were subjected to compression or contusion injuries after T10 laminectomy to establish rat models of SCI, which were then divided into SCI group, si-circ group and oe-circ group according to the transfection. There was another sham operation group which received no treatment. There were 10 rats in each group. The Basso-Beattie-Bresnahan scale and HE staining were used to evaluate the changes in neuronal motor function in rats with SCI. TUNEL staining was used to determine the neuronal apoptosis. Flow cytometry was used to measure the changes in HO-induced apoptosis of primary neurons. The activities of myeloperoxidase, malondialdehyde, superoxide dismutase and catalase were measured to evaluate the level of oxidative stress. Western blot was used to measure the expressions of CHOP and CRP78 (which are related to endoplasmic reticulum stress). Expression of circ_014260, miR-384 and THBS1 in tissues and cells was measured by qRT-PCR. RNase R restriction enzyme digestion and chromatin fractionation were used to identify the nature of circ_014260. Dual-luciferase reporter assay and RNA immunoprecipitation were used to verify the targeted binding relationship between circ_014260 and miR-384, as well as between miR-384 and THBS1.

Results: Compared with the sham operation group or the untreated rat primary neurons (control group), increased expression of circ_014260 and THBS1 as well as decreased expression of miR-384 were observed in the spinal cord tissue from rats with SCI and in HO-treated primary neurons (all P<0.05). The results of both and experiments showed that knocking down circ_014260 could reduce neuronal apoptosis and inhibit oxidative stress and endoplasmic reticulum stress in rats with SCI (all P<0.05). Circ_014260 targetedly inhibited miR-384 to up-regulate the expression of THBS1. Both miR-384 inhibitor and THBS1 overexpression vector partially reversed the alleviated neuronal damage by knocking down circ_014260 (both P<0.05).

Conclusion: Circ_014260 promotes neuronal damage in rats with SCI by inhibiting miR-384 to up-regulate the expression of THBS1. Thus, circ_014260 could possibly be a new molecular target of SCI.