Effects of lncRNA HOXA11-AS on Sevoflurane-Induced Neuronal Apoptosis and Inflammatory Responses by Regulating miR-98-5p/EphA4

Abstract

Objective: To explore the molecular mechanism of sevoflurane-induced neurotoxicity and to determine whether lncRNA HOXA11-AS affects sevoflurane-induced neuronal apoptosis and inflammation by regulating miR-98-5p/EphA4.

Methods: Morris water maze (MWM) test was used to detect the learning and memory ability of rats, HE staining was used to observe hippocampal pathology, TUNEL staining was used to detect the level of neuronal apoptosis, and RT-qPCR was used to detect the expression of HOXA11-AS, miR-98-5p, IL-6, IL-1β, and TNF-α. At the same time, the contents of IL-6, IL-1β, and TNF-α in serum were detected by ELISA. The expressions of apoptosis-related proteins EphA4, Bax, Cleaved caspase 3, and Bcl-2 were detected by Western blot. The dual-luciferase gene reporter verified the targeting relationship between HOXA11-AS and miR-98-5p and the targeting relationship between miR-98-5p and EphA4.

Results: The expression of HOXA11-AS was observed in sevoflurane-treated rats or cells and promoted neuronal apoptosis and inflammation. HOXA11-AS was knocked out alone, or miR-98-5p was overexpressed which attenuates neuronal apoptosis and inflammatory inflammation after sevoflurane treatment. Furthermore, knockdown of HOXA11-AS alone was partially restored by knockdown of miR-98-5p or overexpression of EphA4.

Conclusion: Inhibition of lncRNA HOXA11-AS attenuates sevoflurane-induced neuronal apoptosis and inflammatory responses via miR-98-5p/EphA4.

Figure 1

Inhibition of HOXA11-AS alleviates the cognitive impairment caused by sevoflurane in rats. After 3 days of anesthesia, the hippocampal tissue was taken for the following experiments: (a) RT-qPCR detection of HOXA11-AS expression in rat hippocampus. (b, c): MWM test to detect escape latency and the number of platform crossings. (d) HE staining to detect rat hippocampal tissue pathological phenomena. (e) TUNEL staining to detect rat hippocampal tissue nerves apoptosis. (f): ELISA detects inflammatory factor (IL-6, TNF-α, and IL-1β) levels in rat hippocampus. ^∗∗P < 0.01.

Figure 2

Inhibition of HOXA11-AS alleviates sevoflurane-induced neuronal apoptosis and inflammatory response in vitro. (a, d) RT-qPCR for detection of HOXA11-AS and mRNA expression of inflammatory factors. (b) TUNEL staining to detect neuronal apoptosis. (c) Western blot detects apoptosis-related protein expression. (e) ELISA detects the inflammatory factors levels. ^∗∗P < 0.01.

Figure 3

Validation of the correlation between miR-98-5p and HOXA11-AS targeting. (a) Starbase predicts target genes of HOXA11-AS. (b) Dual-luciferase reporter gene verifies the targeting relationship. (c, d) RT-qPCR detects miR-98-5p expression in neuronal cells and rat hippocampus. ^∗∗P < 0.01.

Figure 4

Effects of miR-98-5p on sevoflurane-induced neuronal apoptosis and inflammation in vitro. (a, d) RT-qPCR for detection of HOXA11-AS and mRNA expression of inflammatory factors. (b) TUNEL staining to detect neuronal apoptosis. (c) Western blot detection of apoptosis-related protein expression. (e) ELISA detects the inflammatory factors levels. ^∗∗P < 0.01.

Figure 5

miR-98-5p targets EphA4. (a) Starbase predicts that EphA4 is the target of miR-98-5p. (b) Dual-luciferase reporter gene acknowledged the targeting relationship. (c) Western blot detects EphA4 expression in neurons. (d) Immunohistochemical detection of EphA4 expression in rat hippocampus. ^∗P < 0.05, ^∗∗P < 0.01.

Figure 6

HOXA11-AS regulates sevoflurane-induced neuronal apoptosis and neuroinflammation through the miR-98-5p/EphA4 molecular axis. (a, c) Western blot detection of protein expression in neurons cells. (b) TUNEL staining to detect neuronal apoptosis. (d) RT-qPCR detect the mRNA expression of inflammatory factors. (e) ELISA detects the inflammatory factors levels. ^∗P < 0.05, ^∗∗P < 0.01.

Figure 7

HOXA11-AS alleviates the cognitive impairment caused by sevoflurane in rats through the miR-98-5p/EphA4 molecular axis. After 3 days of anesthesia, the hippocampal tissue was taken for the following experiments: (a, b) MWM test to detect escape latency and the number of platform crossings. (c, f) Western blot detection of protein expression in rat hippocampus. (d) HE staining to detect rat hippocampal tissue pathological phenomena. (e) TUNEL staining to detect rat hippocampal tissue nerves apoptosis. (g) RT-qPCR detect the mRNA expression of inflammatory factors. (h) ELISA detects the inflammatory factors levels in rat hippocampus. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.