miR-23 regulates inflammation and oxidative stress by targeting FIS1 for protecting renal tubular epithelial cell damage in sepsis

Objective  To explore the interaction between microRNA-23 （miR-23） and mitochondrial fission protein 1 （FIS1） in human kidney-2 （HK-2） cells and examine the effect of miR-23 on inflammatory response and oxidative stress by targeting FIS1.

Methods  HK-2 cells were cultured in vitro and induced by lipopolysaccharide （LPS） for modeling of sepsis. Dual luciferase reporter assay was utilized for verifying the interaction between miR-23 and FIS1. HK-2 cells were randomized into groups of miR-23 mimic/inhibitor and eukaryotic expression vector and small interfering RNA of FIS1 alone or in a combination. Cell proliferation was detected by cell counting kit-8 （CCK-8） assay and apoptosis and reactive oxygen species （ROS） were examined by flow cytometry. Malondialdehyde （MDA） was detected by MDA kit and the levels of tumor necrosis factor t （TNF-α）, interleukin （IL）-1β and IL-6/10 were measured by enzyme-linked immunosorbent assay（ELISA）.

Results The miR-23 mRNA in HK-2 cells induced by LPS was (0.60±0.12) folds that of control group(P<0.05) while FIS1 mRNA was (2.16±0.21) folds that of control group(P<0.05), FISI protein level was (2.69±0.28) folds that of control group(P<0.05). And miR-23 could form complementary binding to 3'-UTR of FIS1 and negatively regulate FIS1. TNF-α, IL-1 and IL-6 of miR-23 mimic group were (0.021±0.003), (0.0310±0.007) and (0.017±0.006) mg/g protein and (0.015±0.004), (0.043±0.007) and (0.020±0.008) mg/g protein in FIS1 siRNA group. TNF-α, IL-1 and IL-6 in miR-23 mimic and FIS1 siRNA groups were lower than those of blank control group (P<0.05). The levels of IL-10 in miR-23 mimic and FIS1 siRNA groups were (0.325±0.011) and (0.376±0.018) mg/g protein and both were higher than those of control group (P<0.05). TNF-α and IL-6 of miR-23 inhibitor group were (0.117±0.015) and (0.096±0.007) mg/g protein versus (0.168±0.024) and (0.147±0.030) mg/g protein in pcDNA3.1-FIS1 group. TNF-α and IL-6 of miR-23 inhibitor and pcDNA3.1-FIS1 groups were higher than those of blank control group (P<0.05). IL-10 of miR-23 inhibitor and pcDNA3.1-FIS1 groups was (0.149±0.012) and (0.121±0.024) mg/g protein and both were lower than those of blank control group (P<0.05). TNF-α, IL-1 and IL-6 of miR-23 mimic+pcDNA3.1-FIS1 group were (0.060±0.010), (0.084±0.009) and (0.048±0.008) mg/g protein and they were higher than (0.021±0.003), (0.0310±0.007) and (0.017±0.006) mg/g protein of miR-23 mimic group(P<0.05) while IL-10 (0.149±0.025) mg/g protein was lower than (0.325±0.011) mg/g protein of miR-23 mimic group(P<0.05). TNF-α, IL-1 and IL-6 were (0.049±0.007), (0.056±0.008) and (0.037±0.006) mg/g protein in miR-23 inhibitor+FIS1 siRNA group and they were lower than (0.117±0.015), (0.085±0.015) and (0.096±0.007) mg/g protein of miR-23 inhibitor group(P<0.05). And IL-10 (0.325±0.027) mg/g protein was higher than (0.149±0.012) mg/g protein of miR-23 inhibitor group(P<0.05). ROS of miR-23 mimic and FIS1 siRNA groups were (1328±84) and (1300±70) and both were lower than those of blank control group(P<0.05). ROS of miR-23 inhibitor and pcDNA3.1-FIS1 group were (1825±80) and (1930±105) and both were higher than those of blank control group(P<0.05). ROS of miR-23 mimic+pcDNA3.1-FIS1 group was (1538±96) was higher than (1 328±84) of miR-23 mimic group(P<0.05). ROS of miR-23 inhibitor+FIS1 siRNA group was (1490±70) and it was lower than (1825±80) of miR-23 inhibitor group(P<0.05). MDA of miR-23 mimic and FIS1 siRNA groups were (17.34±2.18) mol/L and (12.05±2.47) mol/L and both were lower than those of blank control group(P<0.05). MDA of miR-23 inhibitor and pcDNA3.1-FIS1 groups were (52.20±7.48) mol/L and (46.25±6.50) mol/L and both were higher than those of blank control group(P<0.05). MDA of miR-23 mimic+pcDNA3.1-FIS1 group was (35.44±3.03) mol/L and it was higher than (17.34±2.18) mol/L of miR-23 mimic group(P<0.05). MDA of miR-23 inhibitor+FIS1 siRNA group was (40.26±4.87) mol/L and it was lower than (52.20±7.48) mol/L of miR-23 inhibitor group(P<0.05). Proliferation rate of miR-23 mimic and FIS1 siRNA group were (183±14)% and (171±11)% and both were higher than those of blank control group (P<0.05). Apoptotic rates were (7.89±1.21) % and (11.22±2.98)% and both were lower than those of blank control group(P<0.05). Proliferation rate of miR-23 inhibitor and pcDNA3.1-FIS1 groups were (134±10)% and (127±9)% and both were lower than those of blank control group(P<0.05). Apoptotic rate was (23.22±2.54)% and (28.15±3.12)% and it was higher than that of blank control group(P<0.05). Proliferation rate of miR-23 mimic+pcDNA3.1-FIS1 group was (148±12)% and it was lower than (183±14)% of miR-23 mimic group(P<0.05). Apoptotic rate was (17.02±2.29)% and it was higher than (7.89±1.21)% of miR-23 mimic group(P<0.05). Proliferation rate of miR-23 inhibitor+FIS1 siRNA group was (160±8)% and it was higher than (134±10)% of miR-23 inhibitor group(P<0.05). Apoptotic rate was (14.27±2.00)% and it was lower than (23.22±2.54)% of miR-23 inhibitor group (P<0.05).

Conclusion  Abnormal LPS-induced expression of miR-23/FIS1 is relevant to inflammatory response and oxidative stress process in renal tubular epithelial cells. And miR-23 may reduce the inflammation and oxidative stress by negatively regulating FIS1 in HK-2 cells. It plays a protective role in renal tubular epithelial cell injury in sepsis.