DNA Methylation of miR-122 Aggravates Oxidative Stress in Colitis Targeting SELENBP1 Partially by p65NF-κB Signaling

Aberrant microRNA (miRNA) expressions contribute to the development and progression of various diseases, including Crohn's disease (CD). However, the accurate mechanisms of miRNAs in CD are definitely unclear. We employed colonic tissue samples from normal volunteers and CD patients, an acute mice colitis model induced by 2,4,6-trinitro-benzene-sulfonic acid (TNBS), and a cellular oxidative stress model induced by H2O2 in HT-29 cells to determine the effects of oxidative stress on expressions of miR-122, selenium-binding protein 1 (SELENBP1, SBP1), p65 nuclear factor κB (p65NF-κB) signaling, and DNA methylation. We found that SBP1 was mainly located on epithelial cells and was significantly increased in patients with active CD. SBP1 was the target gene of miR-122. miR-122 expression was downregulated while SBP1 expression was upregulated under TNBS-induced colitis or oxidative stress. Pre-miR-122 or siRNA SBP1 (si-SBP1) treatment ameliorated acute TNBS-induced colitis and H2O2-induced oxidative stress. Cotreatment of pre-miR-122 and si-SBP1 enhanced these effects. Besides, pre-miR-122 and si-SBP1 obviously activated the p65NF-κB signaling by phosphorylation of IκBα. Bisulfite sequencing of the CpG islands in the promoter region of miR-122 showed that CpG methylation was significantly increased under oxidative stress. Treating cells with 5′-AZA which was well known as a DNA-demethylating agent significantly increased miR-122 expression. Our results suggest that oxidative stress-induced DNA methylation of miR-122 aggravates colitis targeting SELENBP1 partially by p65NF-κB signaling and may promote the progression of CD.


Introduction
Crohn's disease (CD), which is involved in inflammatory bowel disease (IBD), is a chronic refractory gastrointestinal colitis. It usually appears on juvenile and brings global burden to all over the world [1]. The annual incidence of CD soars to about 12.7/100,000 in Europe and 5/100,000 in Asia [2,3]. However, although there are some effective treatments on CD at present, patients still need long-term medication and even undergo surgery [4,5]. To some extent, it is a huge challenge to explore the pathogenesis and develop new therapeutic targets of CD.
Oxidative stress has been found to play a key role in the pathogenesis and progression of CD [6]. In a normal intestinal mucosa, there is a balance between oxidative stress factors such as reactive oxygen species (ROS), hydrogen peroxide (H 2 O 2 ), superoxide anion (O 2-), and antioxidant molecules such as glutathione (GSH), superoxide dismutase (SOD), and glutathione-S-transferase (GST) [7][8][9]. In the intestinal mucosa of CD, infiltrating inflammatory cells release excessive ROS, while SOD and other antioxidant enzymes decrease, leading to oxidative stress enhancement, lipid peroxidation, which ultimately causes intestinal mucosal damage [10,11]. Enhanced oxidative stress can result in serious cell damage directly as well as activating nuclear factor κB (NF-κB) signaling and the following proinflammatory factors (IL-6, IL-8, etc.) and intracellular adhesion molecules (ICAM) [12,13].
Selenium-binding protein 1 (SELENBP1, SBP1), a member of the selenoprotein family, widely distributes in a variety of tissues, with a high level in the heart and a moderate level in the liver, lung, kidney, and intestinal tissues [14]. A few studies have reported the anticancer effect of SBP1 in renal cell carcinoma, head and neck cancers, and prostate cancer [15][16][17]. However, the role of SBP1 in oxidative stress and CD remains largely unknown.
To our knowledge, microRNAs (miRNAs) are a series of endogenous small molecules, regulating the pathogenesis of many diseases, such as inflammation, autoimmune diseases, cancer, and CD [18][19][20][21]. The mature miRNAs bind to the target mRNA through RNA-induced silencing complex (RISC) and complement to the 3 ′ -untranslated region of target mRNA, causing degradation or translation inhibition of target mRNA [22]. A number of abnormal expressed miR-NAs have been selected in CD, which not only participate in the pathogenesis of CD but also attract more and more attention in the progression of the diagnostic and therapeutic potentials [21,23]. miR-122, known as a liver-specific miRNA, involved in the pathology of liver diseases, such as hepatitis and liver cancer [24,25]. Antisense oligonucleotide miravirsen targeting miR-122, as the first microRNA-targeted drug, has been used in clinical trials for the treatment of hepatitis C virus (HCV) infection [26].
Until now, we have confirmed that miR-122 is closely related to oxidative stress in a rat liver injury model [27]. However, whether miR-122 mediates oxidative stress in CD and its mechanisms has not been reported. Therefore, this study was designed to investigate the potential role of miR-122 on oxidative stress and inflammation in CD, which would provide a theoretical basis for an effective target of diagnosis and treatment of CD.

Materials and Methods
2.1. Mucosal Biopsy Specimens. Biopsy tissues were obtained from inflamed and noninflamed mucosa of patients with active CD (n = 6) who were diagnosed according to clinical and macroscopic criteria. Control samples were collected from healthy volunteers (n = 6). They were fixed in formalin and embedded in paraffin for an immunohistochemistry assay.

Establishment of an Acute CD Model and Assessment.
Female Balb/c mice aged 7 weeks were purchased from the Laboratory Animal Center of Nanjing Medical University (Nanjing, China). The acute CD model was induced with TNBS (Sigma, USA) as published previously [28]. Briefly, 3 mg of TNBS in 100 μL of 50% ethanol was slowly administered via a transrectal polyethylene catheter inserted 4 cm from the anus (TNBS, n = 20). An equivalent volume of 50% ethanol was instilled into the control group (ethanol, n = 20).
Mice were killed by cervical dislocation on days 1, 3, 5, 7, and 10 to get the most severe one. To determine the effect of pre-miR-122 in CD, mice were treated with pre-miR-122 or siRNA (5 mg/kg) for 12 h after TNBS injection and killed on day 3. To evaluate the degree of colitis, the disease activity index [29] was calculated involving body weight (ranged from 0 to 4), fecal consistency (ranged from 0 to 4), and the presence of a bloody stool (ranged from 0 to 4) daily after TNBS treatment. Meanwhile, hematoxylin and eosin staining on the lowest 4 cm colon was performed, and the histology score was calculated involving edema (ranged from 0 to 3) and depth of inflammatory cell infiltration (ranged from 0 to 3) on the bowel wall.

Ectopic
Expression of miR-122. Synthetic miR-122 precursor (pre-miR-122, GenePharma, Shanghai, China) and scrambled negative control RNA (NC) were added to HT-29 cells with Lipofectamine 2000 (Invitrogen, USA) when cells were approximately 70% confluent. Twenty-four hours after transfection, the cells were used for the special experiments.
2.5. SBP1 Inhibition. Specific SBP1 siRNA (si-SBP1, Gene-Pharma, Shanghai, China) and negative control siRNA (si-NC) were transfected, respectively, when cells were approximately 50% confluent. Lipofectamine 2000 (Invitrogen, USA) and siRNA (80 nM/L) were used in a total serum-free medium of 2 mL per well. After 8 h, 2 mL of culture medium was added to each well for another 40 h.
2.6. Cell Viability Assay. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8, Dojindo, Japan) following the manufacturer's instructions. Twenty-four hours after treatment with H 2 O 2 (0, 200, 400, 600, 800, and 1000 μM), approximately 2 × 10 3 cells were seeded into each well of 96-well plates. The value of each sample was measured on a microplate reader (Bio-Rad, USA) at three different time points.
2.7. Cytokine Enzyme-Linked Immunosorbent Assays. HT-29 cells were then stimulated with H 2 O 2 (0, 100, 200, 300, 400, 500, and 1000 μM) for 24 h or left untreated. The levels of MDA, SOD, and ROS were measured in culture supernatants using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA). Absorbance was measured at 450 nm and compared with the respective standard curve of the cytokines.
As to the expressions of 8-OHdG, GSH, MDA, ROS, IL-6, and IL-8 in the TNBS-induced colitis model, full-thickness colonic tissues of mice were homogenized in protease inhibitors (KeyGen Biotech, Nanjing, China) supplemented with 1 mM phenylmethane sulphonyl fluoride (PMSF) to obtain the supernatants, which were finally tested for protein level. To measure these cytokines, mouse double-antibody sandwich ELISA kits (R&D Systems) were developed.
2.8. RNA Isolation and qRT-PCR. Total RNA was isolated and extracted with the miRNeasy Mini Kit (Qiagen, Germany) as the manufacturer's instructions. The complementary DNA (cDNA) was prepared from total RNA using the Omniscript RT kit (Qiagen, Germany). The levels of miR-122 and SBP1 were conducted with the miScript SYBR Green PCR kit (Qiagen, Germany) on a 7500 Real-Time PCR system (Applied Biosystems, USA). U6 and GAPDH were normalized for the expressions of miR-122 and SBP1, respectively. Primer pairs used in this study were   p-IκBα primary antibodies (Abcam, MA, USA), ICAM, and GAPDH (Santa Cruz, CA, USA) primary antibodies were incubated overnight at 4°C. The protein levels were normalized to GAPDH. Alpha Innotech (San Leandro, CA) imaging software was used to quantify these data.
2.10. Luciferase Reporter Assay. SBP1 3 ′ -untranslated region (3 ′ -UTR) was mutated using the mutagenesis kit (Promega, USA). Wild-type and mutant sequences were amplified and inserted into the vector to construct luciferase reporter plasmids following the manufacturer's instructions (Promega, USA). Cells were seeded in 96-well plates and cotransfected with reporter constructions (Wt vector and Mut vector) and miR-122. The luciferase activity was measured using the dual-luciferase reporter assay (Promega, USA).
To amplify the CpG islands in the promoter region of the miR-122 gene, the sequence was amplified using the primers (F) ATTTTAATTTATGGGAGTAGAACGA and (R) ATCT TACTTTAACCCTAAAACCGAC. The bisulfite-treated PCR products were purified with a commercially available extraction kit (Magen, China) and cloned into the pMD-18T vector (TaKaRa, Japan). Ten clones from each sample were sequenced.
2.12. Statistical Analysis. GraphPad Prism 5 software (GraphPad Software, San Diego, CA) was performed to carry out all statistical analyses. One-way analysis of variance was used for multiple group comparison. When only two groups were compared, Student's t-test was performed. p values of less than 0.05 (p < 0 05) were considered to be significant. All quantitative data were expressed as the mean ± SEM.
2.13. Ethical Considerations. All animals received appropriate care according to the requirements of the Animal Care and Use Committee of Nanjing Medical University. All patients and normal control individuals signed a written informed consent form.

SBP1 Expression Was Increased in Patients with
Active CD. Strong SBP1 staining was observed in the samples from patients with active CD while relatively weak SBP1 staining was observed in the normal group. SBP1 mainly located in the cytoplasm of IECs (Figure 1).

TNBS Induced Severe Colitis and SBP1 Was Increased in
This Colitis. The TNBS-induced colitis group showed more obvious mean body weight loss compared with controls (50% ethanol). The mean body weight loss was 8.2% over 24 h and reached a peak of 18.8% at 72 h (Figure 2(a)). The mean DAI was 7.9 in the TNBS group while it was 3.67 in the control group at 72 h (Figure 2(b)). The pathology assay also showed the most severe colitis at day 3 ( Figure 2(c)). The SBP1 level was higher in the TNBS group compared with the control group via the Western blotting assay (Figure 2(d)) and immunohistochemical staining (Figure 2(e)). SBP1  shared the same location with E-cadherin which indicated its expression on epithelial cells (Figure 2(f)).

Oxidative Stress
Affected miR-122 and SBP1 Levels. As demonstrated by CCK-8 viability assays, the HT-29 cell viabilities were markedly declined in the H 2 O 2 group (400, 500, and 1000 μM but not 100, 200, and 300 μM) compared to the control group (Figure 3(a)). HT-29 cells administrated with H 2 O 2 (300, 400, 500, and 1000 μM) succeeded in a remarkably higher level of MDA and ROS accompanied by a decreased level of SOD with the ELISA assay (Figures 3(b)-3(d)). Thus, we selected H 2 O 2 (300 μM) for the following experiments because it could activate oxidative stress but not affect the viability of HT-29 cells at this concentration. Besides, we also found that HT-29 cells succeeded in the lower level of miR-122 expression and the higher level of SBP1 mRNA in the H 2 O 2 group than the control group (Figures 3(e) and 3(f)). We next determined SBP1 expression after H 2 O 2 treatment by Western blotting and found that the H 2 O 2 -induced SBP1 expression occurred in a dose-dependent manner and showed positive correlation with concentration of H 2 O 2 (Figure 3(g)).

SBP1 Was Verified as a Target of miR-122.
To verify SBP1 as a target of miR-122, we searched the candidate target of miR-122 using the available database (TargetScan, miRanda,   and PicTar) and found that 3 ′ -UTR of SBP1 mRNA contained a high conserved binging site for miR-122. As shown in Figure 3(h), the luciferase activity was decreased in cells cotransfected with pre-miR-122 and 3 ′ -UTR of SBP1 compared with the control group. However, no significant variation in luciferase activity was observed in cells with 3′-UTR-MUT of SBP1 with pre-miR-122 cotransfection. Then, we transfected pre-miR-122 into HT-29 cells to evaluate its efficiency and found significant increased expression of miR-122 compared to the control group (Figure 4(a)) followed by obviously decreased expression of SBP1 mRNA and protein (Figures 4(b) and 4(c)). These results suggested that SBP1 might be a direct target for miR-122. Meanwhile, we transfected si-SBP1 into HT-29 cells to evaluate its efficiency and found significant decreased expression of SBP1 compared to the control group (Figure 4(d)).

Pre-miR-122 or/and si-SBP1 Protected H 2 O 2 -Induced
Oxidative Stress In Vitro. Then, cells pretransfected with pre-miR-122, si-SBP1, and both pre-miR-122 and si-SBP1 were then stimulated with H 2 O 2 (300 μM) for the indicated times. Subsequently, we found a higher level of ROS, MDA, 8-OHdG, IL-6, and IL-8 in the H 2 O 2 group than the normal group, while a lower level of them in the pre-miR-122 and si-SBP1 group than the H 2 O 2 group. Cotreatment of pre-miR-122 and si-SBP1 could intensify this phenomenon (Figure 4(e)).
3.6. Pre-miR-122 or si-SBP1 Treatment Ameliorated TNBS-Induced Colitis In Vivo. We established TNBSinduced colitis as a murine model of CD as our previous study ( Figure 5(a)). First, we examined the SBP1 expression and found lower levels of SBP1 in the pre-miR-122 or si-SBP1-added TNBS group than the TNBS group ( Figure 5(b)). Pre-miR-122 or si-SBP1 could obviously ameliorate the DAI and histology scores compared to the TNBS group (Figures 5(c)-5(e)). Meanwhile, we found lower levels of 8-OHdG, MDA, IL-6, and IL-8 and higher levels of GSH and SOD in the pre-miR-122 or si-SBP1-added TNBS group than the TNBS group ( Figure 5(f)). At last, we also found that pre-miR-122 or si-SBP1 obviously inhibited the levels of       ( Figure 6(d)). Subsequently, we examined the levels of ICAM and p65NF-κB signaling related to oxidative stress and found significant activation of p65NF-κB signaling and ICAM under H 2 O 2 stimulation. Moreover, we found higher levels of p-p65, p65, and ICAM and lower levels of GPX1 and p-IκBα in the miR-122 inhibitor-added H 2 O 2 group than the H 2 O 2 group (Figure 6(e)).

Inhibition of miR-122 Aggravated TNBS-Induced Colitis
In Vivo. We established TNBS-induced colitis as a murine model of CD as our previous study (Figure 7(a)). First, we examined the SBP1 expression and found a higher level of SBP1 in the miR-122 inhibitor-added TNBS group than the TNBS group (Figure 7(b)). Inhibition of miR-122 could obviously aggravate the DAI and histology scores compared to the TNBS group (Figures 7(c)-7(e)). Meanwhile, we found high levels of 8-OHdG, MDA, IL-6, and IL-8 and lower levels of GSH and SOD in the miR-122 inhibitor-added TNBS group than the TNBS group (Figure 7(f)). At last, we also found that inhibition of miR-122 obviously promoted the levels of p-p65, p65NF-κB, and ICAM and decreased the expression of GPX1 and p-IκBα in vivo (Figure 7(g)).
In order to investigate whether DNA methylation regulated the expression of miR-122 under oxidative stress conditions, we compared the levels of DNA methylation. We found that the promoter region of miR-122 under oxidative stress conditions was overmethylated compared to the control group (Figure 8(a)). When 5′-AZA was present, the percentage of miR-122-methylated DNA sites was decreased significantly and the miR-122 level was increased (Figure 8(b)). Moreover, we also found that the level of SBP1 was decreased in the 5 ′ -AZA group compared to the control group (Figure 8(c)).

Discussion
Crohn's disease (CD) is a chronic recurrent inflammatory disorder of the gastrointestinal tract with a high recurrence rate, canceration risk, and poor prognosis [30]. Although the treatment of CD has been improved to some extent, the etiology is still uncovered. Therefore, it is crucial to study the pathogenesis and develop new therapeutic targets of CD.