Downregulation of circ-ZNF644 alleviates LPS-induced HK2 cell injury via miR-335-5p/HIPK1 axis.

Circular RNA (circRNA) has been confirmed to be involved in regulating sepsis-induced acute kidney injury (AKI). Our research aims to explore circ-ZNF644 role in the development of sepsis-induced AKI. Lipopolysaccharide (LPS) was used to induce kidney tubular epithelial cell (HK2) injury. ELISA assay was performed to measure the concentrations of inflammation factors. Cell functions were determined by cell counting kit 8 assay, EdU assay and flow cytometry. Protein expression was evaluated by Western blot analysis. Quantitative real-time PCR was used to detect relative expression of circ-ZNF644, miR-335-5p and homeodomain-interacting protein kinase 1 (HIPK1). RNA interaction was confirmed by dual-luciferase reporter assay and RIP assay. LPS enhanced HK2 cell inflammation, oxidative stress, apoptosis, and reduced proliferation. Circ-ZNF644 was overexpressed in sepsis-induced AKI patients. Circ-ZNF644 knockdown suppressed LPS-induced HK2 cell injury, and this effect could be revoked by miR-335-5p inhibitor. MiR-335-5p was sponged by circ-ZNF644, and its expression was downregulated in sepsis-induced AKI patients. HIPK1 was targeted by miR-335-5p, and its expression could be suppressed by circ-ZNF644 knockdown. MiR-335-5p had an inhibition effect on HK2 cell injury induced by LPS, and HIPK1 overexpression could reverse this effect. Circ-ZNF644 knockdown relieved LPS-induced HK2 cell injury through the miR-335-5p/HIPK1 axis, confirming that circ-ZNF644 contributed to sepsis-induced AKI.

Gong J ，Zhao S ，Luo S ，Yin S ，Li X ，Feng Y ... -  《-》

Resveratrol Inhibits circ_0074371-related Pathway to Alleviate Sepsis-induced Acute Kidney Injury.

Resveratrol has a protective effect on sepsis-induced acute kidney injury (AKI). Circ_0074371 has been confirmed to inhibit sepsis-induced AKI process, but whether resveratrol inhibits sepsis-induced AKI by regulating circ_0074371-related pathway remains unclear. In this study, lipopolysaccharide (LPS)-induced renal tubular epithelial cells (HK2) were used to mimic AKI cell models. Quantitative real-time PCR was used to detect relative expression of circ_0074371, microRNA (miR)-145-5p and inositol polyphosphate multikinase (IPMK). Cell proliferation and apoptosis were detected by cell counting kit 8 assay, EdU assay and flow cytometry. The levels of inflammation factors were measured by ELISA assay, and MDA level and SOD activity were examined to assess oxidative stress. Protein expression of IPMK was evaluated by western bolt analysis. The relationship between miR-145-5p and circ_0074371 or IPMK was confirmed by dual-luciferase reporter assay. It was showed that circ_0074371 was upregulated in AKI patients and LPS-induced HK2 cells, and silencing of circ_0074371 promoted proliferation and inhibited apoptosis, inflammation and oxidative stress in LPS-induced HK2 cells. In terms of mechanism, circ_0074371 sponged miR-145-5p to positively regulate IPMK. IPMK overexpression could reverse the relieving effect of circ_0074371 knockdown on LPS-induced HK2 cell injury. Moreover, resveratrol suppressed LPS-induced apoptosis, inflammation and oxidative stress in HK2 cells, and circ_0074371 overexpression also reversed the protective effect of resveratrol against LPS-induced cell injury. Our data suggested that resveratrol alleviated LPS-induced HK2 cell injury by inactivating the circ_0074371/miR-145-5p/IPMK axis.

Zhu D ，Wu X 《-》

KNOCKDOWN OF CIRC_0114428 ALLEVIATES LPS-INDUCED HK2 CELL APOPTOSIS AND INFLAMMATION INJURY VIA TARGETING MIR-215-5P/TRAF6/NF-ΚB AXIS IN SEPTIC ACUTE KIDNEY INJURY.

Background: Sepsis is a systemic inflammatory disease that can cause multiple organ damage. Circular RNAs (circRNAs) have been reported to play a regulatory role in sepsis-induced acute kidney injury (AKI); however, the role of circ_0114428 has not been studied. Methods: In this study, HK2 cells were treated with different concentrations of LPS to induce cell damage, and then the expressions of circ_0114428, microRNA-215-5p (miR-215-5p), and tumor necrosis factor receptor-associated factor 6 (TRAF6) were detected by quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot examined the Bax and cleaved-Caspase-3 proteins. Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) assay. In addition, cell apoptosis was detected by flow cytometry, and the levels of inflammatory factors were detected by enzyme-linked immunosorbent assay. Results: After LPS treatment with different concentrations, we found that LPS at 10 μg/mL had the best effect on HK2 cells. Circ_0114428 was highly expressed in sepsis-AKI patients and LPS-treated HK2 cells. Knockdown of circ_0114428 restored the effects of LPS treatment on proliferation, apoptosis, and inflammatory response of HK2 cells. MiR-215-5p was a target of circ_0114428, and TRAF6 was a downstream target of miR-215-5p. Circ_0114428 regulated TRAF6 expression by sponging miR-215-5p in LPS-treated HK2 cells. Circ_0114428 regulated LPS-induced NF-κB signaling in HK2 cells by targeting miR-215-5p/TRAF6 axis. Conclusion: Circ_0114428 knockdown abolished the cell proliferation, apoptosis, and inflammatory damage in LPS-induced HK2 cells by targeting miR-215-5p/TRAF6/NF-κB.

Li Y ，Zhang C ，Zhao Z 《-》

Circ_0006944 aggravates LPS-induced HK2 cell injury via modulating miR-205-5p/UBL4A pathway.

Zhou F ，Liu D ，Ye J ，Li B ... -  《-》

Silencing circ_0074371 inhibits the progression of sepsis-induced acute kidney injury by regulating miR-330-5p/ELK1 axis.

Sepsis-induced acute kidney injury (AKI) is a common in clinic. Circular RNAs (circRNAs) play significant roles in ameliorating AKI. The purpose of this study was aimed to identify the role of circ_0074371 and the potential action mechanism in sepsis-induced AKI. AKI patients and healthy individual serum samples were collected and the relative expression of circ_0074371 was measured by real-time polymerase chain reaction (RT-PCR). HK2 cells were treated with different dose (0, 2.5, 5 and 10 μg/ml) lipopolysaccharide (LPS) to establish the AKI cell model. The cell viability and apoptosis of HK2 cells were detected using cell counting kit-8 (CCK-8) and flow cytometry, respectively. The contents of malondialdehyde (MDA), and superoxide dismutase (SOD) were evaluated using the relative commercial kits. The IL-1β and TNF-α levels in cell culture supernatants were measured by ELISA. The interaction relationship between miR-330-5p and circ_0074371 or ELK1 was predicted by Targetscan database and further confirmed by the dual-luciferase reporter assay system. The circ_0074371 expression was up-regulated in sepsis patients and LPS-induced HK2 cells. Silencing circ_0074371 promoted HK2 cells viability and inhibited the HK2 cells apoptosis. miR-330-5p inhibitor weakened circ_0074371 inhibitor-induced cell viability, apoptosis and oxidative stress. Further mechanism analysis showed that circ_0074371 acted as a sponge for miR-330-5p to increase ELK1 expression level. Importantly, miR-330-5p downregulation or ELK1 upregulation reversed the action of circ_0074371 knockdown on LPS-induced HK2 cells. Knockdown of circ_0074371 ameliorated LPS-induced HK2 cells apoptosis, inflammation and oxidative stress via regulating miR-330-5p/ELK1, opening a new window into the pathogenesis AKI.

Wang QY ，Zhang RR ，Cui L ，Sun YP ... -  《-》