Functional role of microRNA-135a in colitis

Mice were fed with 4% (wt/vol) DSS (MW 36–50 KDa, MP Biomedical LLC, Solon, OH) dissolved in sterile distilled water (vehicle control) ad libitum for the duration of the experiment (days 0–8). Mouse weight was recorded as the initial weight before administration with DSS. After administration with DSS, the body weight of mice was recorded daily, and also recorded whether mice had the following symptoms: hematochezia, diarrhea, bloody diarrhea and weight loss. In order to confirm whether the mouse colitis model was successfully induced, the serum samples and colonic tissue samples were collected from the mice. Histopathologic examination was performed and relative miR-135a expression was detected. All animal work was approved by the Institutional Animal Care and Use Committee of Huaihe Hospital of Henan University and the local Experimental Ethics Committee.

Colon segments were fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin (H&E) for histopathological detection of severity of inflammation, extent of injury and crypt damage.

TUNEL (deoxynucleotidyl transferase–mediated deoxyuridine triphosphate) (Beyotime, China) assay using paraffin-embedded tissues was conducted as manufacturer’s instructions. Slides were observed under fluorescent microscopy (Nikon Eclipse 80i, Japan).

In situ hybridization was performed with 5′-locked digoxigeninlabeled LNA™ miR-135a probe complementary to mouse mature miR-135a and LNA™ U6 snRNA as positive control (Exiqon, Vedbaek, Denmark). Briefly, colon tissues were deparaffinized and deproteinized with protease K for 15 min at 37 °C. Slides were then washed twice with PBS and dehydrate with ethanol. Hybridization was performed at 37 °C for 28 h, followed by blocking with 0.3% BSA in PBS for 30 min. The probe-target complex was detected immunologically by incubating with a digoxigenin Ab conjugated to alkaline phosphatase acting on the chromogen NBT/5-bromo-4-chloro-3-indolyl phosphate (Sigma) for 16 h. Slides were counterstained with nuclear fast red, examined and photographed (Nikon Eclipse 80i, Japan).

Colonic epithelial cells were isolated. Briefly, the colon was removed, everted, incubated for 30 min at 37 °C in a Ca-free Krebs-Henseleit buffer containing 2.5 mM dithiothreitol, 5 mM EDTA, and antibiotics (2.5 μg/ml amphotericin, 100 μg/ml kanamycin monosulfate, 250 U/ml penicillin G, and 250 μg/ml streptomycin sulfate). The epithelium was subsequently forced from the connective tissue with a pressurized stream of Krebs-Henseleit buffer containing 5 mM dithiothreitol and 0.025% bovine serum albumin. Epithelial cells were washed and resuspended in the buffer solution.

miR-135a overexpresison vector was constructed using pcDNA3. The recombinant vector pcDNA3 and miR-135a were constructed and after screening and identifying the recombinant, it was transfected into colonic cells. Antisense oligonucleotides (ASO)-miR-135a and its corresponding negative control (NC), i.e. ASO-NC, were all purchased from Genepharma (Shanghai, China). The siRNA of Hif1α (5’-AGGAAGAACTATGAACATAAA-3′) and the corresponding negative control were designed and synthesized by GenePharma. They were referred as to siRNA-Hif1α and siRNA-control. The cell transfection was performed using Lipofectamine 3000 (Invitrogen Life Technologies, Carlsbad, CA, USA) following the manufacturer’s protocol. After 48 h of transfection, cells were collected for forthcoming analyses.

Culture supernatant was collected and the productions of inflammatory factors were determined by specific ELISA kits. Quantitative determination of IL-8, IL-6, and TNF-α was performed by following the manufacturer’s recommendations (R&D Systems, Minneapolis, MN). The lower detection limits were 31.2 pg/ml for IL-8, 9.4 pg/ml for IL-6, and 15.6 pg/ml for TNF-α.

For western blotting, cells were lysed in RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) with protease inhibitor cocktail (Roche, Basle, Switzerland) on ice for 30 min and cleared by centrifugation. 20 μg lysates were boiled at 100 °C for 5 min in 4 × SDS loading buffer and the samples were separated by SDS–PAGE, and transferred to a PVDF membrane. The membranes were blocked in 5% non-fat milk blocking buffer (Thermo Scientific, Shanghai, China) and then incubated overnight at 4 °C with primary antibody and detected by immunoblotting analysis with the indicated antibodies using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Massachusetts, America). Band intensities were quantified by densitometric analyses using NIH ImageJ. All the experiments were performed at least three times and the most representative results were shown.

Total RNA was extracted from cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. For miR-200a detection, cDNA was synthesized by Taqman MicroRNA Reverse Transcription Kit and PCR was performed by using Taqman Universal Master Mix II (Applied Biosystems, Foster City, CA, USA). For Hif1α detection, the cDNA was synthesized with PrimeScript 1st Strand cDNA Synthesis Kit (Takara, Japan) and the expression of Hif1α gene encoding was quantified by qPCR with SYBR Green Real time PCR Master Mix (TOYOBO, QPK-201). Amplification of cDNA was performed on an Applied Biosystems 7900HT Fast Real-Time PCR System. Cycling parameters were 95 °C for 1 min and then 40 cycles of annealed at 60 °C for 15 s, extended at 72 °C for 1 min. U6 was used as the endogenous control for miR-135a expression. β-actin was used as the endogenous control for Hif1α expressions. The relative expression was calculated using (2^−ΔΔCt) method. The primer sequences were:

miR-135a, forward, 5’-ACACTCCAGCTGGGTATGGCTTTTTATTCCT-3’, and reverse, 5’-GGTGTCGTGGAGTCGGCAA-3’;

Hif1α, forward, 5’-ACTGCCACCACTGATGAATCAAAAACAG-3′, and reverse, 5′- TTCCATTTTTCGCTTCCTCTGAGCATTC-3′.

Cell apoptosis was examined by flow cytometry through Annexin V-FITC/Propidium Iodide (PI) double staining assay. After different treatment, colonic epithelial cells were harvested, washed with cold phosphate buffered saline (PBS) and suspended in binding buffer containing 10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl₂ at a concentration of 10⁶ cells/mL. Then the cells were stained with Annexin V-FITC and PI followed and analyzed on the flow cytometer to evaluate the percentage of apoptotic cells. Experiment was repeated three times and data were analyzed with Cell Quest Software.

3’UTR luciferase reporter plasmids and either miR-135a mimics or ASO-miR-135a were cotransfected in colonic epithelial cells. Cells were harvested at 48 h after transfection and luciferase activity was assayed with Dual-Luciferase Reporter Assay System on a luminometer (Berthold, LB9507, Germany) according to the manufacturer’s protocol (Promega). All experiments were performed in triplicate. The relative ratio of Renilla luciferase activity to Firefly luciferase activity was calculated for each well.

Our experimental results revealed that the mice was presented with symptoms of diarrhea and significant weight loss after 8 days of administration of 4% DSS. After 8 days, the loss of body weight was about 30% compared to control group mice (Fig. 1a), indicating that DSS treatment resulted in a significant weight reduction. The pathological examination of tissue sections with H&E staining confirmed that the colonic epithelium and mucosal tissue of the mice were severely damaged and accompanied by a large number of inflammatory cell infiltration after 8 days of administration of 4% DSS (Fig. 1b). To investigate the consequence in cell apoptosis, we performed a TUNEL assay. After induction of colitis, mice model displayed a significantly higher number of apoptotic epithelial cells compared with normal mice (Fig. 1c). At the same time, we measured the expression level of miR-135a in mice by qRT-PCR and in situ hybridization (Fig. 1d and e). Consistently, miR-135a was significant down-regulated in colon of DSS-treated mice relative to control mice (p < 0.001).

Apoptosis is one of the important reasons for initiation of inflammatory colitis. In order to explore the role of miR-135a in colitis, we performed flow cytometer analysis (Fig. 2a) to detect the effect of miR-135a on apoptosis of colonic epithelial cells. Next, we performed qRT-PCR and western blot to detect the expressions of apoptosis-related genes (Fig. 2b and c). The results showed that miR-135a could significantly inhibit the expression of Bax, the apoptosis-promoting factor, and promote the expression of Bcl-2, the anti-apoptotic factor. At the same time, we determined the levels of TNF-α, IL-8, and IL-6 (measured by ELISA), and found that miR-135a significantly inhibited the levels of IL-6 (p < 0.05), IL-8 (p < 0.01), and TNF-α (p < 0.05) (Fig. 2d).

We first predicted the candidate target gene by using TargetScan, and then confirmed that Hif1α was a target gene of miR-135a (Fig. 3a) and was also negatively regulated by miR-135a (confirmed by luciferase reporter assay, qRT-PCR, and western blot), (Fig. 3b-e).

Knockdown of Hif1α led to the suppression of apoptosis and inflammation in the DSS-induced murine colitis model (Fig. 4a). Western blot analysis revealed significant fall (p < 0.05) in Bax, a pro-apoptic factor and significant increase (p < 0.01) in the level of Bcl-2, anti-apoptic factor, (Fig. 4b and c) similarly to those seen in cells overexpressing miR-135a. Again, similar to the effects of miR-135a, knockdown of Hif1α led to suppression of expressions of inflammatory mediators namely, IL-6 (p < 0.05), IL-8 (p < 0.01), and TNF-α (p < 0.05) compared to control group of cells (Fig. 4d).