The Circ_35953 induced by the NF‐κB mediated the septic AKI via targeting miR‐7219‐5p/HOOK3 and IGFBP7 axis

Abstract A few studies suggested that CircRNAs were involved in the development of septic AKI. However，the role and regulation mechanism of CircRNA_35953 in septic AKI remains unclear. Here, we found that Circ_35953 was induced by LPS via activation of NF‐κB signal in BUMPT cells. Functionally, Circ_35953 mediated the LPS induced the apoptosis in BUMPT cells. Moreover, we demonstrated that Circ_35953 sponged miR‐7219‐5p to upregulate the expression of HOOK3 and IGFBP7. Finally, we verified that knock down of Circ_35953 alleviated the progression of CLP‐induced AKI via targeting the miR‐7219‐5p/HOOK3 and IGFBP7 signal. Collectively, the data suggested that Circ_35953 /miR‐7219‐5p/HOOK3 and IGFBP7 axis mediated the septic AKI, which also revealed a potential mechanism of septic AKI.


| INTRODUC TI ON
Sepsis, a life-threatening clinical syndrome, is the key cause of acute kidney injury (AKI) and responsible for nearly half of all AKI patients. [1][2][3] Recent more studies focused on the mechanism of SA-AKI (septic AKI), 4 the pathophysiological mechanism of it remains unknown, which leads to the no available and nonspecific therapy. Hence, it is the key step to unravel the pathophysiological mechanism of SA-AKI progression for the development of effective therapeutic way. 2 Circular RNAs (circRNAs), a class of non-coding RNAs that do not have 5′ end caps or 3′ end poly (A) tails, has cell and tissue-specific expression patterns. 5 Mechanistically, circRNAs usually performed the pivotal biological function via multiple ways of microRNA sponges, translation templates, protein regulators and gene expression regulators. [6][7][8] Furthermore, circRNAs have been implicated in diseases such as diabetes mellitus, neurodegenerative, ocular diseases, cardiovascular and cancer. [9][10][11][12][13] More recent studies reported that CircRNAs was involved in the progression of SA-AKI. For example, several CircRNAs of CIRC-Ttc3, CircTLK1, Circ_0114428, CircHIPK3, circ-FANCA and circ-BNIP3L mediated the progression of SA-AKI. [14][15][16][17][18][19] By the contrast, CircRNAs of CircVMA21, Circ_0068888 and Circ_0091702 protested against the development of SA-AKI. [20][21][22][23] The above-mentioned findings revealed the function of parts of cirRNAs. The Circ_35953 was one of circRNAs, localized in the position of cttnbp2nl. The role and mechanism of it in SA-AKI remains largely unknown.
In current study, we demonstrated that both LPS and CLP induced the expression of Circ_35953 in vitro and vivo. The Circ_35953 mediated the LPS-induced apoptosis in the BUMPT cells. Mechanistically, Circ_35953 sponged endogenous miR-7219-5p to increase the expression of HOOK3 and IGFBP7. Finally, silencing of Circ_35953 attenuated the progression of CLP-induced AKI. Our findings found a novel regulation mechanism of Circ_35953 in SA-AKI progression.

| Luciferase reporter assays
For the assessment of the microRNA (miRNA) activity, miRNA target insertion of sites was inserted at the end of the firefly luciferase gene (luc2) of the pmirGLO dual-luciferase miRNA target expression vector (Promega). The luciferase vectors of

| Renal function, morphological studies and apoptosis
The levels of BUN and creatinine were evaluated according to instruction of renal function examination KIT (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China), haematoxylin and eosin staining was performed to assess morphology, and assessed by the renal injury score. 25 TUNEL staining was also used to examine renal cell apoptosis, and then quantified by counting positive staining cells according to the previous described. 26 An Olympus microscope equipped with UV epi-illumination was applied to analysis the stained samples. FCM procedures were carried out according to the manufacturer's protocol.

| ChIP analysis
Chromatin immunoprecipitation (ChIP) assay the binding site of NF-κB interaction with promoter of Circ_35953 was performed using commercial kit (Millipore). RT-qRCR was used to analyse the precipitated DNA. The following specific primers were used for NF-κB

| Immunoblot analysis
Equal amounts of proteins were separated by SDS-PAGE and then transferred to a nitrocellulose membrane (Amersham). The membrane was incubated with primary antibodies against HOOK3, IGFBP7, cas-pase3, cleaved caspase3 and β-actin followed by incubation with secondary antibody. β-actin was used as an internal loading control.

| Fluorescence in situ hybridisation (FISH)
The fluorescence probes of Circ_35953 and miR-7219-5p were synthesized by Ruibo, U6 and 18S were considered as nucleus and cytoplasmic control, respectively. Briefly, the slides of BUMPT cells and mice kidney were hybridized overnight with respective probes and subsequently stained with 4′,6-diamidino-2-phenylindole (DAPI). A laser-scanning confocal microscope was applied for fluorescence imaging analysis.

| Statistical analyses
Two-tailed Student's t tests were used for comparing two groups.
One-way anova was performed for multiple group comparison.
Quantitative data were expressed as mean ± standard deviation (SD). The Spearman rank correlation coefficient was used to assess the correlations between variables. All statistical analyses were carried out with the SPSS package (SPSS) and GraphPad Prism software (GraphPad Prism Software). p < 0.05 was considered statistically significant.

| Circ_35953 is induced by LPS and CLP in vitro and vivo
We explored whether Circ_35953 was induced by LPS and CLP in BUMPT cells and C57BL/6 mice, respectively. Here, the RT-qPCR analysis results showed that the expression of Circ_35953 was induced by LPS at 12 h, and reached a peak at 24 h ( Figure 1A).
Furthermore, the RT-qPCR analysis results also indicated that the expression of Circ_35953 was increased by CLP at 9 h, and attained a peak at 18 h ( Figure 1B). The results of renal function and TUNEL staining ( Figure 1E,F) showed that CLP-induced the increasing of levels of BUN, Creatinine and apoptosis at 9 h, and reached a peak at 18 h ( Figure 1C,D). The correlation analysis indicated that the renal cell apoptosis rate is highly associated with the expression of Circ_35953 foldchange (R = 0.9172; Figure 1G). The fluorescent in situ hybridisation (FISH) analysis indicated that Circ_35953 is in the cytoplasm of BUMPT cells ( Figure 1H). The data showed that Circ_35953 was associated with the apoptosis.

| NFκ B mediated the expression of Circ_35953 during LPS treatment
To investigate the potential regulation mechanism of the expression of Circ_35953 caused by the LPS treatment, we focused on the NF-κB signal pathway. First, the immunoblot results showed that LPS induced the activation of NF-κB in BUMPT cells at 12 h and reached a peak at 24 h ( Figure 2A,B). Second, we found that two potential NF-κB binding sites existed at the gene promoter of Circ_35953 by JASPAR Database (http://jaspar.gener eg.net/), and then named as sites 1 and 2 (Table 1). Third, RT-qPCR analysis demonstrated that TPCA-1, a specific NF-κB inhibitor, markedly suppressed the LPSinduced the expression of Circ_35953 in BUMPT cells ( Figure 2C).
Finally, the ChIP assay indicated that LPS enhanced the binding of NF-κB and the site 2 but not site 1 sequence of the gene promoter of Circ_35953 ( Figure 2D). However, we found that knockdown of Circ-35953 did not affect the activation of NF-κB ( Figure S3). The data suggested that NF-κB directly upregulates the expression of Circ_35953 during LPS treatment.

| Knockdown of Circ_35953 expression ameliorated LPS induced BUMPT cell apoptosis
Although the above-mentioned data suggested that Circ_35953 might be involved apoptosis induced by LPS in BUMPT cells. The

| Overexpression of Circ_35953 enhanced apoptosis caused by LPS in BUMPT cells
To confirm the effect of Circ_35953 on apoptosis, the Circ_35953 ex- confirmed that Circ_35953 is an apoptosis driver during LPS treatment.

| Circ_35953 sponged the miR-7219-5p
As we known, circRNAs perform as competitive endogenous RNAs show that miR-7219-5p is a direct target of Circ_35953.

| MiR-7219-5p mimic alleviated LPS-induced apoptosis in BUMPT cells
Here, the RT-qPCR analysis showed that miR-7219-5p mimic markedly increased the expression of it under untreated and LPS treatment ( Figure 6A). The FCM results showed that the miR-7219-5p mimic inhibited the LPS induced apoptosis in BUMPT cells ( Figures 6B,C), which was verified by the immunoblot of the expression of cleaved caspase3 (Figures 6D,E) Hence, the data showed that miR-7219-5p protected from the apoptosis caused by LPS.

| HOOK3, a target gene of miR-7219-5p, mediated LPS-induced apoptosis
One study reported that Hook microtubule-tethering protein 3(HOOK3) is an apoptosis inducer. 27 We predicated that HOOK3 is a potential target gene of miR-7219-5p using the miRBase database ( Figure 7A). The luciferase reporter gene assay demonstrated that the miR-7219-5p mimic inhibited the luciferase activity of HOOK3-WT but not HOOK3-MUT1 and HOOK3-MUT2 ( Figure 7B). The RT-qPCR and immunoblot results indicated that the miR-7219-5p mimic markedly suppressed mRNA and protein levels of HOOK3 ( Figure 7C,D). The FCM results showed that HOOK3 siRNA noticeably inhibited the LPS-induced apoptosis in BUMPT cells ( Figure 7E,F), which was confirmed by the immunoblot detection of the expression of HOOK3 and cleaved caspase-3 ( Figure 7G,H). Altogether, the data showed that HOOK3 was a direct target gene of miR-7219-5p.

| MiR-7219-5p was a mediator of pro-apoptosis effects of Circ_35953
To further confirm if miR-7219-5p mediates the proapoptotic effects of Circ_35953 during LPS treatment. RT-qPCR analysis showed that Circ_35953 siRNA suppressed the expression of Circ_35953 while miR-7219-5p inhibitor also inhibited the expression of miR-7219-5p ( Figure 8A,B). The FCM analysis showed that Circ_35953 siRNA alleviated the LPS-induced apoptosis, which was reversed by the miR-7219-5p inhibitor ( Figure 8C,D).
Immunoblot analysis of cleaved caspase3 and HOOK3 further verified the findings of FCM ( Figure 8E,F). The data confirmed that Circ_35953 mediated the LPS-induced apoptosis by targeting of the miR-7219-5p.

| Circ_35953 knockdown suppressed the progression of AKI caused by CLP by regulation of the miR-7219-5p/HOOK3 and IGFBP7 axis
To confirm the function of Circ_35953 in CLP-induced AKI in vivo, C57BL/6J mice were preinjected with the Circ_35953 siRNA-cy3 via images showed circ_35953-cy3 siRNA was successfully transfected into tubular cells of mice kidney ( Figure S4). The renal function results suggested that Circ_35953 knock down reduced the CLPinduced the increasing of both BUN and creatinine ( Figure 9A,B). ( Figure S2C,D). These data verified that NF-κB is an apoptosis inducer during LPS treatment.

| DISCUSS ION
Recent studies suggested that several CircRNAs play a pivotal role in SA-AKI. 14-23 However, the function of more CircRNAs needed to be investigated. In current study, we for the first time reported  was demonstrated by the LPS and CLP injury models in vitro and vivo. Collectively, we verified that Circ_35953 was one of drivers of SA-AKI.
The studies indicated that the programmed cell death of tubular epithelial cells played a key role in SA-AKI. [28][29][30] CircRNAs were involved in the renal cell apoptosis. For example, the CIRC-Ttc3, circVMA21, and Circ_0091702 suppressed LPS induced the tubular cell apoptosis. 14,20,21,23 However, circTLK1, circ_0114428, circHIPK3, and circ-FANCA promoted LPS-induced tubular cell apoptosis. [15][16][17][18] In present study, we found that Circ_35953 mediated the LPS-induced the apoptosis in BUMPT cells, which was demonstrated by the knock down and overexpression of Circ_35953 experiments (Figures 3 and 4). Furthermore, the evidence was supplied by the vivo finding that silencing of Circ_35953 also attenuated the CLP-induced the tubular cell apoptosis (Figure 9). Altogether, the data verified that Circ_35953 was an apoptosis driver in SA-AKI.
How about the regulation mechanism of Circ_35953 for the tubular cell apoptosis? We verified that Circ_35953 located in the cytoplasm of BUMPT cells (Figure 1). Previous study reported that circRNAs in the cytoplasm could act as microRNA to regulate the expression of target mRNAs. 31 The multiple ways of bioinformatics analysis, Dual-luciferase, RNA-FISH and RT-qPCR demonstrated that the miR-7219-5p was a direct target of Circ_35953 ( Figure 5).
Collectively, the data strongly suggested that miR-7219-5p was a direct target of Circ_35953.
The function and target gene of miR-7219-5p remains unclear.
Here, we for the first time demonstrated that miR-7219-5p mimics markedly suppressed the LPS-induced apoptosis and the increasing of cleaved caspase3 using the detection of FCM and immunoblot ( Figure 6). We further verified that HOOK3 mRNA is a direct target of miR-7219-5p by the miRtarget prediction website and dual luciferase reporter assay ( Figure 7A,B). The results RT-qPCR and western blot confirmed that miR-7219-5p noticeably suppressed the mRNA and protein expression of HOOK3 ( Figure 7C,D). Knockdown HOOK3 decreased the BUMPT cells apoptosis and caspase3 activation in vitro ( Figure 7E-H), which is consistent with the previous finding in cardiomyocyte apoptosis caused by the I/R. 27 We also revealed that silence Circ_35953 expression decreased LPS-induced renal cell apoptosis, and these effects were reversed by miR-7219-5p inhibitor ( Figure 8). Moreover, Circ_35953 siRNA suppressed CLP-induced kidney injury via miR-7210-5p axis (Figure 9).
NF-κB is a protein family consisting of 5 dimers, RelA (p65), RelB, c-Rel, p50 (generated from p105), and p52 (generated from p100), which can form a variety of homodimers or heterodimers. Normally, NF-κB dimers are inactivated through interacting with the inhibitor of κB (IκB). 32 As one of the most important components of the pathogenesis, systematic inhibition of NF-κB affects the severity of AKI. In a disease model induced by folic acid, inhibition of NF-κB mitigates AKI-injury by reduction of RelA and NFκB2 activation. 33 In our study, we found NF-κB inhibitor suppressed the progression of BUMPT apoptosis and SA-AKI caused by LPS and CLP (Figures S1 and S2).
HOOK3, the human hook microtubule tethering proteins family comprises, 27 a well-established dynein-activating adaptor. 34 Previous studies have reported that HOOK3 can serve as a fusion partner in gastrointestinal stromal tumour (GIST) and papillary F I G U R E 9 (Continued) thyroid carcinoma. The chimeric HOOK3-FGFR1 fusion protein contains the coiled-coil domain from HOOK3, indicating its potential leukaemogenesis role in EMS. 35 IGFBP7, insulin-like growth factor binding protein 7, a member of the insulin-like growth factor (IGF)-binding protein (IGFBP) family.
Kashani et al. 36 reported that IGFBP7 and TIMP-2 were identified as septic AKI biomarkers. Furthermore, IGFBP7 mediated the podocyte apoptosis caused by high glucose. 37 Interestingly, recent study verified that IGFBP7 promoted LPS-induced renal proximal tubular cell apoptosis in septic AKI. 38 Here, we found that IGFBP7 was another proapoptotic target gene of miR-7219-5p ( Figure S5). Finally, we demonstrated that knockdown of Circ_35953 also suppressed the expression of IGFBP7 in mice CLP-induced septic AKI model ( Figure 9).
In summary, we found that Circ_35953 accelerates CLP-induced AKI via the miR-7219-5p/HOOK3 and IGFBP7 axis, which presenting a new mechanism of SA-AKI development.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

CO N S E NT FO R PU B LI C ATI O N
All authors agree to publish in Journal of Cellular and Molecular Medicine.