Hsa-miR-494-3p attenuates gene HtrA3 transcription to increase inflammatory response in hypoxia/reoxygenation HK2 Cells

The occurrence of cardiac surgery-associated acute kidney injury (CSA-AKI) increases hospital stay and mortality. MicroRNAs has a crucial role in AKI. This objective of the current study is to explore the function of hsa-miR-494-3p in inflammatory response in human kidney tubular epithelial (HK2) cells with hypoxia/reoxygenation. According to KDIGO standard, patients after cardiac surgery with cardiopulmonary bypass were divided into two groups: AKI (n = 10) and non-AKI patients (n = 8). HK2 were raised in the normal and hypoxia/reoxygenation circumstances and mainly treated by overexpression ofmiR-494-3p and HtrA3. The relationship between miR-494-3p and HtrA3 was determined by dual-luciferase reporter assay. Our result showed that Hsa-miR-494-3p was elevated in the serum of patients with CSA-AKI, and also induced in hypoxic reoxygenated HK2 cells. Hsa-miR-494-3p also increased a hypoxia-reoxygenation induced inflammatory response in HK2 cells. Moreover, as a target gene of miR-494-3p, overexpression of HtrA3 downregulated the hypoxia-reoxygenation induced inflammatory response in HK2 cells. Overexpression of hsa-miR-494-3p-induced inflammatory response was inhibited by overexpression of HtrA3. Collectively, we identified that hsa-miR-494-3p, a miRNA induced in both circulation of AKI patients and hypoxia-reoxygenation-treated HK2 cells, enhanced renal inflammation by targeting HtrA3, which may suggest a possible role as a new therapeutic target for CSA-AKI.

Acute kidney injury (AKI) is a common and serious complication of hospitalized patients, which results in oliguria, anuria and elevated creatinine, and apparently increases the mortality of hospitalized patients [1][2][3] . Cardiac surgery with cardiopulmonary bypass (CPB) is the second most common cause of AKI after sepsis 4 . At present, CPB is considered to be the main cause of AKI after cardiac surgery, and ischemia-reperfusion injury is considered the main pathway of CPB associated-kidney injury [5][6][7][8] . Cardiac surgery-associated acute kidney injury (CSA-AKI) patients are the ideal AKI study population because the time of damage is known, and CPB provides a non-physiological condition of ischemia and hypothermia, it is a high risk factor for kidney damage.
MicroRNAs (miRNAs) are small non-coding RNAs with a length of about 20-24 nucleotides. By binding to the 3′-non-coding region of targeted mRNA 9 , miRNAs inhibit the translation of targeted proteins and achieve negative regulation of gene expression at the post-transcriptional level 10 . MiRNAs is the most frequently studied non-coding RNA for AKI 11 . Current studies on animal models and some types of AKI, such as AKI after ischemia-reperfusion injury 12 , kidney transplantation 13 and application of nephrotoxic drugs 14 , have accumulated evidence that a variety of miRNAs may be involved in the pathogenesis of AK [15][16][17] , showing its potential clinical value, providing a new way to study the molecular mechanism of CSA-AKI. MicroRNAs of CSA-AKI is secreted into circulation or urine, so can be used as highly sensitive and specific biomarkers to study the occurrence, development and prognosis of CSA-AKI 18 . The study on upregulation of miR-494 has been identified as an oncogenic miRNA and is associated with poor prognosis in several types of human cancer [19][20][21] . However, miR-494 is found downregulated and suppressed cell proliferation in some cancer cells [22][23][24] . At present, upregulation of hsa-miR-494-3p promotes acute kidney injury by negatively regulating ATF3 protein synthesis, because ATF3 plays a protective role in renal ischemia-reperfusion injury 25 . However, the network regulation of hsa-miR-494-3p on renal injury is the same complex as on cancer. It is unclear whether hsa-miR-494-3p affects the severity of kidney injury by regulating other proteins, so we try to further study hsa-miR-494-3p on this point.

Results
Hsa-miR-494-3p is elevated in the serum of patients with CSA-AKI. According to KDIGO standard 26 [serum creatinine increased by more than 0.3 mg/dL (26.5 mmol/L) within 48 h after cardiac surgery or more than 1.5 times higher than the preoperative baseline within 7 days after cardiac surgery] 26 , patients after cardiac surgery with cardiopulmonary bypass were divided into two groups: AKI and non-AKI patients (Fig. 1A). The changes of serum creatinine during perioperative period between the two groups were shown in Fig. 1B. The onset time of CSA-AKI was concentrated between the first day and the third day after operation. The serum creatinine of AKI patients in this study all recovered to the normal level around seven days after operation. Hsa-miR-494-3p in the serum of patients with CSA-AKI is elevated significantly at an hour after back into ICU, and returns to its baseline level at 12 h after back into ICU (Fig. 1C).

Discussion
A kind of miRNAs can regulate the translation of various protein mRNAs. At present, the negative regulation mechanism of hsa-miR-494-3p on transcription factor 3 (ATF-3, a protein that protects kidney against stress response) protein synthesis has been clearly studied. miR-494-3p can be detected in renal tissue and urine, and it is upregulated during ischemia-reperfusion injury, which significantly inhibits the activation of ATF3, thus promoting the production of inflammatory mediators IL-6, MCP-1 and P-selectin in AKI model. Transfection of antisense miR-494-3p into mice improved renal injury induced by ischemia-reperfusion, reduced the production of pro-inflammatory cytokines and inhibited the apoptotic activity of caspase-3. This study also laid a solid foundation for the pathophysiological role of miR-494-3p in AKI model 25 . The study on cyclosporine (CsA)-induced nephrotoxicity showed kidney injury in mice was associated with an early increase in expression of miR-494, and it also explored tubular epithelial cell epithelial-mesenchymal transition induced by CsA toxicity resulted in the upregulation of miR-494 and a decrease in PTEN levels in vitro. miR-494 directly targeted Pten and negatively regulated its expression 27 .
In addition to targeting protein ATF3 and PTEN, our data show hsa-miR-494-3p attenuates gene HtrA3 transcription to increase inflammatory response in hypoxia/reoxygenation HK2. Like ATF3, HtrA3 proteins also play a protective role in ischemia-reperfusion kidney injury. The expression of HtrA3 protein may inhibit inflammatory response through nuclear factor-kappa B (NF-kappa B) signaling pathway which promotes renal inflammation, apoptosis and renal damage. The human HtrA family of ATP independent serine proteases (HtrA1, HtrA2, HtrA3, and HtrA4) are the key enzymes associated with pregnancy and closely related to the development and progression of many pathological events 28,29 , and they promote cell death in stress conditions and play a role www.nature.com/scientificreports/ as tumor suppressors. HtrA3 is a trimeric PDZ bearing propapoptotic serine protease, which is also involved in various diseases including pre-eclampsia and cancer 30 . A recent study on Endometrial cancer demonstrated hypoxia reduced the content of HtrA3, and silencing HtrA3 promoted Endometrial cancer cell migration, and as the degree of hypoxia increases in Endometrial cancer. Hypoxia leads to a decrease in HtrA3, which may promote cancer progression. These results indicated that HtrA3 plays an adaptive role in endometrial cancer in anoxic regions 31 . The human HtrA3 protease is involved in development and progress of many physiological events, including placentation, stimulation of apoptosis and considered as a tumor suppressor. The molecular mechanism of HtrA3's function is unclear now, and knowledge of its cellular targets is limited 32 . Our study is the first to explore the role of HtrA3 in renal inflammation. It also explained that HtrA3 can reduce the renal inflammatory response induced by hsa-miR-494-3p, but cannot completely block or even reverse the kidney injury. So our results also suggested that hsa-miR-494-3p may play a complex network regulatory role in kidney injury (Supplementary information). Studies have shown that urinary levels of miR-494-3p in AKI patients in comprehensive ICU increased before changes in serum creatinine and urea nitrogen 25 ; however, there was no difference in serum samples, and their causes of AKI in the comprehensive ICU were inconsistent. Our study showed hsa-miR-494-3p is elevated in serum of CSA-AKI patients. Because some patients may have no urine in clinic, we did not test hsa-miR-494-3p in urine samples. However, the difference between these two studies in the detection of miR-494-3p in serum may be related to the time of kidney injury, and may also be related to prognosis. The pathogenesis of CSA-AKI is not clear 6 , and there are no effective preventive and therapeutic measures. At present, the indicators representing the loss of renal function cannot timely diagnose the occurrence of AKI. Because miRNAs regulated transcription of proteins, miRNAs should be more sensitive and earlier to be detected, compared with proteins, for example SCr, and miR-494-3p rose for 1 h after the surgery and returned to its baseline level at 12 h after the surgery. So hsa-miR-494-3p can be used as a molecular biological marker for early diagnosis of CSA-AKI patients. Hsa-miR-494-3p may participate in the early stage of the occurrence of CSA-AKI, and may also predict a good prognosis of CSA-AKI.
We showed that miR-494-3p was induced in response to hypoxia/reoxygenation injury which indicated AKI following ischemia-reperfusion injury. MiR-494-3p directly bound to 3′-UTR of HtrA3, which increased an anoxia-reperfusion induced inflammatory response. The expression of HtrA3 protein may inhibit inflammatory response through NF-kappa B signaling pathway. According to our knowledge, this is the first study that showed

Materials and methods
Detection of hsa-miR-494-3p in human serum. Adult patients undergoing on-pump valvular surgery or simultaneous coronary artery bypass grafting were selected. There were no chronic complications before operation and no organ damage except the kidney after operation. CSA-AKI diagnosis is based on the 2012 KDIGO criteria for improved global prognosis of kidney disease [serum creatinine increased by more than 0.3 mg/dL (26.5 mmol/L) within 48 h after cardiac surgery or more than 1.5 times higher than the preoperative baseline within 7 days after cardiac surgery] 26 .
Target prediction analysis. We use the public prediction platform TargetScan (TargetScan Release 7.

RNA extraction and Real-time PCR.
According to the manufacturer's instructions, the total RNA was extracted from the HK2 cells by using TRIzol reagent (Invitrogen, USA). The abundance of miRNA was measured by quantitative real-time PCR (RT-qPCR) using One-Step miR RT-qPCR reagent kits and validated primers (Biomics, China) and the abundance of mRNA was measured by RT-qPCR using the system with SYBR-Green Master Mix (TaKaRa, Japan). The fold changes in the RNA expression were analyzed via the 2 −ΔΔCt method, and the level of the endogenous U6 RNA and β-actin performed as the normalized control. N (the replicate for these Real-time PCR analyses) = 3.
Western blotting analysis. Total protein from cultured HK2 cells was harvested and lysed in RIPA lysis buffer (Beyotime, China) and protease inhibitor cocktail (Sigma, USA) for 30 min at 4 °C. The lysate was centrifuged for 15 min at 12,000 rpm and 4 °C, the upper supernatant was collected, and the protein was separated by 10% SDS-PAGE and transferred onto 0.45-μM polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) 33 . The membranes were blocked with 5% non-fat milk for 60 min and then washed in TBS-Tween 20 buffer for 15 min three times and incubated with specific primary antibodies at 4 °C overnight. Then, the membranes were washed three times with TBSTween 20 buffer and incubated with secondary antibody, horseradish peroxidase-conjugated anti-mouse antibody or anti-rabbit antibody (ZSGB-BIO, 1:10,000), at 25 °C for 1 h, and then developed onto the chemiluminescence Western blotting detection system using an ECL chemiluminescent kit (ECL-plus, Thermo Scientific). Quantitative densitometric analyses of immunoblotting images were performed using Image J software (NIH, Bethesda, USA). And the experiment was repeated three times 33    Luciferase reporter assay. The HtrA3 3′UTR carrying a putative miR-494-3p binding site was generated by PCR and then cloned into pMiR-report vector to establish the luciferase reporter constructs HtrA3-wildtype and HtrA3-mutated-type. Subsequently, cells were co-transfected with the reporter constructs HtrA3-wildtype or HtrA3-mutated-type and miR-494-3p vector or scramble using transfection reagent (Hanbio, shanghai, China) according to the manufacturer's instructions. For determination of the luciferase activity, reporter assays were done using the dual-luciferase assay system 34 . N (the replicate for these Luciferase reporter assays) = 3.

Statistical analysis.
Data are expressed as the mean ± SEM. Statistical significance was analyzed by twotailed unpaired t test or one-way analysis of variance (ANOVA), followed by Tukey post hoc tests using Graph-Pad Prism 6 software 35 .
Ethics approval and consent to participate. This study was approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University and informed consent of patients was obtained, and all experimental protocols used in the present study were in compliance with the guidelines of the Declaration of Helsinki.

Data availability
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.