Lipopolysaccharide enhances ADAR2 which drives Hirschsprung's disease by impairing miR‐142‐3p biogenesis

Abstract Researches over the past decade suggest that lipopolysaccharide is a dominant driver of gastrointestinal motility and could damage the enteric neuron of rat or porcine. However, it remains poorly defined whether LPS participates in Hirschsprung's disease (HSCR). Here, we discovered that LPS increased in HSCR tissues. Furthermore, LPS treatment suppressed the proliferation and differentiation of neural precursor cells (NPCs) or proliferation and migration of human 293T cells. ADAR2 (adenosine deaminase acting on RNA2)‐mediated post‐transcriptional adenosine‐to‐inosine RNA editing promotes cancer progression. We show that increased LPS activates ADAR2 and subsequently regulates the A‐to‐I RNA editing which suppresses the miR‐142 expression. RNA sequencing combined with qRT‐PCR suggested that ADAR2 restrain cell migration and proliferation via pri‐miR‐142 editing and STAU1 up‐regulation. In conclusion, the findings illustrate that LPS participates in HSCR through the LPS‐ADAR2‐miR‐142‐STAU1 axis.

Accumulated evidence suggests that adenosine deaminase acting on RNA (ADAR) enzymes, such as ADAR1 and ADAR2, accelerates tumour development. 12,13 The ADAR enzyme is a double-stranded RNA (dsRNA) binding protein that regulates adenosine into inosine (A to I) at the post-transcriptional level. 14 ADAR2, an indispensable enzyme for brain development and function, 15 is enough to inhibit glioblastoma proliferation or cancer progress. 16 Then, we speculated that ADAR2 could be associated with the development of the enteric nervous system (ENS). Recently, it has been shown that ADARs may edit miRNA precursors, thereby altering its maturation step. 17 Here, we demonstrated that increased concentration of LPS enhanced the activity of ADAR2. Then, ADAR2-mediated A-to-I editing suppressed the development of ENS by impairing miR-142-3p biogenesis, resulting in unopposed STAU1 expression. The experiments were conducted to unravel the biological roles of LPS and underlying regulative mechanism between LPS-ADAR2-miR-142-3p-STAU1, which may contribute to the pathogenesis of HSCR.

| Clinical tissue samples
The Nanjing Medical University Ethics Committee approved the study. HSCR and control tissues were obtained from Nanjing Children's Hospital from 2011 to 2016 and then stored at À80°C. In this study, control samples refer to samples confirmed no HSCR or other intestinal nerve malformations. HSCR sample was confirmed by examination of intestinal ganglia. The clinical data of the patients are summarized in Table 1.

| Neural precursor cell culture
The Institute of Cancer Research (ICR) E16.5 mice were killed by cervical dislocation. Then, we separate the outer layer of the intestinal tract under a stereoscopic microscope. The remaining tissue was triturated to 0.5-1 mm 3 and incubated 60 minutes in 1 mg/mL collagenase. Then, the small pieces were centrifuged and filtered through a 200-lm filter to produce a single-cell suspension using DMEM-F12 supplemented with N2 (1%), B27 (2.0%), bFGF (20 ng/mL), EGF (20 ng/mL). The cells were placed in 37°C, 5% CO2 incubator with water-saturated atmosphere.

| RNA Extraction and qRT-PCR
Total RNA was isolated from bowel tissue and cell line using the TRIzol reagent according to the manufacturer's protocol (Life Technologies, CA, USA). Then, the OD260, OD280 and OD260/OD280 ratios of total RNA were measured using a Beckman DU-800 UV spectrophotometer. 500 ng RNA was reverse-transcribed into cDNA by Reverse Transcription Kit (Takara, Tokyo, Japan) for polymerase chain reaction. The PCR primer sequences for these genes are shown in Table 2 and were synthesized by Realgene (Nanjing, China). PCR amplification was processed as follows: 1 9 (95°C, 30 seconds); 40 9 (95°C, 5 seconds; 60°C, 30 seconds);1 9 (95°C, 15 seconds; 60°C, 60 seconds; 95°C, 15 seconds). All experiments were repeated three times.

| RNA editing site-specific qPCR (RESSqPCR)
In order to implement a rapid and cost-effective method to detect an RNA editing fingerprint, we devised an RNA editing site-specific primer design strategy that is compatible with SYBR green qRT-PCR protocols (RESSqPCR). We have previously developed qRT-PCR primers that specifically recognize A-to-I editing, and here, we employed a similar approach in designing RESSqPCR primers. 18  to stain the proliferating cells. Fluorescence staining was determined using a confocal microscope (Olympus, Tokyo, Japan). All experiments were repeated three times independently.

| Transwell assay
Cells were cultured on 6-well plates and treated differently. About

| Plasmid constructs
The ADAR2 overexpression plasmid was purchased from Gene-Pharma (Shanghai, China). The binding site of STAU1 was inserted into the KpnI and SacI sites of pGL3 promoter vector (Realgene, Nanjing, China) to get the pGL3-STAU1-Wild plasmid, and the binding site was mutated antisense to clone to the pGL3 vector called pGL3-STAU1-Mut.

| Sanger sequencing
The amplification products were inserted into a T-vector for Sanger sequencing to detect the A-to-I RNA editing. The primers of pri-miR-142 were designed: 5 0 -TGGAGCAGGAGTCAGGAGG-3 0 (sense) and 5 0 -GCCGAGGAAGATGGTGG-3 0 (antisense) and were synthesized by Tsingke (Nanjing, China). The Sanger sequencing was performed by Tsingke too.
T A B L E 2 Sequences of primers for qRT-PCR-and siRNA-related sequence Name Sequence

| Statistical analysis
The experiments were performed in triplicate independently. Chisquare tests and Student's t test were used to evaluate statistical differences in demographic and clinical characteristics. Data were considered statistically significant as follows: *P < .05, **P < .01 and ***P < .001.

| LPS inhibits the cell proliferation and differentiation
Previous studies suggest that the lipopolysaccharide (LPS) 19,20 has a function of neuroprotection and can regulate the GI motility. Initially, the concentration of LPS was detected in intestinal tissue, and the relative LPS concentration of HSCR segment was higher than control segment as shown in Figure 1A. Then, we tested the mRNA level of

| LPS increases ADAR2 expression in cells
As the LPS dramatically causes rise to ADAR1 editing activity and promotes ADAR1-mediated A-to-I editing as demonstrated before, 11 we first tested the ADAR1 expression in HSCR and control tissues, out of our expectation,there was no significant statistical difference ( Figure S1A). But still, A-to-I editing could also regulate by ADAR2, which belongs to ADAR enzymes. We have been suggested that LPS contributed to activation of ADAR2-mediated A-to-I editing. Then, we detected the expression of ADAR2 in control and HSCR bowel tissues, and the result showed that ADAR2 increased in HSCR tissues (Figure 2A,B). Our study suggested that ADAR2 expression enhanced NPC and 293T cells treated with LPS at different concentrations (0, 1, 10 lg/mL) ( Figure 2C,D). The protein level of ADAR2 also increased in 293T cells treated with LPS at a concentration of 10 lg/mL ( Figure 2E). Furthermore, the ADAR2 0 s editing activity measured by RNA editing site-specific qPCR (RESSqPCR) is shown in Figure 2F. 18 In summary, these data suggest that LPS strengthens ADAR2-mediated A-to-I editing.

| ADAR2 edits the pri-miR-142-3p and impairs cell proliferation and migration
To determine whether ADAR2 is required for cell function, the gain and loss function of ADAR2 was carried out in 293T cells to test the proliferation and migration. The ADAR2 was increased by plasmid ( Figure 3A ( Figure 3E). Conversely, down-regulation of ADAR2 in 293T cells increased mature miR-142-3p expression ( Figure 3E). In addition, the numbers of proliferous and migrated cells were significantly enhanced in cells when ADAR2 was suppressed by ADAR2 siRNA ( Figure 3C,D).
The qRT-PCR products from the post-transfection cells were processed to detect A-to-G changes editing events in the cDNA sequences of pri-miR-142. The qRT-PCR products from ADAR2 overexpressing vector-infected cells were collected for TA-cloning; 50 clones were selected for sequencing subsequently. Figure 3F illustrates the sites that nucleotide changes when comparing with the sequence of samples treated with negative control relative to the mature miR-142-3p. This change did not appear in cells transfected with negative control but in the cells overexpressing ADAR2 or at a high concentration of LPS ( Figure 3G), which favour the editing events regulated by ADAR2. Nucleotide changes of miR-142 precursors evaluate in the 293T cells overexpressing ADRA2 were U-to-C changes because ADAR2-mediated A-to-I editing may occur on RNA transcripts complementary to pri-miRNAs. 22

| ADAR2 is associated with the nucleotide changes in pri-miRNAs in HSCR samples
Then, we tested whether the clinical specimens also appeared the ADAR2-mediated nucleotide changes in 293T cells. The qRT-PCR products of cDNA from 20 HSCR and control samples were sequenced to detect the editing sites of pri-miR-142-3p. Two HSCR samples showed a U-to-C change at the sequence of pri-miR-142-3p ( Figure 3H) and such change did not occur on that of the control tissues. Interestingly, the editing event occurred on HSCR tissues seemed in accordance with the nucleotide change appeared in 293T cells overexpressed ADAR2. As our experiments suggested that Ato-G editing could be induced by ADAR2, we would have to observe whether ADAR2 was enhanced in HSCR tissues which have been detected by nucleotide change. The results demonstrated that the ADAR2 in HSCR was significantly higher than that in the control group ( Figure 3I). All these results suggested that HSCR was associated with ADAR2-mediated A-to-I editing caused by ADAR2 elevation.

| ADAR2-mediated editing on pri-miR-142-3p functionally affects the target gene of miR-142-3p
To further study the impact of the editing event mediated by ADAR2 on miR-142-3p targets, the bioinformatic prediction (DIANA, miRanda, PicTar, PITA, TargetScan) was used to point out RAC1, STAU1, CCNT2 as putative miR-142-3p target genes. MiR-142-3p mimics were transfected to 293T cells, which lead to a reduction in the level of STAU1 ( Figure 4A), but not RAC1 and CCNT2 (Figure S1D,E). Moreover, overexpression of ADAR2 or adding LPS at the concentration of 10 lg/mL to 293T cells also caused an increase in STAU1 ( Figure 4B,C), which demonstrated that the ADAR2- The nucleotide residues changes were labelled with red arrows. The number above the arrows indicates the positions relative to that of the mature miRNAs. I, The protein expression of ADAR2 in the two HSCR tissues was examined by Western blot. *indicates significant difference (P < 0.05). **indicates remarkable difference (P < 0.01). ***indicates statistical significant differences at P < 0.001. mediated editing event affects miR-142-3p target gene. In clinical specimens, STAU1 mRNA level was obviously increased in HSCR patients in contrast to control patients. The protein level of STAU1 was in parallel with the mRNA levels ( Figure 4D,E).
To determine whether miR-142-3p acts on STAU1, the 293T cells were transfected with plasmid pGL3-STAU1 and pGL3-STAU1-Mut, which contains the predicted binding site and mutant binding site ( Figure 4F). The luciferase reporter assay elucidated that miR-142-3p mimics inhibited luciferase activity in pGL3-STAU1-transfected cells. Meanwhile, there were no changes in the luciferase activity of pGL3-STAU1-Mut-transfected cells between miR-142-3p mimics and control group ( Figure 4G). In general, our findings proved that STAU1 is a direct target gene of miR-142-3p. The STAU1 was decreased by STAU1 siRNA and increased by STAU1 plasmid both in mRNA level and in protein level ( Figure S2C,D). The Transwell and CCK8 assay showed that STAU1 overexpression decreased the ability of cells to proliferate and migrate while the ability was inhibited by STAU1 siRNA (Figure S2E,F).

| LPS-ADAR2-miR-142-3p is critical for cellular functions
We next checked whether miR-142-3p participated in cell migration or proliferation. MiR-142-3p mimics and inhibitor were added to 293T cells to observe the migration and proliferation. The Transwell and CCK8 assay showed that miR-142-3p overexpression enhanced the number of proliferative or migratory cells ( Figure 5A,B). In contrast, the cell proliferation or migration ability could be reversed by miR-142-3p inhibitor, which was consistent with the cells treated by LPS and ADAR2 plasmid ( Figure 5A,B). However, the accelerative

| DISCUSSION
Lipopolysaccharide has emerged as the main driving force of gastrointestinal motility 8 and could damage the enteric neuron of rat or porcine. 9,10 The dysplasia of ENS is responsible for many disorders including HSCR. Moreover, a recent report described that the A-to-I editing events conferred by ADAR activation was enhanced by LPS. 11 Further more,another previous study reported that gain or loss of ADAR contributes to cancer progression by regulating the maturity of several miRNAs modulated by RNA editing. 23 Taken together, our study provides a novel link between LPS-and ADAR2-mediated miRNA maturity in HSCR.
Our results showed that high concentration of LPS inhibited the proliferation and differentiation of NPC and impeded the migration of 293T cells, while some researchers showed that lowdose LPS induced neuroprotection. 20 It seems that the LPS affects the NPC in a dose-dependent manner. Significantly, enhanced F I G U R E 5 LPS-ADAR2-miR-142-3p is critical for cell functions A, Cell proliferation of 293T cell lines transfected with miR-142-3p mimics and inhibitor. B, Cell migration was detected using the Transwell assays. C and D, ADAR2 siRNA with or without LPS was transfected into 293T cells; the mRNA level (C) and protein level (D) of STAU1 were evaluated by qRT-PCR(C) and Western blot, respectively (D). E, 293T cells were transfected with miR-142-3p inhibitor with or without ADAR2 siRNA and qRT-PCR was used to detect the relative mRNA levels of STAU1. F, Relative protein level of STAU1 in 293T cells when transfected with miR-142 inhibitor or miR-142 inhibitor plus ADAR2 siRNA. G and H, The proliferation and migration ability of 293T cells were detected by CCK8 and Transwell assays after treated with ADAR2 siRNA with or without LPS. I and J, CCK8 assay and Transwell assays were performed to detect the proliferation and migration of cells transfected by miR-142 inhibitor and treated with miR-142 inhibitor plus ADAR2 siRNA. *indicates significant difference (P < 0.05). **indicates remarkable difference (P < 0.01). ***indicates statistical significant differences at P < 0.001.
ADAR1 activity was observed in LPS-stimulated alveolar macrophages. 24  According to previous research, miR-142-3p is involved in cell migration, proliferation and apoptosis in renal cell carcinoma. 27 Moreover, lower expression of miR-142-3p inhibited the cell migration and proliferation, which was just consistent with the representation of overexpression of ADAR2. Predicted target genes for miR-142-3p from DIANA, miRanda, PicTar, PITA and TargetScan identified 3 genes (RAC1, STAU1, and CCNT2) as potential targets for miR-142-3p. Only STAU1 was inhibited by miR-142-3p mimics.
STAU1 encodes a protein that forms granules initially thought to be neuronal active macromolecular structures, 27 and in colorectal cancer, the expression of Staufen gene was significantly lower than that in corresponding distal normal tissue at mRNA level. 28 When LPS was in high concentration and ADAR2 was highly expressed, STAU1 levels increased, most probably owing to the suppression of miR-142-3p. Thus, miR-142-3p accelerated cell migration by targeting STAU1.
The key progress of this research is that ADAR2 editase activity

CONFLI CTS OF INTEREST
None.