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. 2022 May 5;13(5):11388–11401. doi: 10.1080/21655979.2022.2061293

circSTRN3 aggravates sepsis-induced acute kidney injury by regulating miR-578/ toll like receptor 4 axis

Qiuying Gao 1, Yan Zheng 1, Hui Wang 1, Limin Hou 1, Xingxing Hu 1,✉
PMCID: PMC9275965  PMID: 35510365

ABSTRACT

Sepsis is a systemic inflammatory response caused by infection, and severe sepsis is commonly associated with the development of acute kidney injury (AKI). Accumulating evidence has revealed the implication of circular RNAs in AKI. In this study, we explored the potential engagement and the underlying mechanism of hsa_circ_010157 (circSTRN3) in sepsis-induced AKI. CircSTRN3 levels in HK2 cells and serum samples of patients were determined by RT-PCR. The protein levels of TLR4 (Toll Like Receptor 4), bax (Bcl-2-associated X protein), cleaved caspase 3 and bcl-2 (B-cell lymphoma-2) were detected by Western blotting (WB), and the levels of proinflammatory cytokines were detected by ELISA. The molecular interactions between mir-578/TLR4 and circSTRN3/miR-578 were analyzed by dual luciferase reporter assay as well as RNA pull-down experiment. Lipopolysaccharide (LPS) treated HK2 cells were used as an in vitro model to investigate the functional interaction of circSTRN3/miR-578/TLR4 axis. We found that the expression level of circSTRN3 in patients with sepsis-induced AKI and LPS-induced HK2 cells was higher. Silencing cicrSTRN3 alleviated LPS-induced cell proliferation, and suppressed the inflammatory response and apoptosis in LPS-treated HK2 cells. In contrast, the overexpression of circSTRN3 aggravated the cellular damages induced by LPS treatment. CircSTRN3 targeted miR-578/TLR4 axis to influence the damage effect induced by LPS. miR-578 inhibitor or TLR4 overexpression impaired the rescue effect of circSTRN3 knockdown. These results indicate that circSTRN3 upregulation in sepsis-induced AKI modulates miR-578/TLR4 axis to promote the pathogenesis of AKI, which could serve as future therapeutic targets for AKI treatment.

KEYWORDS: circSTRN3, miR-578/TLR4, sepsis, acute renal injury, inflammatory responses

Graphical abstract

graphic file with name KBIE_A_2061293_UF0001_OC.jpg

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Highlights

  • CircSTRN3 is upregulated in the patients of sepsis-associated AKI

  • Silencing circSTRN3 alleviates sepsis-induced kidney injury

  • CircSTRN3 sponges miR-578 and miR-578 negative target TLR4

  • miR-578/TLR4 axis mediates the effect of circSTRN3 in AKI

Introduction

Sepsis is a systemic inflammatory response caused by microbial infection, and severe sepsis could lead to multi-organ dysfunction [1]. About 60–70% of patients with severe sepsis develop the symptoms of acute kidney injury (AKI) [2]. AKI is generally characterized by intrarenal blood flow reorganization and low perfusion in the kidney, as well as kidney tubular injury, apoptosis, fibrosis and inflammatory responses [3,4]. The onset of AKI reduces glomerular filtration rate and is accompanied with the disturbance of urinary electrolytes and proteins, which can eventually lead to renal failure and mortality [5–7]. Sepsis has been recognized as the main cause of AKI [8], and AKI is a common complication of sepsis [9]. Previous studies suggest that the mortality rate of septic patients with AKI is significantly higher [10–12], and several risk factors including age, chronic kidney disease, cardiovascular disease, diabetes, hypoproteinemia, and chronic liver disease are associated with higher incidence of AKI in septic patients [13–15]. The pathophysiology of sepsis-associated AKI is multifactorial and complex, involving hemodynamic changes, microcirculation malfunction, inflammatory responses and severe fibrosis in kidney tissues [16]. In addition, endothelial glucocorticoid receptor and endothelial Sirtuin 3 have been implicate in the onset of renal fibrosis regulation [17,18]. Potential drugs alleviating the fibrosis and kidney injury have been reported. For example, in the mouse model of diabetic kidney injury, DPP-4 inhibitor linagliptin, JAK-stat inhibitors, glycolysis inhibitors, ACE inhibitors, and peptide AcSDKP have been shown to have beneficial roles in protecting renal fibrosis [19].

Although genetic factors could also contribute to the susceptibility of sepsis induced AKI [20], non-coding RNAs (ncRNAs) have been implicated in the regulation of inflammatory responses and other pathological conditions in the occurrence and development of acute renal injury [21–23]. ncRNAs, including microRNAs, circular RNAs (circRNAs) and long noncoding RNAs (lncRNAs), are involved in gene expression regulation at transcriptional and post-transcriptional levels. The dysregulation of ncRNAs can affect cell differentiation and the development of a variety of diseases such as kidney fibrosis [24,25]. For example, the crosstalk between miR-29s and miR-let-7s have been shown to regulate epithelial-to-mesenchymal transition program and plays an important antifibrotic role [26,27]. Due to the versatile roles of ncRNAs in the renal fibrosis [28], understanding the mechanism by which ncRNA regulates fibrotic responses in AKI could provide insights into the development of strategies to ameliorate the symptoms of AKI.

In this study, we first demonstrated that circSTRN3 was significantly upregulated in patients with sepsis-associated AKI. Silencing circSTRN3 attenuated the cell proliferation inhibition, inflammatory responses and apoptosis in LPS-induced HK2 cells, which was also validated in a mouse mode of LPS induced AKI. Since circRNAs could interact with other miRNAs to regulate sepsis-induced AKI [29], we further search for the downstream target of circSTRN3. We found that circSTRN3 interacted with miR-578 to regulate the expression of TLR4. Targeting miR-578/TLR4 axis could modulate the effect of circSTRN3 knockdown on LPS-induced HK2 cell model. Our study suggests that circSTRN3 upregulation in sepsis-induced AKI regulates miR-578/TLR4 axis to aggravate AKI, which could serve as potential therapeutic targets for AKI treatment.

Materials and methods

Cell culture and cell model

Human renal tubular epithelial HK2 cells were acquired from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and cultivated within RPMI-1640 medium (Gibco Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (FBS, Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 1% Penicillin-Streptomycin Solution (Solarbio, P1400-100 ml, CHINA) in a humidified incubator under 37°C and 5% CO2. The renal injury cell model was established by treating HK2 cells with lipopolysaccharide (LPS) at 2 μg/mL for 48 hours.

Clinical samples

The serum samples from 30 healthy controls and patients diagnosed with sepsis-associated AKI were collected from 2020 June to 2020 Dec at the Department of Hematology, Shaanxi Provincial People’s Hospital, China. All the participants signed the informed consent. The study was approved by the ethic committee of Shaanxi Provincial People’s Hospital.

Cell transfection

HK2 cells (1 × 105/well) were seeded in 6-well plates overnight under 37°C till 60% confluency. serum free medium. The transfection was performed using Lipofectamine® 2000 (Invitrogen) in serum-free medium. The following molecules were synthesized and purchased from RiboBio (Guangzhou, China): si-NC: 5′-ACGUGACACGUUCGGAGAATT-3′, si-circSTRN3#1: 5'-GGTGAAGAG CCCGGATTGCAT-3', si-circSTRN3#2: 5'-AAGGGGTGAAGAGCCCGGATT-3', si-circSTRN3#3: 5'-TGAAGAGCCCGGATTGCATTT-3', miR-578 inhibitor, miR-578 mimic, miR-NC pcDNA3.1 vector and pcDNA3.1-TLR4 expression vector. Cells were transfected with 100 nM of microRNA mimic or inhibitor or 6 ug of pcDNA3.1-TLR4 plasmid according to manufacturer’s instruction for 8 h. Afterward, medium containing 10% FBS was used to culture cells for additional 48 h before further experiments.

Cell proliferation assay

Cells proliferation was measured by CCK-8 Cell Proliferation and Cytotoxicity Assay Kit (Solarbio, China). Cells were seeded in a 96-well plate (5 × 103 cells/well) and cultured at 37°C for 24 h, 48 h and 72 h. 10 μL CCK8 reaction solution was added to each well at indicated time point and incubated for 3 h in a humidified incubator. The light absorption value (OD value) at 450 nM in each condition was captured on a Synergy H1 microplate reader.

Dual luciferase reporter assay

To validate functional interactions between two molecules, the sequence containing wild type (WT) binding site and the sequence with mutated binding site (MUT) were cloned into a firefly luciferase reporter plasmid (Promega, USA). The reporter plasmid and Renilla luciferase (hRlucneo) plasmid were co-transfected into cells in the presence of miR-578 mimic or miR-NC. 48 h after transfection, the relative luciferase activities were examined using the Dual-Luciferase Reporter Assay Kit (Promega, USA) on a luminescence plate reader. The firefly luciferase activity was normalized to that of Renilla luciferase.

RNA Pull down assay

HK2 cell lysates were collected by IP lysis buffer (Beyotime, China) and incubated with 200 nM biotinylated miR-578 or miR-NC probe. 10% of total cell lysates was reserved as the input. The mixture was then incubated with 100 µL M-280 streptavidin magnetic beads (Sigma-Aldrich, Germany) at 4°C for 4 h, and a magnetic bar was used to precipitate the magnetic beads. After 4 times washes with the lysis buffer, the samples in the beads were purified with Trizol reagent (Invitrogen, USA) according to the manufacturer’s protocol. RT-qPCR was then performed to determine the relative level of precipitated circSTRN3 in each sample by normalizing to that of the total input.

RNA isolation, cDNA synthesis and RT-PCR

HK2 cells (5 × 104/well) were cultured in 6-well plates and subjected to indicated treatment. The total RNA was isolated using iPrep™ Trizol™ Plus RNA Kit (Thermo Fisher Scientific, USA). 2 µg of total RNA was reverse transcribed into cDNA using RNase Inhibitor and High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA). Gene expression was determined using FastFire qPCR PreMix (Tiangen, China) in a ProFlex™ PCR system through standard curve method. The PCR condition was as follows: initially denatured at 95°C 2 min; 10s at 95°C, 15s at 60°C, and 30s at 72°C for 40 cycles.

RNase R digestion assay

Rnase R (TaKaRa, Maebashi, Japan) is used to degrade linear RNA. The RNA sample was divided equally into two portions: one was used for Rnase R digestion (Rnase R group), and the other was used as control (Mock group). The two portions of samples were incubated at 37°C for 25 min. The relative amount of STRN3 mRNA and circNMD3 in each sample was detected by RT-qPCR.

For RNA stability assay, the transcription was blocked by 3 μg/mL actinomycin D (Sigma) for 4 h and RNA samples were collected by TRizol reagent. The stability of circNMD3 and NMD3 mRNA was analyzed by RT-qPCR by comparing to that in the samples before treatment (Control).

Apoptosis assay

Annexin V-FITC/PI apoptosis Kit (Elabscience, E-CK-A211, China) was used for apoptosis analysis by flow cytometry. In brief, 5 µl of Annexin V and 3 µl PI dye was added to 1 ml cell suspension in annexin-V binding buffer for 20 min staining. Stained cells were centrifuged and washed twice with 1xPBS and resuspended in 400 μL PBS. The percentage of apoptotic cells was detected by BD FACS CantoTM II Flow Cytometer (BD Biosciences). Cells were gated on single cells using FSC-A and FSC-H parameters.

Western blotting (WB) analysis

HK2 cells were lysed using RIPA Lysis-Buffer (Solarbio, R0010, China) which contained Protease Inhibitor Cocktail (Roche Applied Science, Pleasanton, CA, USA). BCA Protein Detection kit (Solarbio, PC0020, China) was used to measure total protein concentration. 10 ug of total protein was used for SDS-PAGE electrophoresis. Separated protein in SDS_PAGE gel was transferred onto the PVDF membrane. After blocking with 5% skimmed milk for 1 hour, the membrane was incubated with primary antibodies at 4 oC overnight: anti-TLR4, bax, cleaved-caspase 3, bcl-2 and β-actin (all the antibody purchased from Abcam, dilution 1:1000). The membrane was washed 3 times with TBST for 5 minutes each. After wash, the membrane was further incubated with HRP-linked secondary antibody (1:3000; Boster, BA1112, China). Then, the membrane was washed 4 times with TBST buffer and the protein bands were visualized using an enhanced chemiluminescence kit (Santa Cruz, USA) and photographed on a gel imager system. The densitometry analysis was performed with Image J software (Bethesda, MD, USA).

ELISA

The determination of interleukin (IL)-1β, interleukin (IL)-6 and tumor necrosis factor (TNF)-α was performed by corresponding ELISA kit (Sigma, St. Louis, MO, USA). Briefly, supernatant was added to the capture-antibody-coated plate. After a wash step to remove unbound material, biotin-labeled detection antibody was added, which is followed by streptavidin–HRP. Chemiluminescent detection reagents were added for signal development and the optical density of samples and standards was measured at 450 nm using a microplate reader (Infinite 200 PRO; Tecan). The concentration of each cytokine was measured based on the linear regression of the standards.

Mouse model of AKI

In the AKI induction group, 8-week-old male mice were injected intraperitoneally with 30 mg/kg LPS. The control group was injected with equal volume of PBS, and the rescue group was injected with LPS and 0.2 mg/kg si-circSTRN3. Each group contains 5 mice. After 48 h, blood samples were collected from the tails in heparinized tubules for measuring IL-1β, IL-6 and TNF-α. Mice were sacrificed 120 h after the induction of the AKI. This animal study was approved by the Ethics Committee at the Shaanxi Provincial People’s Hospital.

H&E staining

Hematoxylin and Eosin (H&E) Staining was performed using H&E Stain Kit (ab245880, Abcam). Kidney tissue sections were incubated in adequate Hematoxylin solution for 5 min. The section was rinsed twice with distilled water to remove excess stain. Then, adequate Bluing Reagent was applied for 30 sec. After washing with distilled water, the section was dehydrated in absolute alcohol, followed by the counter staining with Eosin Y Solution for 2–3 min. The section was rinsed using absolute ethanol for three times and then mounted to a slide. The images were collected under an inverse microscope.

Statistical analysis

Statistical analysis was completed by SPSS22.0. The data of CCK8, PCR, Western blot, ELISA and RNA pull down were expressed as mean ± SD. The statistical difference between two groups was analyzed by unpaired student’s t tests. Comparisons among multiple groups were examined using one-way analysis of variance (ANOVA) with Tukey’s post hoc test. P < 0.05 indicated statistical significance.

Results

CircSTRN3 was upregulated in the serum samples of patients with sepsis-associated AKI. Silencing circSTRN3 attenuated the cell proliferation inhibition, inflammatory responses and apoptosis in LPS-induced HK2 cells, which was also validated in a mouse mode of LPS induced AKI. Since circRNAs could interact with other miRNAs to regulate sepsis-induced AKI [29], we further search for the downstream target of circSTRN3. We found that circSTRN3 interacted with miR-578 to regulate the expression of TLR4. Targeting miR-578/TLR4 axis could modulate the effect of circSTRN3 knockdown on LPS-induced HK2 cell model.

CircSTRN3 was highly expressed in the serum sample of sepsis-associated AKI patients and LPS-induced AKI model

We first collected the serum samples from 30 healthy controls and patients diagnosed with sepsis-associated AKI. RT-qPCR analysis revealed the upregulation of circSTRN3 in the serum of patients with AKI (Figure 1a). CircSTRN3 expression was also significantly increased in HK2 cells with LPS induction (Figure 1b). To demonstrate the stability of circSTRN3 as a circular RNA, we performed RNase R digestion assay in HK2 cells, which revealed that STRN3 mRNA decreased after RNase R treatment, while there was no significant change in the expression level of circSTRN3 (Figure 1c). Furthermore, the RNA stability assay by Actinomycin D treatment showed that cicrSTRN3 was more stable than STRN3 mRNA (Figure 1d).

Figure 1.

Figure 1.

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Expression level of CircSTRN3 in serum of SA-AKI patients. A: RT-qPCR was used to detect the expression level of circSTRN3 in the serum samples of healthy controls and patients with SA-AKI (n = 30). B: RT-qPCR analysis of the expression of circSTRN3 HK2 cells treated with LPS. C: The expression levels of STRN3 mRNA and circSTRN3 in the total RNA samples of HK2 cells with or without RNase R treatment. D: The expression levels of STRN3 mRNA and circSTRN3 in the total RNA samples of HK2 cells after actinomycin D treatment. Data was normalized to the control samples treated with DMSO. ***P < 0.001.

CircSTRN3 knockdown attenuated LPS-induced sepsis in cell and mouse model

To investigate the functional role of circSTRN3 in sepsis and AKI, we applied circSTRN3 siRNAs (si-circSTRN3#1, si-circSTRN3#2, si-circSTRN3#3) in HK2 cells. RT-qPCR analysis showed that si-circSTRN3#1 had the greatest silencing effect in HK2 cells, which was used in the following experiment (Figure 2a). We then performed CCK8 proliferation assay to examine the effect of circSTRN3 silencing upon LPS treatment in HK2 cells. LPS treatment suppressed cell proliferation, and the transfection of si-circSTRN3 partially rescued the proliferation (Figure 2b). Consistently, the apoptosis-induced by LPS was also partially inhibited by circSTRN3 knockdown (Figure 2c). These results indicate that in the cell mode of sepsis, silencing circSTRN3 shows protective effect against LPS-induced damage.

Figure 2.

Figure 2.

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The knockdown of CircSTRN3 rescues LPS-induced damages in HK2 cells and mouse model. A: The expression level of circSTRN3 in HK2 cells after the transfection with si-NC, si-circSTRN3#1, si-circSTRN3#2, si-circSTRN3#3. B: CCK8 proliferation assay in HK2 cells upon LPS treatment and si-circSTRN3 transfection. C: Apoptosis analysis in HK2 cells upon LPS treatment and si-circSTRN3 transfection. D: H&E staining of kidney medulla tissues in mice treated with PBS (con), LPS, or LPS and si-circSTRN3 (n = 5 in each group, scale bar = 800 μm). E-G: ELISA analysis of inflammatory cytokines (TNF- α, IL-1 β and IL-6) in the blood samples of mice treated with PBS (con), LPS, or LPS and si-circSTRN3 (n = 5 in each group). H. Western blot analysis of protein levels of collagen I, bax, cleaved caspase-3, and bcl2 in the kidney tissues of each group. ***P < 0.001; **P < 0.01; *P < 0.05.

We next investigated the role of circSTRN3 silencing in the mouse model of AKI. Mouse were intraperitoneally injected with PBS (Control), LPS or LPS+si-circSTRN3. H&E staining revealed the tubular cell vacuolation and tubular dilation and distortion after LPS treatment, and si-circSTRN3 partially suppressed these symptoms in the kidney (Figure 2d). ELISA analysis in the serum samples demonstrated that the increased level of proinflammatory cytokines (interleukin (IL)-1β, interleukin (IL)-6 and tumor necrosis factor (TNF)-α) upon LPS treatment were significantly reduced by si-circSTRN3 (Figure 2 e-g). We further performed Western blot to analyze the protein levels of apoptosis-related proteins bax, cleaved caspase 3 and bcl-2, as well as collagen I (fibrosis marker) in the kidney tissues. As expected, LPS treatment increase the level of pro-apoptotic proteins (bax and cleaved caspase 3) and fibrotic marker (collagen I), but reduced the level of anti-apoptotic protein (bcl-2). The presence of si-circSTRN3 partially suppressed the increases of pro-apoptotic protein and fibrotic marker (Figure 2h). Together, these results indicate that the upregulation of circSTRN3 contributes to the sepsis-induced AKI in the mouse model.

CircSTRN3 overexpression aggravates LPS-induced cell damages in HK2 cells

To further demonstrate the contribution of circSTRN3 to sepsis-induced kidney cell damages, we transfected JK2 cells with circSTRN3 overexpression vector, which could increase the level of circSTRN3 by 3 folds (Figure 3a). The overexpression of circSTRN3 exacerbated the inhibitory effect of LPS induced cell proliferation (Figure 3b). CircSTRN3 overexpression also promoted the apoptosis (Figure 3c) and the proinflammatory cytokine production induced by LPS (Figure 3d-f). Meanwhile, circSTRN3 overexpression enhanced the production of collagen I after LPS treatment (Figure 3g). Overall, these data suggest that circSTRN3 upregulation could exacerbate the cellular damages and fibrosis induced by LPS.

Figure 3.

Figure 3.

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CircSTRN3 overexpression aggravates the effects of LPS treatment in HK2 cells. A:HK2 cells was transfected with empty vector and pcDNA3.1-circSTRN3 plasmid, and RT-qPCR was performed to detect the expression level of circSTRN3 after transfection. B: CCK8 proliferation assay in HK2 cells upon LPS treatment and circSTRN3 overexpression. C: Apoptosis analysis in HK2 cells in HK2 cells upon LPS treatment and circSTRN3 overexpression. D-F: ELISA analysis of inflammatory cytokines (TNF- α, IL-1 β and IL-6) in the cell culture supernatant of HK2 cells upon LPS treatment and circSTRN3 overexpression. G: Collagen I expression in HK2 cells upon LPS treatment and circSTRN3 overexpression. ***P < 0.001; **P < 0.01; *P < 0.05.

CircSTRN3 negatively regulates mir-578 in HK2 cells

To identify the downstream target of circSTRN3, we search the Circinteractome online database and miR578 was identified as the top ranked target with potential binding site to circSTRN3 (Figure 4a). We then performed dual luciferase reporter assay using WT reporter containing the wild type binding site and the MUT reporter containing mutated binding site. The presence of miR-578 significantly inhibited the luciferase activity of WT reporter, while no effect was observed in MUT reporter (Figure 4b). To further validate their physical interaction, we performed RNA-pull down assay using biotin-labeled miR578 probe, with biotinylated miR-NC as the control. Compared with miR-NC probe, miR-578 probe could significantly enriched circSTRN3 in the pull-down assay (Figure 4c).

Figure 4.

Figure 4.

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CircSTRN3 targets miR-578. A. The binding site between miR-578 of circSTRN3 was predicted by Circinteractome. B: Dual luciferase reporter assay in HK2 cells to verify the interaction. C: RNA pull-down assay in HK2 lysate with biotinylated miR-578 probe or miR-NC probe. The precipitated circSTRN3 level was quantified by RT-qPCR and the data was normalized to the level of input. D: RT-qPCR was used to detect the expression level of miR-578 in serum of healthy controls and patients with SA-AKI (n = 30). E: Correlation analysis showed that there was a negative correlation between the expression of circSTRN3 and miR-578 in the serum samples of patients with SA-AKI. F: The expression levels of miR-578 in HK2 cells treated with LPS or LPS+si-circSTRN3 were detected by RT-qPCR. ***P < 0.001; **P < 0.01.

The interaction between circRNA and miRNAs may affect the expression of miRNAs. We next examined the expression level of miR-578 in serum samples of healthy controls and patients with sepsis-associated AKI. In contrast to cicrSTRN3, the level of miR-578 was significantly reduced low in patients with AKI (Figure 4d). Correlation analysis showed that there was a negative correlation between the expression of circSTRN3 and miR-578 in the serum samples of patients with AKI (Figure 4e). In the cell model of HK2, LPS treatment suppressed the expression of miR-578, and the silencing of circSTRN3 rescued its expression (Figure 4f). Together, our data indicate that circSTRN3 negatively regulates mir-578 in HK2 cells.

MiR-578 targets TLR4 in HK2 cells

To search for the mRNA target of miR-578, we relied on the TargetScan database and found that there was a binding site of miR-578 at the 3’ UTR of TLR4 mRNA, and the dual luciferase reporter assay showed that miR-578 mimic could specifically suppressed the luciferase activity of WT reporter (Figure 5a). The overexpression of miR-578 by miR-578 mimic transfection could significantly reduce the protein level of TLR4 (Figure 5b). On the contrary, inhibition of miR-578 by the transfection of miR-578 inhibitor significantly increased the level of TLR4 (Figure 5c). We also examined the effect of circSTRN3 overexpression on TLR4 expression. Western blot revealed a significant increase of TLR4 level after the transfection of circSTRN3 expression plasmid (Figure 5d). Together these results suggest that miR-578 negatively regulate TLR4 expression in HK2 cells.

Figure 5.

Figure 5.

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miR-578 negatively regulates TLR4. A: The miR-578 binding site in the 3’ untranslated region of TLR4 mRNA was predicted by TargetScan, and the luciferase reporter assay was carried out in HK2 cells. B: Protein level of TLR4 in HK2 cells transfected with mir-NC, miR-578 mimic. C: Protein level of TLR4 in HK2 cells transfected with NC inhibitor or miT-578 inhibitor. D: Protein level of TLR4 in HK2 cells upon circSTRN3 overexpression. ***P < 0.001; **P < 0.01.

CircSTRN3 regulates LPS-induced cell injury by targeting miR-578/TLR4 axis

Based on the above observation, we hypothesized that miR-578/TLR4 axis mediates the effect of circTRN3 in LPS-induced cell damages. To test this hypothesis, we first applied TLR4 expression vector to overexpress TLR4 in HK2 cells (Figure 6a). We then transfected HK2 cells with si-circSTRN3, si-circSTRN3+ miR-578 inhibitor or si-circSTRN3+ TLR4 expression vector upon LPS treatment. CCK-8 proliferation assay showed that in the presence of miR-578 inhibitor or TLR4 overexpression, the rescue effect of circSTRN3 silencing was largely abrogated (Figure 6b). Consistently, the rescue effect of si-circSTRN3 on apoptosis was attenuated by miR-578 inhibitor or TLR4 overexpression (Figure 6c). miR-578 inhibitor or TLR4 overexpression also increased the expression of proapoptotic proteins as well as collagen I. In the meanwhile, miR-578 inhibitor or TLR4 overexpression impaired the effect of circSTRN3 silencing on suppressing inflammatory cytokine (Figure 6e). Together, these data indicate that miR-578/TLR4 axis mediates the effect of circSTRN3 in LPS-induced kidney cell damages.

Figure 6.

Figure 6.

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CircSTRN3 regulates LPS-induced KK2 cell injury by targeting miR-578/TLR4 axis. A: Protein level of TLR4 HK2 cells after the transfection with TLR4 expression vector. B: CCK8 proliferation assay in HK2 cells (Control, LPS, LPS+si-circSTRN3, LPS+si-circSTRN3+ miR-578 inhibitor, LPS+si-circSTRN3+ TLR4). C: Apoptosis analysis in HK2 cells of the indicated treatment group. D: Western blot was performed to detect the protein levels of collagen I, cleaves caspase 3, bax and bcl-2 in indicated groups. E: ELISA analysis of inflammatory cytokines (TNF – α, IL-1 β and IL-6) in indicated groups. ***P < 0.001; **P < 0.01; *P < 0.05.

Discussion

Sepsis is a systemic syndrome in which the tissues are infected by pathogenic bacteria [30]. The pathogen-derived toxins circulate in the blood system and induces systemic inflammatory responses [31]. Among them, acute kidney injury (AKI) is a common clinical complication as a consequence of sepsis [32], which could increase the mortality and aggravate the physical and psychological burden of the patients [33–35]. The pathogenesis of AKI caused by sepsis is complicated, which is related to the functional disorder caused by hemodynamic changes, microcirculation malfunction, inflammatory responses and severe fibrosis in kidney tissues [36,37]. Therefore, attenuating the inflammatory responses and fibrosis have been recognized key strategies in ameliorating the damages in AKI.

Recent studies have highlighted the critical roles of circRNAs in a variety of pathogenic conditions, and the dysregulation of circRNAs has been proposed as clinical marker for certain diseases [38]. Recent findings suggest that circRNAs are implicated in regulating immune responses against different pathogens, and the deregulation of circRNAs has been reported in sepsis [39]. In this study, we found that the level of hsa_circ_0101574 (circSTRN3) in the serum samples of patients with sepsis-associated AKI and in the mouse model of AKI was significantly upregulated. The upregulation of circSTRN3 was also induced in HK2 upon LPS treatment. Importantly, the silencing of circSTRN3 in HK2 cells and in the mouse model of AKI could attenuate the inflammatory responses, apoptosis and fibrosis induced by LPS. In contrast, in HK2 cells circTRN3 overexpression exacerbates the damaging effect of LPS by promoting cell death, inflammatory responses and upregulating fibrotic marker. Therefore, we conclude that circSTRN3 upregulation contributes to the progression of sepsis-induced AKI.

We also demonstrated that circSTRN3 targets miR-578 and suppresses the expression of miR-578. Since there is a negative correlation between circSTRN3 and miR-578 levels in the serum samples of patients with AKI, indicating circSTRN3 and miR-578 plays opposite roles in AKI. This is consistent with previous studies in which circular RNAs sponge miRNAs and negatively regulate their activities [40,41]. We further showed that miR-578 interacts with TLR4 mRNA and suppresses TLR4 expression. Since miR-578 inhibitor or TLR4 overexpression could attenuate the effect of circSTRN3 silencing in GK2 cells, our study indicates that circSTRN3 regulates LPS-induced kidney cell injury by targeting miR-578/TLR4 axis.

Our study also suggests that the knockdown of circSTRN3 protects LPS-induced kidney cell injury by suppressing apoptosis, inflammatory responses and fibrosis. These effects are mediated by miR-578/TLR4 axis. On the contrary, the over expression of circSTRN3 aggravates apoptosis, inflammatory responses and fibrosis induced by LPS treatment. Recent studies highlighted the roles of ncRNAs such as miRNAs and lncRNAs in kidney disease. For example, the crosstalk between miR-29s and miR-let-7s regulates epithelial-to-mesenchymal transition program and plays an important antifibrotic role in diabetes-related kidney fibrosis [26,27]. LncRNA SNHG5 relieves sepsis-induced AKI by regulating the miR-374a-3p/TLR4 axis [42].

As a critical mediator of innate immunity and inflammation, TLR4 plays a significant role in inflammation-associated disease. Increased TLR4 expression has been reported to aggravate sepsis-induced inflammation in mouse model [43]. In LIGHT (the 14th member of the tumor necrosis factor superfamily) knockout mice, the TLR4-Myd88-NF-κB signaling axis in the kidney is dramatically tuned down, and LIGHT deficiency protects against kidney damages in LPS-induced AKI mouse model [44]. Our study also suggests that the upregulation of circSTRN3 could promotes TLR4 expression by suppressing miR-578. Therefore, targeting miR-578/TLR4 axis could serve as potential therapeutic approach to ameliorate kidney damages in sepsis-induced AKI.

Conclusion

In summary, we reported that circSTRN3 is upregulated in patients with sepsis-associated AKI, as well as in the mouse model of sepsis-induced AKI. The upregulation of circSTRN3 suppresses kidney cell proliferation, promotes apoptosis, inflammatory responses and fibrosis by targeting mir-578/TLR4 axis. Future work is needed to fully elucidate the mechanism how circSTRN3 is upregulated in sepsis-associated AKI, which can provide insights into the development of therapeutic strategy to target circSTRN3 in AKI.

Funding Statement

The author(s) reported there is no funding associated with the work featured in this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability

The data is available from the corresponding author on reasonable request.

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Data Availability Statement

The data is available from the corresponding author on reasonable request.

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