Knockdown of circ-FANCA alleviates LPS-induced HK2 cell injury via targeting miR-93-5p/OXSR1 axis in septic acute kidney injury

Abstract

Background: Sepsis is life-threatening disease with systemic inflammation and can lead to various diseases, including septic acute kidney injury (AKI). Recently, diverse circular RNAs (circRNAs) are considered to be involved in the development of this disease. In this study, we aimed to elucidate the role of circ-FANCA and the potential action mechanism in sepsis-induced AKI.

Methods: HK2 cells were treated with lipopolysaccharide (LPS) to establish septic AKI cell model. The expression of circ-FANCA, microRNA-93-5p (miR-93-5p) and oxidative stress responsive 1 (OXSR1) mRNA was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability was assessed using cell counting kit-8 (CCK-8) assay. Cell apoptosis and cell cycle distribution were measured by flow cytometry. The inflammatory response was monitored according to the release of pro-inflammatory cytokines via enzyme-linked immunosorbent assay (ELISA). The activities of oxidative indicators were examined using the corresponding kits. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were applied to validate the interaction between miR-93-5p and circ-FANCA or OXSR1. Protein analysis was conducted through western blot.

Results: Circ-FANCA was upregulated in septic AKI serum specimens and LPS-treated HK2 cells. Functionally, circ-FANCA knockdown facilitated cell proliferation and restrained apoptosis, inflammation and oxidative stress in LPS-triggered HK2 cells. Further mechanism analysis revealed that miR-93-5p was a target of circ-FANCA and circ-FANCA modulated LPS-induced cell damage by targeting miR-93-5p. Meanwhile, miR-93-5p overexpression repressed LPS-treated HK2 cell injury by sponging OXSR1. Furthermore, circ-FANCA regulated OXSR1 expression by sponging miR-93-5p. Besides, exosome-derived circ-FANCA was upregulated in LPS-induced HK2 cells, which was downregulated by GW4869.

Conclusion: Circ-FANCA knockdown attenuated LPS-induced HK2 cell injury by regulating OXSR1 expression via targeting miR-93-5p.

Keywords: AKI; OXSR1; Sepsis; circ-FANCA; miR-93-5p.

Fig. 1

Circ-FANCA was overexpressed in septic AKI patients and LPS-treated HK2 cells. a The level of circ-FANCA was examined by qRT-PCR in serum samples of septic AKI patients (n = 19) and healthy controls (n = 19). b The expression of circ-FANCA in HK2 cells treated with different concentrations of LPS for 24 h was detected by qRT-PCR. c QRT-PCR assay was conducted for circ-FANCA expression in nuclear and cytoplasmic fractions. d After RNase R treatment, the levels of circ-FANCA and linear FANCA mRNA were determined using qRT-PCR. *P < 0.05, ****P < 0.0001

Fig. 2

Circ-FANCA knockdown promoted proliferation and suppressed apoptosis and inflammation in LPS-treated HK2 cells. a The efficiency of siRNA-mediated circ-FANCA knockdown was examined by qRT-PCR in LPS-treated HK2 cells. b Cell viability in HK2 cells treated with different concentrations of LPS for 24 h was measured by CCK-8 assay. HK2 cells were divided into 4 groups: Control, LPS, LPS + si-NC and LPS + si-circ-FANCA. c Cell viability in LPS-treated HK2 cells with different transfection or not was measured by CCK-8 assay. d, e Cell apoptosis and cell period distribution were evaluated through flow cytometry in the above groups. f The release levels of inflammatory cytokines IL-1β and TNF-α were examined via ELISA using corresponding kits. g, h The levels of SOD and MDA in HK2 cells were measured by matched kits to assess oxidative stress. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

Circ-FANCA directly interacted with miR-93-5p. a The complementary sequence between circ-FANCA and miR-93-5p were exhibited. b The overexpression efficiency of miR-93-5p in HK2 cells was evaluated by qRT-PCR. c, d The targeted relationship between circ-FANCA and miR-93-5p was validated by dual-luciferase reporter assay and RIP assay. e The expression level of miR-93-5p in the serums of septic AKI patients (n = 19) and healthy controls (n = 19) was examined by qRT-PCR assay. f Spearman’s correlation coefficient analysis was performed to estimate the correlation between circ-FANCA and miR-93-5p in the serums of septic AKI patients. g The level of miR-93-5p in LPS-stimulated HK2 cells was determined by qRT-PCR assay. h The expression level of circ-FANCA in LPS-induced HK2 cells transfected with circ-FANCA or pCD5-ciR was determined by qRT-PCR assay. i MiR-93-5p level was determined through qRT-PCR in LPS-induced HK2 cells following the transfection of si-NC, si-circ-FANCA, pCD5-ciR or circ-FANCA. **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

Circ-FANCA regulated LPS-induced HK2 cell injury by targeting miR-93-5p. a The knockdown efficiency of anti-miR-93-5p was analyzed by qRT-PCR. HK2 cells were divided into 6 groups: Control, LPS, LPS+ si-NC, LPS+ si-circ-FANCA, LPS+ si-circ-FANCA +anti-miR-NC and LPS+ si-circ-FANCA+ anti-miR-93-5p. b The qRT-PCR was performed for miR-93-5p expression detection in the above groups. c–e The measurement of cell viability (c), cell apoptosis (d), cell cycle distribution (e) was carried out via CCK-8 assay or flow cytometry in the above groups. f The release of IL-1β and TNF-α was investigated by ELISA. g, h The activities of SOD and MDA in these cells were determined using the corresponding kits. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

OXSR1 was a direct target of miR-93-5p. a The potential binding sites between miR-93-5p and OXSR1 were exhibited. b, c Dual-luciferase reporter assay and RIP assay were performed to verify whether OXSR1 could interact with miR-93-5p. d The mRNA level of OXSR1 in the serums of septic AKI patients (n = 19) and healthy controls (n = 19) was measured by qRT-PCR assay. e The correlation between OXSR1 mRNA and miR-93-5p in the serums of septic AKI patients was analyzed by Spearman’s correlation coefficient analysis. f The protein expression of OXSR1 in HK2 cells treated with different concentrations of LPS was quantified by western blot. g The protein level of OXSR1 in LPS-induced HK2 cells with miR-93-5p knockdown or overexpression was tested by western blot. **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 6

Overexpression of miR-93-5p alleviated LPS-induced cell injury in HK2 cells by targeting OXSR1. a The protein level of OXSR1 in HK2 cells transfected with pcDNA or OXSR1 was measured by western blot. b–h LPS-treated HK2 cells were transfected with miR-NC, miR-93-5p, miR-93-5p+pcDNA or miR-93-5p+OXSR1. b The protein level of OXSR1 in LPS-induced HK2 cells after transfection was tested by western blot. c Cell viability in these cells was assessed by CCK-8 assay. d, e Cell apoptosis and percentage of cells at different stages were monitored by flow cytometry assay. f The concentration of IL-1β and TNF-α was examined by ELISA using corresponding kits. g, h The levels of SOD and MDA in these cells were examined by corresponding specific kits. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 7

Circ-FANCA positively modulated OXSR1 expression by targeting miR-93-5p. a, b After transfected with si-circ-FANCA, si-circ-FANCA+anti-miR-93-5p or corresponding controls, the mRNA and protein levels of OXSR1 in LPS-induced HK2 cells were measured by qRT-PCR and western blot assays. ***P < 0.001, ****P < 0.0001

Fig. 8

Exosomes derived from LPS-induced HK2 cells mediated the release of circ-FANCA. a Electron micrograph of exosomes isolated from the culture medium of LPS-treated HK2 cells or untreated HK2 cells. The scale bar represents 100 nm. b Western blot analysis for exosome markers (CD9 and CD63) in HK2 cells. c QRT-PCR assay for the expression level of exo-circ-FANCA in HK2 cells treated with LPS. d QRT-PCR assay for the expression of circ-FANCA in HK2 cells co-incubated with exosomes derived from LPS-induced HK2 cells or PBS. e QRT-PCR assay for the expression of exo-circ-FANCA in LPS-treated HK2 cells co-incubated with GW4869 or not. **P < 0.01, ***P < 0.001, ****P < 0.0001