The establishment of a bupivacaine-induced neurotoxicity model
To analyze the bupivacaine-induced neurotoxicity process, we first verified the model. Apoptosis and cell viability were analyzed via caspase3 activity and MTT assays. Caspase3 activity increased with increasing concentrations of bupivacaine and reached its highest level at 2.0 mM, the relative increase was about 1.01±0.1, 1.79±0.08, 1.89±0.05, 2.72±0.08, 3.11±0.07. (Fig. 1A). Cell viability was significantly reduced compared with that in the control group. In addition, 2.0 mM bupivacaine reduced cell viability by almost 50%, the relative decrease was about 1.00±0.1, 0.82±0.08, 0.75±0.09, 0.63±0.07, 0.53±0.08. (Fig. 1B). Thus, our results confirmed that bupivacaine induced cell apoptosis.
Expression of lincRNA Gm14012 in bupivacaine-induced neurotoxicity
We first detected the expression of lincRNA Gm14012 to investigate the possible change. As shown in Fig. 1C, the expression of lincRNA Gm14012 increased with increasing bupivacaine concentration and reached a maximum of an almost 16-fold increase at 2.0 mM, the relative increase was about 1.03±0.1, 1.92±0.18, 4.13±0.24, 8.92±0.28, 17.05±0.27. Similarly, we also detected the expression of lincRNA Gm14012 after exposure to 1.0 mM for different durations. Our results showed that the expression of lincRNA Gm14012 increased with time, the relative increase was 1.01±0.12, 1.33±0.19, 1.87±0.2, 3.77±0.26, 7.09±0.3. (Fig. 1D).
Function of lincRNA PADNA in bupivacaine-induced neurotoxicity
We constructed a knockdown vector to assess the function of lincRNA Gm14012. As shown in Fig. 2A, the expression of lincRNA Gm14012 was significantly downregulated, the relative decrease was about 0.66, 0.48. Next, we used the MTT assay to assess the function of lincRNA Gm14012. Interestingly, knockdown of lincRNA Gm14012 significantly promoted cell death and reduced cell viability after exposure to bupivacaine, the relative decrease was about 0.64±0.1, 0.43±0.08 (Fig. 2B). To date, there has been no report on lincRNA Gm14012 in bupivacaine-induced neurotoxicity. Thus, we named this lincRNA PADNA (protect cell death RNA). To further explore the function of lincRNA PADNA in bupivacaine-induced neurotoxicity, we performed comprehensive analyses, such as TUNEL assay, caspase3 activity assay, and western blotting. TUNEL assays were used to assess cell apoptosis. There was no significant difference between the two groups under normal conditions; however, when cells were exposed to 1.0 mM bupivacaine, knockdown of lincRNA PADNA significantly increased the cell apoptosis rates, suggesting that knockdown of lincRNA PADNA promoted cell death, the relative increase was about 0.41±0.07, 0.63±0.06 (Fig. 2C). In addition, we measured caspase3 activity using a kit. Our results suggested that downregulation of lincRNA PADNA significantly increased caspase3 activity, the relative increase was about 0.34±0.09, 0.56±0.08 (Fig. 2D). Similar results were obtained from western blotting (Fig. 2E). Overall, our experiments demonstrated that lincRNA PADNA played a protective role in bupivacaine-induced neurotoxicity.
Preliminary analysis of the mechanism of lincRNA PADNA
Long noncoding RNAs often act as sponge RNAs to bind with miRNAs to mediate different functions in numerous processes. To investigate the possible mechanism of lincRNA PADNA, we first used miRDNA to predict the potential binding targets of lincRNA PADNA. As shown in Fig. 3A, miR-194 was predicted to bind with lincRNA PADNA. Next, we used dual-luciferase reporter assays to verify whether lincRNA PADNA can bind with miR-194. Our results suggested that the relative luciferase activity of miR-194 was significantly reduced in the wt-lincRNA PADNA group, while no significant difference was detected in the mut-lincRNA PADNA group, the relative decrease was about 1.0±0.1, 0.34±0.05 (Fig. 3B). Thus, the above results preliminarily identified miR-194 as a target of lincRNA PADNA. Next, we detected the expression of lincRNA PADNA under different conditions. Overexpression of miR-194 markedly reduced the expression of lincRNA PADNA and knockdown of miR-194 increased the expression of PADNA, the relative values are 1.05±0.08, 0.41±0.04, 1.00±0.09, 6.04±0.13 (Fig. 3C). Similar results were also obtained in HEK293 cells, the relative values are 1.02±0.1, 0.56±0.04, 1.03±0.08, 12.04±0.21 (Fig. 3D). Moreover, we also analyzed the expression of miR-194 in bupivacaine-treated cells. Our results revealed that the expression of miR-194 was clearly reduced with increasing concentrations of bupivacaine and reached its lowest level at 2.0 mM bupivacaine, the relative decrease was about 1.01±0.07, 1.00±0.07, 0.75±0.06, 0.51±0.05, 0.36±0.06 (Fig. 3E). The above results revealed that lincRNA PADNA could negatively regulate the expression of miR-194.
FBXW7 is the direct target of miR-194
To further study the mechanism of miR-194 involved in bupivacaine-induced neurotoxicity, we used StarBase2.0 to predict the target of miR-194. We found that FBXW7 was predicted as the direct target of miR-194. Previous studies have also shown that FBXW7 is regulated by miR-194. However, the role of miR-194 and FBXW7 in bupivacaine-induced neurotoxicity remains unknown. Thus, we performed a comprehensive analysis to analyze their relationship. The binding sequence is shown in Fig. 4A. A dual-luciferase reporter assay revealed that the relative luciferase activity of wt-FBXW7 was significantly reduced in the miR-194 group, the relative values are 1.0±0.09, 0.2±0.04 (Fig. 4B). Overexpression of miR-194 markedly reduced the expression of FBXW7 and while silencing miR-194 reduced its expression, the relative values are 1.00±0.05, 0.32±0.03, 1.00±0.1, 3.41±0.35 (Fig. 4C). We also performed a western blot assay to analyze the protein level of FBXW7. Our results suggested that the protein level of FBXW7 was decreased in the miR-194 overexpression group (Fig. 4D). Fig. 4E shows the negative correlation between miR-194 and FBXW7. In addition, we also analyzed the expression of FBXW7 in bupivacaine-treated cells. The expression of FBXW7 was increased with the increase in concentration and time course of bupivacaine and reached the highest level at 2.0 mM bupivacaine, the relative values are 1.01±0.12, 1.06±0.11, 1.00±0.1, 1.82±0.13, 2.83±0.17 (Fig. 4F, Fig. 4G). Thus, our study provides evidence that miR-194 negatively regulates the expression of FBXW7.
The lincRNA PADNA/miR-194/FBXW7 axis in bupivacaine-induced neurotoxicity
We performed comprehensive analysis to further verify the lincRNA PADNA/miR-194/FBXW7 pathway. The expression of FBXW7 was downregulated in the lincRNA PADNA knockdown group, as demonstrated by qPCR, the relative values are 1.01±0.1, 0.31±0.07 (Fig. 5A). Similar results were obtained by western blotting suggesting that lincRNA PADNA positively regulates FBXW7 (Fig. 5B). Considering the above results, we knocked down miR-194 and PADNA and then detected the expression of FBXW7. Interestingly, the expression of FBXW7 was increased in the cotransfection group as demonstrated qPCR and western blot assays, the relative values are 1.00±0.1, 0.35±0.06, 0.95±0.07 (Fig. 5C, Fig. 5D). Thus, knockdown of miR-194 could block the effect of lincRNA PADNA. Therefore, a rescue experiment was performed to verify the relationship between miR-194 and FBXW7. TUNEL assays revealed that cotransfection of miR-194 and PADNA could reduce the apoptotic effect compared with knockdown of lincRNA PADNA, the relative values are 0.11±0.01, 0.35±0.04, 0.6±0.06, 0.41±0.04 (Fig. 5E). In addition, caspase3 activity and cleaved caspase3 levels were decreased, the relative values are 0.11±0.02, 0.46±0.04, 0.61±0.06, 0.45±0.02 (Fig. 5F, Fig. 5H). Moreover, cell viability was increased in the cotransfection group compared with the lincRNA PADNA knockdown group, the relative values are 1±0.11, 0.66±0.04, 0.43±0.06, 0.59±0.09 (Fig. 5G). Through rescue experiments, we proved that the lincRNA PADNA/miR-194/FBXW7 axis plays an important role in bupivacaine-induced neurotoxicity.