Abstract:

Objective

To investigate the role and mechanisms of LncRNA NEAT1 （NEAT1）on lipopolysaccharide （LPS）-induced renal tubular epithelial cell damage. Methods Human renal tubular epithelial cells HK-2 were cultured in vitro， LPS was used to treat HK-2 cells under different concentrations. Flow cytometry was used to determine the apoptosis rate of cells treated with different concentrations of LPS. The expression of NEAT1， miR-129-5p， phosphorylated （p-） β-Catenin and β-Catenin were detected by RT-qPCR and Western blot. Experimental groups-1 were mimic NC group， miR-129-5p mimic group； miR-NC group， miR-129-5p inhibitor group. The above four groups were co-transfected with NEAT1-WT or NEAT1-MUT. Dual luciferase reporter gene experiment was used to analyze and verify the direct sequence interactions and negative regulatory relationships between NEAT1 and miR-129-5p in the above groups of cells. Experimental group-2 were the blank control group， si-NC group and si-NEAT1 group. si-NC and si-NEAT1 were transfected，respectively. The relative expression levels of NEAT1 and miR-129-5p were detected in the above three groups of cells. Cells were treated with 1 μg/mL LPS after transfection， and co- treated for 24 h. Cells were divided into LPS group （1 μg/mL LPS）， si-NC+miR-NC group （co- transfected with si- NC+miR- NC）， si- NEAT1+miR-NC group （co-transfected with si-NEAT1+miR- NC），si-NC+miR-129-5p inhibitor group （co-transfected with si-NC+miR-129-5p inhibitor），si- NEAT1+miR-129-5p inhibitor group （co-transfected with si-NEAT1+miR-129-5p inhibitor）.CCK-8 and flow cytometry experiment were used to detect cell viability， cell cycle and apoptosis rate； the expression of Bcl-2， Bax， Cleaved caspase-3， β-catenin， p-β-catenin， cyclin D1 and c-myc proteins in cells were detected by Western bot. One-way ANOVA analysis of variances was used for multi-group comparisons， and pairwised comparisons between groups were performed by post hoc Turkey's test. P ＜ 0.05 was considered statistically significant.

Results

Compared with LPS group（0 μg/ mL）， the cell viability [（83.70 ± 1.40）% vs （100.00 ± 1.70）%］， the relative expression levels of miR-129-5p （0.81 ± 0.07 vs 1.00 ± 0.13）， and phosphorylated （p-） β-catenin levels（0.78 ± 0.05 vs 1.00 ± 0.14） of HK-2 cells treated by 1μg/mL LPS was decreased， while the relative expression levels of NEAT1 （1.33 ± 0.12 vs 1.00 ± 0.08） and the apoptosis rate [（32.80 ± 1.30）%vs （5.20 ± 0.20） % ］ were increased. Compared with mimic NC group， the expression levels of miR-129-5p was increased （2.34 ± 0.22 vs 1.00 ± 0.06）， the luciferase activity of NEAT1-WT was decreased （0.36 ± 0.03 vs 1.00 ± 0.08）， the luciferase activity of NEAT1-MUT was no significant difference in the miR-129-5p mimic group. Moreover， compared with the si-NC group， the expression of miR-129-5p in the si-NEAT1 group was higher （2.18 ± 0.24 vs 1.04 ± 0.13）. After LPS treatment，compared with si-NEAT1+miR-NC group， cell viability [（67.30 ± 2.40）% vs （147.80 ± 5.60）%］，the proportion of S-phase cells [（19.50 ± 1.70）% vs （40.60 ± 1.60）%］， the proportion of G2/M phase cells [（8.30 ± 0.40）% vs （20.70 ± 0.07）%］， the Bcl-2 protein expression levels （0.21 ± 0.04 vs 1.38 ± 0.14）， the ratio of p-β- catenin/ β-catenin expression levels （0.23 ± 0.03 vs 1.51 ± 1.10），the cyclin D1 protein expression levels （0.17 ± 0.02 vs 0.64 ± 0.08）， the c-myc protein expression levels （0.18 ± 0.02 vs 0.65 ± 0.07） were decreased in si-NC+miR-129-5p inhibitor group，whilethe proportion of G0/G1 phase cells [（71.20 ± 1.30）% vs （43.30 ± 1.90）%］， the cell apoptosis rate[（39.50 ± 2.60）% vs （7.90 ± 1.10）%］， the Bax protein expression levels（1.21 ± 0.11 vs 0.35 ±0.03）and the Cleaved caspase-3 protein expression levels were increased（1.51 ± 0.11 vs 0.28 ± 0.02）.

Conclusion

LPS- induced renal tubular epithelial cell injury may be achieved by up-regulating the NEAT1/ miR- 129-5p axis to inhibit the Wnt/β-catenin pathway.

Key words: Renal tubular epithelial cells, Lipopolysaccharide, LncRNA NEAT1, miR-129-5p