Systematic analysis of the expression profile of non-coding RNAs involved in ischemia/reperfusion-induced acute kidney injury in mice using RNA sequencing

Acute kidney injury (AKI) is a common and serious disease characterized by a rapid decline in renal function and has an unacceptably high mortality rate with no effective treatment beyond supportive care. AKI can be induced by many factors such as ischemia/reperfusion (IR), sepsis, and drug-induced nephrotoxicity. However, the molecular mechanisms of AKI are poorly understood. A non-coding RNA (ncRNA) is a RNA molecule that is not translated into a protein. NcRNAs play multiple roles in cellular processes, and mutations or imbalances of these molecules within the body can cause a variety of diseases. Although growing evidence has supported the key role of ncRNAs in AKI, the specific mechanism remains largely unknown. In this study, the second-generation gene sequencing was performed to investigate the expression patterns of ncRNAs, including microRNA (miRNA), long non-coding RNAs, and circular RNAs, in the kidneys of mice subjected to IR-induced AKI. This information will contribute to future research of the mechanism of ncRNAs in the pathogenesis of AKI and facilitate the identification of novel therapeutic targets of ncRNAs.


INTRODUCTION
Acute kidney injury (AKI) is a major clinical problem without an effective therapy [1,2]. Renal ischemia/ reperfusion (IR) injury, along with sepsis and nephrotoxin injury, is the leading cause of AKI in perioperative patients [3,4]. The prognosis of AKI is poor because there are no currently available therapies to effectively treat or prevent IR-induced AKI [5,6]. However, the mechanism underlying IR-induced AKI has not been fully elucidated. Therefore, it is urgent to explore its pathogenesis to develop an effective treatment for IR-induced AKI.
Non-coding RNAs (ncRNAs) are a family of RNA molecules that typically do not code proteins but regulate gene expression, thus involving themselves in diverse cellular processes such as development, cell differentiation and proliferation, cell cycle, apoptosis, and metabolic function [7][8][9][10]. Based on their size, ncRNAs are subdivided into small ncRNAs (<200 nucleotides long), which encompass microRNAs (miRNAs), long ncRNAs (lncRNAs) with a length between 0.2 and 2 Kb, and circular RNAs (circRNA), which consist of a closed continuous loop [11]. Moreover, emerging data have demonstrated that ncRNAs are critically involved in the pathogenesis of AKI, particularly in IR-induced AKI [12][13][14]. However, the regulatory functions of ncRNAs in AKI and their underlying functional mechanisms have not been systematically described. Therefore, comprehensive estimations and analyses of the ncRNAs underlying the pathogenesis of AKI are essential to develop effective strategies to treat this troublesome disorder and prevent its progression. In this study, we utilized an RNA sequencing approach to investigate ncRNAs in the kidneys of mouse subjected to IR-induced AKI. Our study is designed to systematically identify the expression profiles of noncoding RNAs involved in IR-induced AKI and to provide a valuable resource for exploring their functional roles in AKI therapy, that the raw data in this study can be available in NCBI SRA database.

IR-induced AKI
There is much evidence indicating that IR is the leading cause of AKI [15,16]. To determine the effect of IR on AKI, kidney function was evaluated at 24 hours after IR treatment. Renal function was relatively deteriorated in mice in the IRI group, with blood creatinine and urea nitrogen levels that were markedly higher than those in mice in the CON group ( Figure 1A and 1B). Consistent with the deterioration of kidney function in mice subjected to IR treatment, there was substantial exacerbation in the histological injury of the kidneys as shown by more tubular epithelial cell injury, tubular dilation, and intratubular cast formation in mice in the IRI group compared with mice in the CON group ( Figure 1C, 1D and 1E).

Cell apoptotic in IR-induced AKI
Increasing evidence has indicated that tubular necrosis/apoptosis is an important mechanism underlying IR-induced AKI [17,18]. Therefore, we investigated tubular epithelial cell apoptosis induced by IR to confirm the success of the model. Our results showed that the number of apoptotic tubular cells significantly increased in kidneys from mice subjected to IR treatment as assessed by TUNEL staining ( Figure 1F and 1G).

Differentially expressed (DE) ncRNAs and mRNAs
To determine if ncRNAs are involved in the pathogenesis of IR-induced AKI, we analyzed DE ncRNAs and mRNAs using significance analysis of sequencing technique based on a q-value <0.05. DE ncRNAs and mRNAs in the kidney samples between mice in the IRI group and CON group are shown as a volcano plot, Venn diagram and clustering map. Information regarding the top 20 up-regulated and 20 down-regulated lncRNAs, mRNAs, miRNAs and circRNAs in the kidney tissues of mice in the IRI group compared with the CON group are listed in Tables 1-4

Validation of ncRNAs and mRNAs expression via quantitative polymerase chain reaction (qPCR)
To validate the reliability of the sequencing results and provide the basis for further study, eight RNAs among the DE ncRNA and mRNA transcripts were randomly selected to validate the accuracy of the sequencing data using qPCR, including 2 lncRNAs, 2 circRNAs, 2 miRNAs and 2 mRNAs. Figure 3 shows that all of the selected ncRNA and mRNA transcripts were detected and exhibited significantly different expression in the kidneys of mice subjected to IR. These results were consistent with the RNA sequencing data.

Functional prediction of DE ncRNAs in IR induced-AKI
To ascertain the functions and connections of the differentially expressed genes in IR-induced AKI, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses with an absolute value of correlation greater than 0.95. GO (http://www.geneontology.org/) is the international standard classification system of gene function [19]. According to the distribution of the predicted target genes in the Gene Ontology analysis, the number of genes was statistically analyzed with significant enrichment of each GO term to clarify gene function in biological process (BP), cellular component (CC) and molecular function (MF), and the data are presented as a histogram. Based on the GO analysis of the co-located and co-expressed genes of the DE lncRNAs ( Figure 4A Table 5. These most striking category of gene function will indicate the direction for our further research of ncRNAs. KEGG is a collection of databases with information regarding genomes, biological pathways, diseases, drugs, and chemical substances; these databases can determine significantly enriched pathways among the candidate target genes compared with the entire genome background [20,21]. The top 20 pathways enriched by the candidate target genes are displayed in an enriched scatter diagram, and the degree of KEGG enrichment is reported using the rich factor, q value and number of genes. When the rich factor is greater, q value is closer to zero, and the number of genes is bigger, the enrichment is more significant. Our results showed the most significantly involved pathways in IR-induced AKI based on the KEGG analysis of the intersection of colocalized and co-expressed genes of DE lncRNAs and predicted mRNAs ( Figure 5A-5C) and DE miRNAs and DE circRNAs ( Figure 6A, 6B). The most enriched GO terms and KEGG pathway were listed in Table 5. These main biochemical and signal transduction pathways will be the focus of future studies.

Regulatory network of ncRNAs and mRNAs in IR-induced AKI
To explore the molecular mechanism of ncRNAs involved in the pathogenesis of IR-induced AKI, we conducted an additional regulatory network analysis of ncRNAs and mRNAs. LncRNAs or circRNAs act as miRNA sponges to competitively interact with the binding sites of miRNAs, which play an extensive regulatory role [22,23]. Therefore, a regulatory network of lncRNA-miRNA-mRNA pairs with lncRNA as a decoy, miRNA as the connector, and mRNA as the target is shown in Figure 7. A regulatory network of circRNA-miRNA-mRNA pairs with circRNA as a decoy, miRNA as the connector, and mRNA as the target is shown in Figure 8. The regulatory relationship of ncRNAs and mRNAs in the mechanism of IR-induced AKI was revealed through these regulatory networks. In fact, based on the above results, the regulatory role of ncRNAs in the pathogenesis of IR-induced AKI was so complicated that indepth study should be implemented in the future.

Verification of ncRNAs regulatory network
Apoptosis of renal tubular epithelial cells play an important role in the procession of IR-induced AKI. We found that caspase14 (ENSMUSG035570R105355) was significantly upregulated in the kidneys of mice in the IRI group, which was proved to be an important anti-apoptotic protein [24]. Caspase14 was directly regulated by mmu-miR-874-3p, which was proved that over-expression promoted cellular apoptosis [25]. Therefore, considering that the most common mode of action with ncRNA pairs is sponging effect as ceRNA, we selected two pairs (mmu-miR-874-3p and LNC_000941, mmu-miR-874-3p and mmu_ circ_0004646) to verify in renal tubular epithelial cells of mice with dual-luciferase reporter system. Luciferase assay revealed that mmu-miR-874-3p displayed a sponging effect for LNC_000941 ( Figure 9A) and mmu_circ_0004646 ( Figure 9B) and decreased luciferase activity. The results verified the accuracy of the network interaction of ncRNAs in Figure 7 and 8.

DISCUSSION
IR are the main cause of AKI, which presents as impaired renal function, inflammation activity and apoptosis of the renal tubular epithelium [26][27][28]. In recent years, although there are many studies that have attempted to clarify the etiology and pathogenesis of IR-induced AKI, it is difficult to fully understand the underlying mechanism [29,30]. Therefore, identifying the underlying mechanism is crucial to determine new therapeutic targets and personalize treatment methods. In the present study, this is the first overall report that showed ncRNAs and mRNAs in the kidney that underwent significant changes in response to IR-induced AKI. In addition, we also predicted the potential functions of DE ncRNAs by GO and KEGG analysis and constructed a regulatory network of ncRNAs and mRNAs in the kidneys of mice subjected to IR. With this knowledge, our findings on  the transcription gene analysis provide us with an overall vision of ncRNAs in the pathogenesis of IR-induced AKI as well as useful clues for future and thorough research of the role of ncRNAs in AKI.
We prepared the mouse model of IR-induced AKI to analyze ncRNAs in our study. This model of IR-induced AKI guaranteed the sequencing results. According to the surgery protocol of the IRI model, the serum BUN and creatine levels in mice were tested at 24 hours after either IRI or sham procedures based on the theory that the deterioration of renal function occurs within 24 hours after IRI [31]. In accordance with the pathological results, the results of the renal function verified the successful preparation of this IRI model. In addition, apoptosis of renal tubular epithelial cells has been proven to be an important mechanism of IR-induced AKI by many scholars [29,32]. We also observed TUNEL-positive cells in the kidneys of mice in the IRI and CON groups, which further verified the reliability of the IRI models.
NcRNAs play an important role in several fundamental biological and pathological processes and are associated with a variety of diseases [33,34]. Earlier researchers commonly used microarrays to screen and predict DE ncRNAs in various pathophysiological processes [35]. To better clarify the overall changes and the role of ncRNAs in IR-induced AKI, we adopted the method of the second-generation sequencing. Although there are some limitations in our study, such as a relatively small sample size, we identified novel transcripts aside from annotated transcripts in databases. Moreover, the sensitive detection and reliable quantification of transcripts are the primary advantages of RNA sequencing compared with microarrays, and this method could identify ncRNAs that play important role but are expressed at low levels. In addition, eight DE transcripts identified in the present study were randomly selected to verify the accuracy of the RNA sequencing data by using qPCR. Ultimately, all the results were consistent with the RNA sequencing data, which confirmed again the reliability of our sequencing data and provided a credible base for further study.
Numerous findings have indicated that ncRNAs are involved in the cellular and molecular mechanisms of AKI by inducing multiple pathways [36,37]. Some evidence has shown that the dominant role of miRNAs is to promote the pathological development of IR-induced AKI as determined by microarrays [40,41], and recent studies also showed that lncRNAs were involved in the regulatory process of IR-induced AKI [11,40]. However, there is little comprehensive knowledge regarding ncRNAs (i.e., miRNAs, lncRNAs and circRNAs) in IR-induced AKI. Therefore, we examined the DE ncRNAs and mRNAs in the kidneys of mice subjected to IR-induced AKI. Our results showed that a total of 90 lncRNAs, 8 miRNAs, 56 circRNAs and 993 mRNAs were significantly upregulated or down-regulated in the kidney 24 hours after IR injury. These data are essential and provide the groundwork for a more thorough and comprehensive analysis of potential ncRNAs involved in IR-induced AKI.  A and B) showed significantly different levels at 24 hours in kidneys from mice in the IRI group compared with mice in the CON group. The expression levels of mRNAs (C and D) showed significantly different levels at 24 hours in the kidneys of mice from the IRI group compared with those from the CON group. The expression levels of miRNAs (E and F) showed significantly different levels at 24 hours in the kidneys of mice from the IRI group compared with CON group. The expressions of lncRNAs (G and H) showed significantly different levels at 24 hours in the kidneys of mice from the IRI group compared with CON group. One-way ANOVA followed by Tukey's multiple comparison test. *** P < 0.001. www.impactjournals.com/oncotarget To predict the potential functions of the DE ncRNAs identified in present study, GO and KEGG analyses were performed. GO terms and GO annotations are good predictors of gene function and can elucidate the genetic regulatory networks by forming hierarchical categories organized by molecular function, biological process, and cellular component [41]. The KEGG database is used to understand the high-level functions and utilities of the biological system [42].
The GO functional annotation analysis showed that these DE ncRNAs were enriched in several BPs (response to stimulus, multicellular organismal processes, single-multicellular organism processes, single-organism metabolic processes, stress responses, responses to organic substances, single-organism developmental processes, developmental processes, regulation of metabolic processes, fatty acid catabolic processes, leukotriene  a histogram (B). The GO term of DE miRNAs in the kidneys of mice subjected to IR are shown in (C). The BP and MF GO terms of DE circRNAs in the kidneys of mice subjected to IR are shown in (D). www.impactjournals.com/oncotarget metabolic processes, long-chain fatty acid catabolic processes, fatty acid derivative catabolic processes, icosanoid catabolic processes, leukotriene B4 metabolic processes, leukotriene B4 catabolic processes, and leukotriene catabolic processes), CCs (membrane, partial membrane, intrinsic to membrane, organelles, membranebound organelles, the cytoplasm, whole cell, partial cell and intracellular areas), and MFs (oxidoreductase activity, catalytic activity, transportor activity, binding, protein binding, receptor binding, calcium ion binding,     Figure 7 shows the interactive network of lncRNA-miRNA-mRNAs in the kidneys of mice subjected to IR. mmu-miR-874-3p and LNC_000941 in purple box were verified with dual-luciferase reporter system in Figure 9. organic cyclic compound binding, leukotriene-B4 20-monooxygenase activity, alkane 1-monooxygenase activity and oxidoreductase activity and acting on NADH). Moreover, KEGG analysis showed that the main biochemical and signal transduction pathways were enriched in metabolic pathways, osteoclast differentiation, the TNF signaling pathway, the p53 signaling pathway, proteoglycans in cancer, pathways in cancer, the MAPK signaling pathway, vascular smooth muscle contraction, retinol metabolism, the PPAR signaling pathway, Figure 8: Regulatory network analysis of circRNA-miRNA-mRNAs in the kidneys of mice subjected to IR. Figure 8 shows the interactive network of circRNA-miRNA-mRNAs in the kidneys of mice subjected to IR. mmu-miR-874-3p and mmu_circ_0004646 in purple box were verified with dual-luciferase reporter system in Figure 9.
inflammatory mediator regulation of TRP channels, fatty acid elongation and arachidonic acid metabolism. The above mentioned gene functions and pathways of the predicted ncRNAs in the present data, were also shown in many previous studies about AKI. For example, large amount of previous studies provided evidence that oxidoreductase activity, membrane, etc. were closely associated with AKI [43][44][45]. Dagher PC proposed that activation of p53 are major inducers of apoptotic cell death after ischemic renal injury [46]. Huang W proved that lncRNA PVT1 promote AKI by regulating TNFα and JNK/NF-κB pathways [47]. There are plenty of evidence indicated that MAPK signaling pathway were involved in renal ischemia-reperfusion injury [48]. The conclusion in above previous studies can support our sequencing data. The hypothesis of competing endogenous RNAs reveals a new interactive mechanism of RNA [49]. miRNAs can cause gene silencing by binding mRNAs [50], lncRNA, cirRNA, even mRNA could serve as ceRNA, can competitively bind to miRNAs to regulate gene expression  via miRNA response elements (MREs). The interactive networks of ncRNAs that regulate mRNAs reveal the important role of ncRNA function, which has biological significance [51,52]. Our data respectively showed the interactive network of lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA, which play a regulatory role as observed with mmu-miR-132-3p, mmu-miR-17-5p, mmu-miR-21a-5p, mmu-miR-21a-3p, mmu-miR-20a-5p, mmu-miR-93-5p, mmu-miR-185-5p and mmu-miR-874-3p. It is worth mentioning that miR-21, miR-223-5p, miR-125b and so on proved to be involved in IR-induced AKI, did not appear in our data [53][54][55]. The reason is that all ncRNA sequencing analysized were based on the lncRNA library, not built separate library of miRNA. Therefore, comparing the sequencing method of single building library, DE miRNAs in our data had certain omissions. In future study, combined with previous studies and related database, we can compensate the missing miRNAs in our data. Although little is known about the role of circRNAs in IR-induced AKI, we presented a reliable direction of study for circRNAs. Although evidence has accumulated showing that ncRNAs have significant role in the pathogenesis of AKI in the past few years, the molecular mechanisms underlying the interaction of ncRNAs in AKI remain largely unclear. It has been well demonstrated that miRNAs can function as negative regulators of gene expression in the initiation and/or progression stages of AKI. Therefore, the lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA network of IR-induced AKI were constructed based on the theory of ceRNA, which lncRNAs or circRNAs act as natural miRNA sponges to suppress miRNA function using shared MREs for mutual regulation. These pioneering discoveries might enrich understanding on the mechanisms underlying the role of ncRNAs in the pathogenesis of AKI. For example, miR-132-3p and miR-17-5p were proved to be associated with inflammatory [56,57], miR-185-5p and miR-874-3p are involved in apoptosis in response to damage [25,58]. While it is consensus of experts that inflammatory and apoptosis are imporatant factors in AKI. Therefore, further developed and more targeted study can be done to explore how these ncRNAs mediated AKI by mechanism of ceRNA based on the lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA network in combination with the raw data in SRA.
In conclusion, the present study revealed for the first time that ncRNAs are significantly altered in IRinduced AKI based on second-generation sequencing data. In addition, the data indicated that aberrantly expressed ncRNAs participate in the interaction and regulation of the expression of related genes and are involved in related specific biological processes and pathways that may contribute to the pathogenesis of AKI. While our findings provide newfound and full-scaled information regarding the critical role of ncRNAs in IR-induced AKI, further research is required to fully elucidate the detailed molecular mechanisms underlying the DE ncRNAs in our dataset that have a predicted function.

Animals
Adult male BALB/c mice (10 to 12 weeks old age, body weight 25-30 g, the Laboratory Animal Center of The First People's Hospital of Foshan, Foshan, China) were randomly assigned to either the IRI group or the CON group (6 animals per group). All animal procedures were in accordance with national and international animal care and ethical guidelines and have been approved by the institutional animal welfare committee. The environment was maintained at a constant temperature (22±0.581°C) and relative humidity (60-70%) with a 12-hour light/ dark cycle (lights on at 7 AM). All animals were provided standard laboratory chow and tap water ad libitum. Implementation of the IRI model is described below. Mice were anesthetized by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). Kidneys were exposed through a flank incision and were subjected to ischemia by clamping the renal pedicles using nontraumatic microaneurysm clamps. After 30 min, the clamps were removed, and blood flow was reestablished. Body temperature was maintained at 36.5-37.5°C throughout the entire procedure. Mice in the CON group underwent an identical surgical procedure but without pedicle clamping. All the animals were sacrificed at 24 hours after reperfusion, and the kidneys were harvested.

Measurement of renal function
Serum creatinine was measured using a creatinine assay kit (BioAssay Systems, Hayward, CA) according to the manufacturer's instructions. Blood urea nitrogen was determined fluorometrically as previously described [59].

Detection of apoptotic cells
Apoptotic cell death was determined by using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining with the DeadEnd Colorimetric Apoptosis Detection System (Millipore, Billerica, MA) according to manufacturer's instructions. The number of TUNEL-positive cells per high-power field were counted and analyzed in a blinded fashion.

Quantitative real-time RT-PCR
Total RNA was extracted from kidney tissues using TRIzol reagent (Invitrogen). Aliquots (1 μg) of total RNA were reverse transcribed using SuperScript II reverse transcriptase. Real-time PCR was performed using the IQ SYBR green SuperMix reagent (Bio-Rad, Herculus, CA) with a Bio-Rad real-time PCR machine according to the manufacturer's instructions. The comparative Ct method (ΔΔCt) was used to quantify gene expression, and the relative quantification was calculated as 2 −ΔΔCt . The expression levels of the target genes were normalized to the GAPDH levels in each corresponding sample. The primer sequences are listed in Table 6.

Tissue collection and RNA isolation
We prepared twelve mice for either IR or a sham operation, and all animals were deeply anesthetized with isoflurane at 24 hours after undergoing IRI or the sham operation. Total RNA was extracted from the kidney tissue using TRIzol reagent (Invitrogen, Carlsbad). RNA degradation and contamination was monitored using 1% agarose gels. RNA purity was measured using a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). The RNA concentration was measured using a Qubit® RNA Assay kit and a Qubit® 2.0 Fluorometer (Life Technologies, CA, USA). RNA integrity was assessed using a RNA Nano 6000 Assay kit with a Bioanalyzer 2100 system (Agilent Technologies, CA, USA).

Library preparation for ncRNA sequencing
A total of 3 μg of RNA per sample was used as input material for the RNA sample preparations of lncRNA sequencing. First, ribosomal RNA was removed using a Epicenter Ribo-zero™ rRNA Removal Kit (Epicenter, USA), and rRNA-free residue was washed by ethanol precipitation. Subsequently, sequencing libraries were generated using an rRNA-depleted RNA by NEBNext® Ultra™ Directional RNA Library Prep kit for Illumina® (NEB, USA) following the manufacturer's recommendations. Sequencing libraries of small RNA were generated using an NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA) following manufacturer's recommendations, and index codes were added to the attribute sequences in each sample [61].

Clustering and sequencing of ncRNA
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the libraries were sequenced on an Illumina HiSeq 2500 platform, and 125 bp paired-end and 50 bp single-end reads were generated. The transcription with splicing of each sample were combined and screened as lncRNAs with Cuffmerge Software, and the conditions were as follows: the number of exon≥2, length > 200 bp, FPKM ≥0.5 (Cuffquant) and to eliminate overlapping and coding potential transcription with annotation of database at exon region (Cuffcompare Software). CircRNAs were identified base on the data of lncRNAs with find_circ [62]. Clean reads were screened the lengh of 21-22 nt as miRNA, and located to reference sequence with bowtie. Combined with miREvo Software and mirdeep2 Software to analysis the funtions of new miRNAs. Adopt DESeq2 with negative binomial distribution to analyse differentially expression of ncRNAs. All sequencing program were performed by Novogene Company (China, Beijing).

GO and KEGG analysis
GO and KEGG analysis were applied to investigate the roles of all the DE ncRNAs. In brief, GO analysis was applied to elucidate the genetic regulatory networks of interest by forming hierarchical categories according to the BP, CC and MF of the differentially expressed genes (http://www.geneontology.org). Pathway analysis was performed using KEGG (http://www.genome.jp/kegg/) to explore the significant pathways of the differentially expressed genes.

Analysis of the ncRNA regulatory networks
Interactive networks were built and visualized using Cytoscape software based on the screened lncRNA-miRNA gene pairs and the circRNA-miRNA gene pairs. Different shapes represent the different types of RNA, whereas the different colors represent the regulated relationship. The size of the node was directly proportion to extent of association. In other words, these significant nodes are in a core position in the regulated network and were more associated with IR-induced AKI.

Luciferase assay
A dual-luciferase reporter system E1960 (Promega, Madison, WI, USA) was used to perform luciferase activity assay. In brief, renal tubular epithelial cell of mouse were cultured on 12-well tissue culture plates at a density of 2 × 10 5 cells per well. Cells were cotransfected with the luciferase reporter constructs contain lncRNA (LNC_000941) or cirRNA (mmu_circ_0004646), miRNA(mmu-miR-874-3p) mimics and Renilla luciferase construct for 5h(Lipofectamine® MessengerMAX™ Transfection Reagent, Thermo Fisher Scientific). After 3d culture at 37°C, the transfected cells were lysed by 150 μl of passive lysis buffer. In total, 30 μl of lysates were mixed with 50 μl of LAR II, and then firefly luciferase activity was measured by a luminometer. For the internal control, 50 μl of Stop & Glo reagent was added to the sample.