Circ_UBE2D2 Attenuates the Progression of Septic Acute Kidney Injury in Rats by Targeting miR-370-3p/NR4A3 Axis

Abstract

As circ_UBE2D2 has been confirmed to have targeted binding sites with multiple miRNAs involved in septic acute kidney injury (SAKI), efforts in this study are directed to unveiling the specific role and relevant mechanism of circ_UBE2D2 in SAKI. HK-2 cells were treated with lipopolysaccharide (LPS) to construct SAKI model in vitro. After sh-circ_UBE2D2 was transfected into cells, the transfection efficiency was detected by qRT-PCR, cell viability and apoptosis were determined by MTT assay and flow cytometry, and expressions of Bcl-2, Bax and Cleaved-caspase 3 were quantified by western blot. Target genes associated with circ_UBE2D2 were predicted using bioinformatics analysis. After the establishment of SAKI rat model, HE staining and TUNEL staining were exploited to observe the effect of circ_UBE2D2 on tissue damage and cell apoptosis. The expression of circ_UBE2D2 was overtly elevated in LPS-induced HK-2 cells. Sh-circ_UBE2D2 can offset the inhibition of cell viability and the promotion of cell apoptosis induced by LPS. Circ_UBE2D2 and miR-370-3p as well as miR-370-3p and NR4A3 have targeted binding sites. MiR-370-3p inhibitor reversed the promoting effect of circ_UB2D2 silencing on viability of LPS-treated cells, but shNR4A3 neutralized the above inhibitory effect of miR-370-3p inhibitor. MiR-370-3p inhibitor weakened the down-regulation of NR4A3, Bax and Cleaved caspase-3 and the up-regulation of Bcl-2 induced by circ_UB2D2 silencing, but these trends were reversed by shNR4A3. In addition, sh-circ_UBE2D2 could alleviate the damage of rat kidney tissue. Circ_UBE2D2 mitigates the progression of SAKI in rats by targeting miR-370-3p/NR4A3 axis.

Keywords: Circ_UBE2D2; MiR-370-3p; NR4A3; apoptosis; septic acute kidney injury.

Fig. 1. Effects of circ_UBE2D2 on LPS-induced renal tubular epithelial cell viability and apoptosis.

(A) QRTPCR was used to detect the expression of circ_UBE2D2 in LPS-induced HK-2 cells. (B) The expression of circ_UBE2D2 in cells transfected with sh-circ_UBE2D2 vector was detected by qRT-PCR. (C) MTT assay was used to assess the effect of circ_UBE2D2 on LPS-induced cell viability. (D) Flow cytometry was applied to evaluate the effect of circ_UBE2D2 on LPSinduced apoptosis of HK-2 cells. (E) Western blot was exploited to determine the effect of circ_UBE2D2 on LPS-induced HK- 2 cell apoptosis-related protein and NR4A3 expressions. (**p < 0.01, ***p < 0.001, vs. Control; ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001, vs. LPS+shNC).

Fig. 2. Prediction analysis of circ_UBE2D2 target gene.

(A) StarBase and miRDB predict the binding miRNAs of circ_UBE2D2 and NR4A3, respectively, and the co-targeting miRNA with the highest score (miR-370-3p) was selected as the research object. (B) StarBase was used to predict the targeted binding relationship between circ_UBE2D2 and miR-370-3p. (C) StarBase predicted that NR4A3 have targeted binding sites with miR-370-3p. (D-E) The dual-luciferase reporter assay was used to verify the targeted binding relationship between circ_UBE2D2 and miR-370-3p and between miR-370-3p and NR4A3. (F) The effect of circ_UBE2D2 on the expression of miR-370-3p was detected by qRT-PCR. (***p < 0.001, vs. Control; ⁺⁺⁺p < 0.001, vs. LPS+shNC; ^^^p < 0.001, vs. MC).

Fig. 3. Effects of circ_UBE2D2 on LPS-induced renal tubular epithelial cell viability and apoptosis by targeting miR-370-3p/NR4A3.

(A) The expression of miR-370-3p in the transfected cells was detected by qRT-PCR. (B) The expression of NR4A3 in the transfected cells was quantified by qRT-PCR. (C) MTT assay was conducted to detect cell viability. (D) Flow cytometry was used to measure apoptosis. (E) Western blot was exploited to quantitate the expressions of apoptosis-related proteins in each group. (*p < 0.05, **p < 0.01, ***p < 0.001, vs. LPS+shNC+IC; ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001, vs. LPS+sh-circ_UBE2D2+IC; ^^p < 0.01, ^^^p < 0.001, vs. LPS+shNC+I; ^&&&p < 0.001, vs. LPS+IC+shNR4A3).

Fig. 4. Effects of circ_UBE2D2 on renal tissue injury in CLP rat model.

(A-C) Sh-circ_UBE2D2 lentivirus was injected into CLP rats, and then qRT-PCR was used to detect the expressions of circ_UBE2D2, NR4A3 and miR-370-3p in the rat kidney tissue of each group. (D) HE staining was conducted to observe the damage of the kidney tissue of CLP rats that were injected with sh-circ_UBE2D2 lentivirus. (E) TUNEL staining was conducted to observe the cell apoptosis in CLP rats that were injected with sh-circ_UBE2D2 lentivirus. (F-G) The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were measured in rat serum of each group. (***p < 0.001, vs. Control; ⁺⁺⁺p < 0.001, vs. CLP+shNC).