Knockdown of circ-Gatad1 alleviates LPS induced HK2 cell injury via targeting miR-22-3p/TRPM7 axis in septic acute kidney

Background Sepsis is a life-threatening, systemic inflammatory disease that can lead to a variety of conditions, including septic acute kidney injury (AKI). Recently, multiple circular Rnas (circRNAs) have been implicated in the development of this disease. Methods In this study, we aimed to elucidate the role of circ-Gatad1 in sepsis induced AKI and its potential mechanism of action. High-throughput sequencing was used to investigate abnormal expression of circRNA in AKI and healthy volunteer. Bioinformatics analysis and luciferase reporting analysis were used to clarify the interacted relationship among circRNA, miRNA and mRNA. HK2 cells were treated with lipopolysaccharide (LPS) to establish septic AKI cell model. HK2 cells were employ to analysis the ROS, inflammatory cytokines expression, proliferation and apoptosis under LPS condition. Results The result show that the expression of circ-Gatad1 was increased in septic acute kidney patients. Downregulation circ-Gatad1 suppressed LPS-treated induced HK2 cells injury including apoptosis, proliferation ability, ROS and inflammatory cytokines level. Bioinformatics and luciferase report analysis confirmed that both miR-22-3p and TRPM7 were downstream targets of circ-Gatad1. Overexpression of TRPM7 or downregulation of miR-22-3p reversed the protective effect of si-circ-Gatad1 to HK2 after exposure to LPS (5 µg/ml) microenvironment. Conclusion In conclusion, knockdown of circ-Gatad1 alleviates LPS induced HK2 cell injury via targeting miR-22-3p/TRPM7 axis in septic acute kidney.


Background
Septic shock with acute kidney injury (AKI) is common in critically ill patients [1].Although the in-hospital mortality rate for patients receiving kidney replacement therapy in ICU decreased from 44.9% in 2007 to 36.1% in 2016, the mortality rate for patients with sepsis remained high, at > 50% [2].AKI is a critical and severe clinical disease caused by a variety of factors.Lipopolysaccharide (LPS)-induced sepsis is a primary cause of AKI in critically ill patients [3,4].Therefore, LPS is always utilized to construct septic AKI cell models in renal cells in vitro [5]].Recent studies have confirmed that the pathogenesis of sepsis induced AKI includes a series of complex interactions such as vascular endothelial cell dysfunction, inflammation and renal tubule cell apoptosis [6,7].However, efforts to translate these findings from the laboratory to clinical trials have proved unsuccessful.Therefore, it is necessary to fully understand the pathogenesis of septic kidney injury in order to develop more effective treatment strategies.
Circular ribonucleic acid (circRNA) is a widely available non-coding RNA with a closed-loop structure and high stability [8,9].There is growing evidence that many circrnas are strongly associated with a variety of human diseases, including sepsis [10,11].Previous studies have confirmed that circ-UBE2D2 mitigates the progression of SAKI in rats by targeting miR-370-3p/NR4A3 axis [12].Circ-RASGEF1B promotes LPS-induced apoptosis and inflammatory response by targeting miRNA-146a-5p/ PDK1 axis in septic acute kidney injury cell model [13].Knockdown of circ-FANCA alleviates LPS-induced HK2 cell injury via targeting miR-93-5p/OXSR1 axis in septic acute kidney injury [11].However, the function of circRNA in the progression of sepsis-induced AKI and the underlying mechanisms involved remain largely unknown.
In the current study, we found that the expression of circ-Gatad1 was increased in septic acute kidney.We constructed septic AKI model in HK2 cells with the stimulation of LPS.The expression pattern of circ-Gatad1 and its function were investigated in LPS-treated HK2 cells.Meanwhile, circ-Gatad1 was found to act a sponge for miR-22-3p.Mechanistically, the regulatory function of circ-Gatad1/miR-22-3p/mRNA network was explored, which might provide a new perspective for the pathogenesis of sepsis-related AKI.

Serum samples
The serum specimens were collected from 20 fasting septic AKI patients and 20 fasting healthy subjects at Shanghai Pudong New Area Gongli Hospital.These serum specimens were acquired by centrifugation and then preserved at − 80 °C prior to use.Written informed consent from all participants.The clinicopathologic characteristics of sepsis-AKI patients was listed in Table 1.

Strand-specific high-throughput RNA-Seq library construction
Total RNA from serum of AKI patients were extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA).Then 3 µg total RNA were used remove ribosomal RNA, and retain RNA classes including noncoding RNAs with VAHTS Total RNA-seq (H/M/R) Library Prep kits from Illumina (Vazyme Biotech Co., Ltd, Nanjing, China).We treated RNA through 40 U RNase R (Epicenter) at 37 °C for three hours, followed by TRIzol purification.We prepared RNA-seq libraries via KAPA Stranded RNA-Seq Library Prep kits (Roche, Basel, Switzerland) and used them for deep sequencing (Illumina HiSeq 4000 at Aksomics, Inc., Shanghai, China).

Cell culture, LPS induction and cell transfection
Human renal tubule epithelial cells (Human kidney-2, HK2) were purchased from Procell (Wuhan, China) and cultured in Minimum Essential Medium (MEM; Procell) plus 10% fetal bovine serum (FBS; Procell) and 1% penicillin-streptomycin (Procell) in a 37 °C, 5% CO 2 humid incubator.HK2 cells were treated with LPS at different concentrations (0-15 µg/mL) for 24 h.For the establishment of sepsis-related AKI model in vitro, HK2 cells in the following experiments were exposed to 5 µg/mL LPS (Solarbio, Beijing, China) for 12 h, while the control HK2 cells were cultivated with MEM medium simultaneously.

Cell counting kit-8 (CCK-8) assay
HK2 cells with various treatments were seeded into 96-well plates (1 × 104 cells/well) and incubated for 48 h.Subsequently, 10 µL CCK-8 reagent (5 mg/mL; Beyotime, Shanghai, China) was pipetted into each well, and cells were maintained at 37 °C for another 2 h.The absorbance of each well at 450 nm measured on a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) was utilized for determining cell viability.

Enzyme-linked immunosorbent assay (ELISA)
The release of inflammatory cytokines interleukin-1β (IL-1β), IL-6 and tumor necrosis factor α (TNF-α) in culture medium was determined using the corresponding ELISA kits against TNF-α, IL-1β and IL-6 (Beyotime) according to the user's manual.The colorimetric changes were determined at 450 nm.

RNA isolation and real-time PCR
Total RNA was extracted with TRIzol Reagent (Invitrogen), followed by cDNA synthesis using a TransScript All-in-One First-Strand cDNA Synthesis SuperMix (Transgen Biotech, Beijing, China), was performed.PCR was performed using a Bio-Rad PCR instrument (Bio-Rad, Hercules, CA, U.S.A.) with 2× Taq PCR Master Mix (Solarbio, Beijing, China) following the manufacturer's instructions.The fold changes were calculated by means of relative quantification (2 −△△Ct method).

Dual-luciferase reporter assay
The 3'UTR of HK2 gene and circ-Gatad1 containing the predicated binding sites for miR-22-3p were amplified and inserted into the multiple cloning sites of pMIR-REPORT luciferase reporter vector (Ambion, Austin, U.S.A.).Then, HK2 cells were co-transfected with 0.1 µg of luciferase reporter vectors comprising wild-type or mutant type of 3'UTR of HK2 or circ-Gatad1 and either miR-22-3p mimic or miR-control by Lipofectamine 2000 (Invitrogen, Carlsbad, U.S.A.).Relative luciferase activity was calculated by normalizing the firefly luminescence to the Renilla luminescence using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, U.S.A.) according to the manufacturer's instructions at 48 h post-transfection.

Reactive oxygen species (ROS) activity
Intracellular generation of ROS in HK2 cells or wound skin tissue were detected using Dihydroethidium (DHE) assay (Beyotime, China).Fluorescence was measured at 488 nm excitation and 525 nm emission using a fluorescence microplate reader (PerkinElmer, USA).

Statistical analysis
Continuous variables were denoted by means ± SD (standard deviation).We used one-way variance analysis for the comparisons by GraphPad Prism (version 5.0; GraphPad, La Jolla, USA).P ≤ 0.05 indicated statistical significances.

The expression of circ-Gatad1 was increased in septic acute kidney patients
High-throughput sequencing analysis identified a series of upregulated and downregulated circRNAs between healthy and AKI patient (Fig. 1A).RT-qPCR detection found that circ-Gatad1 expression was increased in septic acute kidney patients (Fig. 1B).Next, the septic AKI model in vitro was established by treating HK2 cells with LPS.And the result show that circ-Gatad1 expression was increased with the concentration increased of LPS (Fig. 1 C). 5 µg/mL LPS was selected for subsequent experiments.Since the localization of RNAs within cells are crucial for their functional display, we further determined the subcellular localization of circ-Gatad1 in HK2 cells.FISH and subcellular fractionation assays revealed that circ-Gatad1 was predominantly located in the cytoplasm (Fig. 1D and E).Meanwhile, RNase R assay showed that the level of linear Gatad1 mRNA was significantly reduced by the digestion with RNase R, while circ-Gatad1 was resistant to RNase R, indicating circ-Gatad1 was a highly stable circular RNA (Fig. 1F).These observations indicated that circ-Gatad1 might be a functional molecule in septic AKI progression.

Both mir-22-3p and TRPM7 were downstream targets of circ-Gatad1
Bioinformatics analysis (http://starbase.sysu.edu.cn/)found that circ-Gatad1 can interacted with a series of miRNA including miR-22-3p.A luciferase reporter analysis confirmed that miR-22-3p inhibited luciferase activity in wild-type cells, but not in mutated cell lines (Fig. 3A and B), suggesting that miR-22-3p was the target of circ-Gatad1.Bioinformatics analysis found that TRPM7 was the downstream target of miR-22-3p.To further confirm the relationship between miR-22-3p and TRPM7, wildtype or mutated 3'UTR-TRPM7 sequences containing the miR-22-3p binding sequence were constructed into a luciferase reporter vector (Fig. 3 C).The luciferase reporter vector was then transfected into HK2 cells combined with or without miR-22-3p mimic.A luciferase reporter analysis found that miR-22-3p inhibited luciferase activity in wild-type cells, but not in mutated cell lines (Fig. 23D), suggesting that TRPM7 was the target of miR-22-3p.

Discussion
Sepsis is a major cause of acute kidney injury (AKI) among patients in the intensive care unit [14].There is substantial evidence that patients diagnosed with septic AKI have a higher risk of death than those with non-septic AKI and generally have longer ICU and hospital stays [15][16][17].Therefore, it is of great significance to identify the specific pathogenesis for finding new therapies for septicemic AKI.Therefore, it is of great significance to identify the specific pathogenesis for finding new therapies for septicemic AKI.In this study, we found that cir-cRNA play an important role in regulation the progress of AKI.High-throughput sequencing analysis found that circ-Gatad1 expression was increased.In vitro simulation experiment show that circ-Gatad1 expression was increased with LPS induction and in dose dependent way.Similar to other circRNAs, circ-Gatad1 is primarily located in the cytoplasm [18,19].
The study also found that downregulation circ-Gatad1 reversed LPS induced proliferation inhibition.And circ-Gatad1 silence also inhibit LPS induced HK2 cells apoptosis, ROS accumulation and inflammatory factor expression.Sepsis caused by lipopolysaccharide (LPS) endotoxin is a common cause and trigger of AKI.Sepsis caused by lipopolysaccharide (LPS) endotoxin is a common cause of AKI and a trigger for inflammatory response [20].Its pathogenesis includes renal endothelial cell injury, renal cell apoptosis, overproduction of multiple inflammatory mediators, superoxide injury and inflammatory immune response [21][22][23].Its pathogenesis includes renal endothelial cell injury, renal cell apoptosis, excessive production of multiple inflammatory mediators, superoxide injury [24].
Further study found that miR-22-3p can interacted with 3'UTR of TRPM7 which was confirmed with luciferase report analysis.Previous studies have confirmed that TRPM7 was upregulated in LPS-treated cells, and knocking improved cell viability, reduced cells apoptosis, inflammation, and oxidative stress [28].The study also found that TRPM7 in renal ischemia-reperfusion injury might provide new mechanistic insights for a potential biomarker as diagnostic and therapeutic target of AKI [29].Our study found that overexpression TRPM7 reversed the protective effect of si-circ-Gatad1 to HK2 after exposure to LPS (5 µg/ml) microenvironment.Suggestion that circ-Gatad1 regulation the progress of AKI by sponging miR-22-3p and promotion TRPM7 expression.

Conclusion
Taken together, our results show that circ-Gatad1 promoted LPS-induced HK2 cell injury via regulating the miR-22-3p/TRPM7 signaling pathway, suggesting that circ-Gatad1 knockdown might be a potential pathway for alleviating sepsis-induced AKI.

Fig. 1
Fig.1The expression of circ-Gatad1 was increased in septic acute kidney.(A) High-throughput sequencing analysis identified a series of upregulated and downregulated circRNAs between healthy and AKI.(B) RT-qPCR detection show that the expression of circ-Gatad1 was increased in septic acute kidney patients.Data are expressed as mean ± SD. *** p < 0.001 vs. Healthy.(C) RT-qPCR detection show that the expression of circ-Gatad1 was increased in HK2 cells after treatment with different dose of LPS (0-15 µg/ml).Data are expressed as mean ± SD. *** p < 0.001 vs. 0 µg/ml.(D and E) Representative FISH images and subcellular fractionation assays showed the subcellular distribution of circ-Gatad1 in HK2 cells.U6 was used as a nuclear control and 18 S rRNA was used as a cytoplasmic control.Scales bar, 20 μm.(F) After RNase R treatment, the levels of circ-Gatad1 and linear Gatad1 mRNA were determined using qRT-PCR.Data are expressed as mean ± SD. *** p < 0.001 vs. Mock

Table 1
Clinicopathologic features of Sepsis-AKI patients