Circ_0023404 sponges miR‐136 to induce HK‐2 cells injury triggered by hypoxia/reoxygenation via up‐regulating IL‐6R

Abstract The significance of circular RNAs (circRNAs) is reported in various kidney diseases including acute kidney injury (AKI). Specific circRNAs have the capacity to function as novel indicators of AKI. Circ_0023404 exhibits an important role in several diseases. Nevertheless, the detailed biological role of circ_0023404 in AKI remains poorly known. The present study aimed to investigate the effect of circ_0023404 on renal ischaemia/reperfusion (I/R) injury in vitro. Here, we evaluated the function of circ_0023404 in HK‐2 cells in response to hypoxia/reoxygenation (H/R). We established a cell AKI model induced by H/R in HK‐2 cells. We found circ_0023404 was significantly increased in AKI. Then, we found loss of circ_0023404 increased cell growth, repressed apoptosis, reduced inflammatory factors secretion and oxidative stress generation in vitro. Besides, circ_0023404 sponged miR‐136. miR‐136 overturned the effects of circ_0023404 on HK‐2 cell injury. We assumed IL‐6 receptor (IL‐6R) as a target of miR‐136 and IL‐6R was activated by circ_0023404 via sponging miR‐136. In conclusion, we revealed circ_0023404 contributed to HK‐2 cells injury stimulated by H/R via sponging miR‐136 and activating IL‐6R.

reported to be implicated in a wide range of diseases including AKI progression. 10 For example, circular RNA can exhibit a protective effect on AKI model. 11 Circ-ciRs-126 can indicate survival in critically Ill AKI patients. 12 CircPRKCI can reduce LPS-triggered HK2 cell injury through inducing the zinc finger E-box-binding homeobox 1 (ZEB2) targeted by miR-545. 13 Additionally, circRNAs are shown to function as sinks for miRNAs and they can control the function of miRNAs. 14 However, it is unknown whether circ_0023404 plays an important influence in AKI and little is reported about its mechanisms.
In the work, we established H/R model using HK-2 cells. We tested expression level of circ_0023404 in AKI patients and HK-2 cells treated with H/R condition. Then, we studied the effects of circ_0023404 on cell proliferation, apoptosis, inflammatory response and oxidative stress. Meanwhile, miR-136 acted as a downstream target of circ_0023404 and IL-6R acted as a target of miR-136. miRt136 has been experimentally verified as new biomarkers in various diseases, including renal diseases. 15 Therefore, it is suggested that miR-136 may exhibit great promise as a crucial biomarker for AKI. We explored the role of circ_0023404/miR-136/IL-6R axis in the development of AKI. This study might indicate that circ_0023404 can function as a novel therapeutic target for AKI via modulating miR-136 and IL-6R.

| Patient samples
The serum samples of 30 AKI cases and aged-equal healthy cases were attained from Affiliated Huai'an Hospital of Xuzhou Medical University. No patients had been given any therapies before enrolment. We obtained the informed consents and medical ethics certification from the Medical Ethics Committee of Affiliated Huai'an Hospital of Xuzhou Medical University. Venous blood was disposed within 1 hour after collection. In brief, serum samples were isolated at centrifugation of 1200 g for 10 minutes. Then, another centrifugation (10 000 g, 10 minutes) was followed to discard residual cellular debris. All centrifugations were carried out at 4°C. All serum samples were maintained in liquid nitrogen for RNA extraction.

| Cells
HK-2 cells were purchased from Shanghai Institutes for Biological Sciences (Shanghai, China). We cultured the cells using DMEM added with 10% FBS and 1% penicillin and streptomycin under a humidified atmosphere consisting of 5% CO 2 and 95% air at 37°C (control group). Hypoxia and reoxygenation (H/R) injury was induced by exposing HK-2 cells to hypoxic conditions (1% O 2 , 5% CO 2 and 94% N 2 ) for 12 hours, followed by reoxygenation for 2 hours in fresh normal medium (H/R group).

F I G U R E 1
The relative expression levels of circ_0023404 in AKI patients and H/R-treated HK-2 cells were increased. A, circ_0023404 expression levels were elevated in AKI serum samples than that in normal serum samples. RT-qPCR was carried out to evaluate circ_0023404 expression in serum samples of patients with AKI (n = 30) and healthy cases (n = 30). B, HK-2 cells were exposed to H/R condition. Circ_0023404 expression was detected using RT-qPCR. Three independent experiments were carried out. Error bars stand for the mean ± SD of at least triplicate experiments. *P < .05, ***P < .001 [Colour figure can be viewed at wileyonlinelibrary.com] F I G U R E 2 Loss of circ_0023404 increased HK-2 cells proliferation and inhibited cell apoptosis. A, After transfection with circ_0023404 siRNA, the transfection efficiency was evaluated using RT-qPCR. HK-2 cells were transfected with circ_0023404 siRNA and then exposed to H/R environment. H/R (B) Cell viability was tested by CCK-8 assay. C and D, Cell apoptosis ratio was assessed by Hoechst 33342 assay. Three independent experiments were carried out. Error bars stand for the mean ± SD of at least triplicate experiments. *P < .05

| Western blot analysis
To obtain the total proteins, RIPA lysis buffer added with PMSF was used at 4°C. The supernatant was obtained after cells were centrifuged at 9500 g/min for 5 minutes. Afterwards, BCA Protein Assay Kit (Beyotime, Shanghai, China) was carried out to measure protein concentration and the protein was boiled for degeneration.
Then, the proteins were loaded on 10% SDS-PAGE and transferred to PVDF membranes. We added the primary antibodies to the membranes for a whole night at 4°C. Primary antibodies, anti-IL-6R and anti-GAPDH (1:1000, Abcam, Cambridge, UK), were used.
After incubated with secondary antibodies for 2 hours, the membranes were exposed to the enhanced chemiluminescence reagent. Finally, the bands were evaluated utilizing ImageJ software.

| Statistical analysis
Three independent experiments were performed, and experimental data were expressed as mean ± standard deviation. Statistical analysis was executed using GraphPad 6.0 software. We calculated P-value using Student's t test or ANOVA. A P < .05 was indicated to be statistically significant.

| Expression of circ_0023404 in serum of AKI patients and H/R-incubated HK-2 cells
Firstly, the relative expression level of circ_0023404 was determined by real-time qPCR in a total of 30 AKI patients. In Figure 1A, circ_0023404 expression was greatly increased in AKI patients.
Then, we investigated the effect of H/R condition on HK-2 cells. HK-2 cells were exposed to H/R environment to set up AKI cell model. In Figure 1B, we displayed that circ_0023404 was up-regulated in H/Rtreated cells. These implied that the expression of circ_0023404 was increased in AKI patients and in HK-2 cells treated with H/R condition.

| Loss of circ_0023404 reduced H/R-triggered injury in HK-2 cells
Next, we investigated whether circ_0023404 participated in HK-2 cell processes. Circ_0023404 siRNA was transfected to modulate circ_0023404 expression in HK-2 cells. It was confirmed circ_0023404 was significantly decreased by circ_0023404 siRNA and circ_0023404 siRNA-02 exerted a best effect in HK-2 cells ( Figure 2A). Then, circ_0023404 siRNA-02 was used in subsequent assays. After exposed to H/R treatment, cell viability and apoptosis were determined. In Figure 2B, we found H/R exposure significantly reduced HK-2 cell viability. HK-2 cell growth was induced by circ_0023404 siRNA. In addition, H/R was able to induce cell apoptosis, which was prohibited by loss of circ_0023404 in vitro ( Figure 2C and D).

| Down-regulation of circ_0023404 repressed inflammatory response
Meanwhile, cell inflammatory response was assessed by detecting inflammatory cytokines. Whether circ_0023404 was involved in inflammation of HK-2 cells under H/R was investigated. We observed that IL-1β, IL-6 and TNF-α protein expression in HK-2 cell culture medium could be triggered by H/R treatment. Decrease of circ_0023404 was able to reverse this process ( Figure 3A-C).
Additionally, IL-1β, IL-6 and TNF-α mRNA levels extracted from HK-2 cells demonstrated a similar tendency as shown in Figure 3D-F.

| Decrease of circ_0023404 reduced H/Rtriggered oxidative stress of HK-2 cells
Next, we further determined whether circ_0023404 regulated oxidative stress in HK-2 cells. We displayed H/R treatment strongly upgraded ROS generation, MDA contents and increased SOD activity ( Figure 4A-C). For another, loss of circ_0023404 was able to repress the oxidative stress process as manifested in Figure 4A-C.
miR-136 expression was changed most significantly. miR-136 was selected miR-136 in our following research. The binding sites between miR-136 and circ_0023404 are shown in Figure 5B. Then, MUT and WT sequence of circ_0023404 were cloned into pGL3 vector to construct circ_0023404 MUT and circ_0023404 WT plasmids. Luciferase reporter assay revealed that the luciferase activity was decreased in cells co-transfected with circ_0023404 WT and miR-136 mimics in Figure 5C. As displayed in Figure 5D, miR-136 was negatively regulated by circ_0023404 in HK-2 cells.

| Circ_0023404 contributed to HK-2 cell injury caused by H/R via sponging miR-136
Furthermore, miR-136 mimics were used to enhance miR-136 expression in HK-2 cells. In Figure 6A, miR-136 was greatly induced by miR-136 mimics, which was reversed by circ_0023404 overexpression plasmid. Transfection with circ_0023404 overexpressing plasmid and miR-136 mimics reduced HK-2 cell viability as displayed in Figure 6B. In addition, in Figure 6C, an enhancement of cell apoptosis was triggered by circ_0023404 up-regulation in vitro. Consistently, we revealed that miR-136 reduced inflammatory cytokines and oxidative stress, which could be enhanced by overexpression of circ_0023404 as demonstrated in Figure 6D-I.

| IL-6R was a target of miR-136
Then, http://starb ase.sysu.edu.cn/ was used to predict the link between miR-136 and IL-6R. In Figure 7A, the binding regions between miR-136 and IL-6R were exhibited. In addition, we constructed luciferase reporter plasmids of WT-IL-6R and MUT-IL-6R. Cotransfection of WT-IL-6R with miR-136 mimics significantly reduced the reporter activity ( Figure 7B). Besides these, miR-136 mimics greatly repressed IL-6R mRNA and protein, which was rescued by overexpression of circ_0023404 in Figure 7C and D.

| D ISCUSS I ON
Acute kidney injury is characterized by sharply renal dysfunction, and it is a common complication in hospitalized patients. 16 Here, in our work, we observed that circ_0023404 was up-regulated in  IL-6 exhibits significant pro-or anti-inflammatory capacity. 32,33 IL-6 functions via binding with IL-6R, which can activate Jak/STAT signalling pathway. 34 Additionally, IL-6/IL-6R has been reported to exert a crucial role in AKI. 35 In our present study, we predicted that IL-6R was a target of miR-136. In our data, we observed that circ_0023404 can regulate IL-6R activation in HK-2 cells under H/R through modulating miR-136. Subsequently, we also demonstrated the direct correlation between them.
In general, this work indicated that circ_0023404 resulted in HK-2 cells injury induced by H/R via sponging miR-136 expression and inducing IL-6R level. Advances in the understanding of circ_0023404-mediated HK-2 cell injury can provide some basic knowledge on the possible therapy for AKI.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.