miR-542-5p targets c-myc and negates the cell proliferation effect of SphK1 in intestinal epithelial cells

Abstract

Intestinal epithelial barrier defects occur commonly during a variety of pathological conditions, though their underlying mechanisms are not completely understood. Sphingosine-1-phosphate (S1P) has been shown to be a critical regulator of proliferation and of maintenance of an intact intestinal epithelial barrier, as is also sphingosine kinase 1 (SphK1), the rate-limiting enzyme for S1P synthesis. SphK1 has been shown to modulate its effect on intestinal epithelial proliferation through increased levels of c-myc. We conducted genome-wide profile analysis to search for differential microRNA expression related to overexpressed SphK1 demonstrating adjusted expression of microRNA 542-5p (miR-542-5p). Here, we show that miR-542-5p is regulated by SphK1 activity and is an effector of c-myc translation that ultimately serves as a critical regulator of the intestinal epithelial barrier. miR-542-5p directly regulates c-myc translation through direct binding to the c-myc mRNA. Exogenous S1P analogs administered in vivo protect murine intestinal barrier from damage due to mesenteric ischemia reperfusion, and damaged intestinal tissue had increased levels of miR-542-5p. These results indicate that miR-542-5p plays a critical role in the regulation of S1P-mediated intestinal barrier function, and may highlight a novel role in potential therapies.

INTRODUCTION

The intestinal epithelium is a rapidly proliferating tissue, undergoing total replacement every 2–3 days; undifferentiated cells replicate in the intestinal crypts and migrate to the villus tips, differentiating as they migrate toward the villus tip, and apoptosis occurs at all areas to maintain a balance in cell numbers and to remove senescent cells (1). Although this balance between proliferation and apoptosis is occurring, the mucosal layer must simultaneously maintain integrity of the barrier. However, the exact mechanisms underlying these processes are not completely understood. Moreover, therapies for pathological defects in barrier integrity are largely conservative, and treatment for injuries such as those induced by intestinal ischemia-reperfusion injuries would be novel.

Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that has shown to be a potent regulator of cell proliferation, apoptosis, and permeability, among other effects (2, 3). It is ubiquitous, as it derives from conversion from sphingolipids that are constituents of eukaryotic cell membranes. Prior studies have demonstrated biological effects through both actions as an intracellular ligand as well as through extracellular G protein-coupled receptors S1P 1–5 that confer resistance to apoptosis, stimulate proliferation in fibroblasts, and augments calcium mobilization (4, 5). In the intestinal epithelia, S1P has been shown to be a critical regulator of cellular barrier function and has been shown to regulate intestinal cell proliferation through c-myc, which is a nuclear transcription factor (6).

Sphingosine is converted to S1P through enzymatic activity of sphingosine kinase 1 (SphK1) that is rate limiting (7). We have shown that increased activity of SphK1 leads to increased levels of S1P and accompanying positive effects on cellular proliferation; this is accompanied by an increase in c-myc levels, also with increased c-myc translational efficiency with increased proliferative index and an increase in G1 to S phase transition (6). The exact mechanisms that control this are not completely understood.

Micro-ribonucleic acids (miRNAs) are small nucleotides that target 3′ untranslated region (UTR) of mRNA causing degradation or inhibition of translation. miRNAs have been shown to influence many physiological and pathological processes, including many intestinal processes (8, 9). miR-542-5p has been shown to be a tumor suppressor by decreasing cell invasiveness and metastasis (10) in lung cancer cells, and also in neuroblastoma (11), and endometrial carcinoma (12). Alternatively, it has been shown to promote tumorigenesis in osteosarcomas (13). It has also been shown to play a role in outcomes from acute lung injury (14) and in hyperglycemia (15). Its role in the intestine has not been reported.

Our prior study demonstrated that ectopic SphK1 clones demonstrated increased c-myc with a higher proliferative index (6). This study was to demonstrate that miR-542-5p plays a critical role in the regulation of c-myc levels that result in the positive regulatory effects of S1P. We also demonstrate that these same moieties are involved in vivo and that an S1P analog can provide a potential therapy for intestinal barrier dysfunction.

MATERIALS AND METHODS

Cell Cultures and Supplies

Dulbecco’s modified Eagle’s medium (DMEM) and dialyzed fetal bovine serum (dFBS) were from Invitrogen (Carlsbad, CA), and biochemicals were from Sigma (St. Louis, MO). The intestinal epithelial cell (IEC)-6 cells lines are derived from normal rat intestinal crypt cells as described previously (16) and were purchased from the American Type Culture Collection, as were HEK cells. IEC-6 cells were maintained in DMEM supplemented with 5% heat-inactivated fetal bovine serum and antibiotics. Antibodies recognizing c-Myc were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Actin purchased from Sigma and SphK1 antibody was purchased from Cell Signaling Technology (Danvers, MA).

Animal Experiments

All experiments were approved according to animal experimental ethical guidelines by the University of Maryland, Baltimore Institutional Animal Care and Use Committee. Mice were housed and handled in a specific pathogen-free area and cared for by trained technicians and veterinarians.

For mesenteric ischemia-reperfusion injury, male C57BL/6 mice were fasted 4–6 h preceding the experiment. The mouse was anesthetized and gavaged orally with FTY720 (S1P agonist) or normal saline following which a midline laparotomy was performed under isoflurane-inhaled anesthesia. The superior mesenteric arteries were clamped at their origin for 20 min using vascular clips (Roboz, Gaithersburg, MD). Twenty minutes later clamps were released and abdominal closure performed. Two hours after reperfusion, the animals were euthanized and mid jejunum harvested for histological and biological examination and permeability studies. Sham operation for controls involved laparotomy without mesenteric ischemia.

Histological Analysis

Dissected intestinal tissues were fixed in formalin and paraffin. Sections were stained with hematoxylin and eosin (H&E) for general histology. Slides were graded in a blinded fashion.

Stable Cell Line Production and Characterization

Human full-length SphK1 plasmid (OriGene Technologies, Rockville, MD) was linearized with the restriction enzyme Not l, sequenced, and then subcloned to an expression vector pCMV6-Neo. Resulting clones were sequenced for the confirmation of successful subcloning. IEC-6 cells were transfected with the SphK1 expression vectors or control vectors containing no SphK1 cDNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. After incubation for 5 h, the transfection medium was replaced by the standard growth medium containing 5% FBS for 2 days before exposure to the selection medium containing 0.6 mg/mL of G418. Clones resistant to the selection medium were isolated, cultured, and screened for SphK1 expression by Western blot analysis with specific anti-SphK1 antibody.

Western Blot

Whole cell lysates were prepared using RIPA Lysis Buffer (No. 20188, Millipore), sonicated, and centrifuged at 4°C for 15 min. The supernatants were boiled for 5 min and size-fractionated by SDS-PAGE. After proteins were transferred onto nitrocellulose filters, the blots were incubated with primary antibodies, Sphk1 (No. 3297S, 1:1,000, Cell signal Technology), C-Myc (N-262,1:500, Santa Cruz Biotechnology), and Actin (No. CP01, 1:2,000, Sigma). All primary antibodies were incubated overnight with blocking solution at 4°C. After incubations with secondary antibodies, secondary antibody conjugated to horseradish peroxidase (HRP) was from Santa Cruz Biotechnology. Relative protein levels were analyzed using the Bio-Rad Chemidoc and XRS system equipped with Image laboratory software (v.4.1).

Reverse Transcription Followed by Polymerase Chain Reaction and Real-Time Quantitative PCR Analysis

Total RNA was isolated from cells after different treatments by using RNeasy mini kit (Qiagen, Valencia, CA) and used in reverse transcription (RT) and PCR amplification reactions as described previously (17). The levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR product were assessed to monitor the evenness in RNA input in RT-PCR samples. Real-time quantitative (q)PCR analysis was performed using 7500-fast real-time PCR systems with specific primers, probes, and software (Applied Biosystems, Foster City, CA).

Cell Cycle Analysis

Cell cycle analysis was performed as described previously (18); after treatment cells were collected by trypsinization and processed using the CycleTES PLUS DNA Reagent Kit (Beckton Dickinson, San Jose, CA) according to the manufacturer’s instructions. Briefly, after trypsinization, the cells were centrifuged, and the cells were washed in a buffer containing sodium citrate, sucrose, and DMSO. Cells were then incubated sequentially for 10 min each in solution A (containing trypsin in a spermine tetrahydrochloride detergent buffer for the enzymatic digestion of cell membranes and cytoskeletons), solution B (containing trypsin inhibitor and ribonuclease A in citrate-stabilizing buffer with spermine tetrahydrochloride to inhibit the trypsin activity and to digest the RNA), and solution C (containing propidium iodide and spermine tetrahydrochloride in citrate stabilizing buffer for the stoichiometric binding of propidium iodide to the DNA at a final concentration of 125 μg/mL). Flow cytometry analysis was carried out to examine the cell cycle distribution in a Beckton Dickinson FACS Calibur analyzer (Becton Dickinson). Data were further analyzed using the software FLOWJOW Ver. 6.1.1 (Tree Star, San Carlos, CA) with the Watson Pragmatic Model.

Biotin Labeling

Biotin-labeled miR-542-5p was transfected into the cells and 24 h later whole cell lysates were collected, mixed with Streptavidin-Dynal beads (Invitrogen), and incubated at 4°C with rotation overnight, as previously described (19). After the beads were washed thoroughly, the beads-bound RNA was isolated and subjected to RT followed by Q-PCR analysis. Input RNA was extracted and served as a control.

Polysome Analysis

Polysome analysis was performed as described previously (20). In brief, SphK1-IEC-6 cells at 70% confluence were incubated for 15 min in 0.1 mg/mL of cycloheximide and then lifted by scraping in 1 mL of PEB lysis buffer (0.3 M NaCl, 15 mM MgCl₂, 15 mM Tris·HCl, pH 7.6, 1% Triton X-100, 1 mg/mL of heparin, and 0.1 mg/mL of cycloheximide) and lysed on ice for 10 min. Nuclei were pelleted (10,000 g; 10 min), and the resulting supernatant was fractionated through a 10%–50% linear sucrose gradient to fractionate cytoplasmic components according to their molecular weights. The eluted fractions were prepared with a fraction collector (Brandel, Gaithersburg, MD), and their quality was monitored at 254 nm by using a UV-6 detector (ISCO, Lincoln, NE). After RNA in each fraction was extracted with 8 M guanidine-HCl, the levels of each individual mRNA were quantified by RT-qPCR in each of the fractions, and their abundance was represented as a percentage of the total mRNA in the gradient.

mRNA Stability

The stability of c-myc mRNA was measured after transfection of SphK1 cells with pre-miR-542-5p and C-siRNA following the addition of actinomycin D (5 μg/mL) to the medium. Total cellular RNA was isolated at the indicated times after exposure to actinomycin D, and the remaining levels of c-myc RNA were measured by qRT-PCR analysis. Nonlinear regression analysis was used to calculate mRNA half-life.

Statistical Analysis

Values are represented as means ± SE from three to six samples. PCR and immunoblotting results were repeated three times. The significance of the difference between means was determined by analysis of variance. The level of significance was determined using the Duncan’s multiple-range test, and for data from multiple groups we performed a multiple comparison adjustment with Tukey–Kramer’s method. P values <0.05 were considered significant.

RESULTS

Stable Overexpression of SphK1 or S1P in Intestinal Epithelial Cells Decreases Expression of miR-542-5p

Intestinal cell lines stably overexpressing SphK1 were established in IEC-6 cells as described previously (1). These cells were shown to have increased expression of S1P. To determine the interaction between SphK1 and microRNAs in the regulation of intestinal mucosal growth, a microarray-based evaluation of global miRNA expression in IEC-6 cells and SphK1-overexpressing clones was performed with differential expression in 20 miRNAs including miR-542-5p. Real-time quantitative PCR (Q-PCR) analysis was used and verified a significant decrease in the levels of miR-542-5p in SphK1 clonal cells (Fig. 1A).

Figure 1.

Sphingosine-1-phosphate (S1P) activity affects expression of miR-542-5p. A: levels of miR-542-5p as examined by quantitative (Q)-PCR analysis in two separate SphK1 stable cell lines that overexpress sphingosine kinase 1 (SphK1). Values are represented as means ± SE of data from three separate experiments. *P < 0.05 compared with control cells and SphK1 cells. B: changes of miR-542-5p levels with varying levels of exogenous S1P treatments of Cdx-2 IEC-6 cells. *P < 0.05 compared with control Cdx-2 cells (n = 5 experiments). C: silencing of S1P activity promotes expression of miR-542-5p. Levels of SphK1 as shown after treatment with control siRNA and with use of siSphK1, demonstrating that SphK1 is effectively reduced by this treatment. Results are from three separate experiments. D: silencing SphK1 demonstrated a marked increase in miR-542-5p levels compared with those treated with control siRNA in five experiments. Control siRNA did not affect miR-542-5p levels, however treating with siSphK1 resulted in a marked increase in miR-542-5p. *P < 0.05 compared with control siRNA and siSphK1. Statistical significance was analyzed by one-way ANOVA followed by Duncan’s test.

To determine whether exposure of intestinal epithelial cells to ectopic S1P could similarly cause significant decreases in the levels of miR-542-5p, cells were treated with various concentrations of S1P. After this exposure, real-time PCR demonstrated a decrease in miR-542-5p expression compared with control (Fig. 1B), however, there was minimal variation with increasing concentrations of S1P.

Silencing of SphK1 in Intestinal Epithelial Cells Increases Expression of miR-542-5p

We next examined the effects of silencing SphK1 on miR-542-5p levels. To reduce SphK1 levels, small interfering RNAs (siRNAs) targeting the coding regions (CRs) of SphK1 mRNA (siSphK1) were used. Transfection with siSphK1 in stable Sphk1-IEC cells significantly reduced SphK1 levels versus transfection with control siRNA. Cells transfected with siSphK1 showed significantly increased levels of miR-542-5p versus control (Fig. 1, C and D). Together, these findings strongly suggest that SphK1 and S1P directly modulate levels of miR-542-5p in the intestinal epithelial cells.

c-Myc Protein Expression in SphK1 Cells is Regulated by miR-542-5p Transfection

We have previously shown (6) that overexpression of SphK1 results in increased expression of SphK1 protein and resulted as well in increased expression of c-myc. Given the results earlier, it was not known if the effects of miR-542-5p were independent to the effects on c-myc or directly affected its expression. To determine this, miR-542-5p was expressed in the SphK1 clonal cells, and as shown its co-expression reduced the expression of c-myc in these cells (Fig. 2A). Conversely, silencing of miR-542-5p further augmented the expression of c-myc (Fig. 2, B and C).

Figure 2.

c-myc expression is regulated by sphingosine-1-phosphate (S1P) activity and miR-542-5p. A: stable sphingosine kinase 1 (SphK1) cell lines were transfected with pre-miR-542-5p vs. control (scramble) for 48 h. Overexpression of miR-542-5p in stable SphK1 cells resulted in decreased levels of c-Myc. *P < 0.05 compared with control cells (n = 3 replicates). B: levels of miR-542-5p were silenced by transfection for 48 h with -miR-542-5p (simiR-542-5p, listed as anti-542-5p) or control siRNA. *P < 0.05 compared with control siRNA (n = 3 replicates). C: silencing of miR-542-5p as shown in (B) resulted in increased levels of c-Myc protein.

Ectopic Expression of miR-542-5p Reduces c-Myc mRNA Levels in SphK1 Cells

As shown earlier, overexpression of SphK1 in cells demonstrates increased expression of c-myc, however concomitant overexpression of miR-542-5p in the intestinal epithelium was associated with decreased c-myc protein expression. To determine the etiology of this suppression, we first measured levels of c-myc mRNA in these conditions (Fig. 3, Aa and Ba) and found that in addition to decreasing c-myc protein expression, miR-542-5p expression also led to reduced c-myc mRNA levels.

Figure 3.

Levels of c-myc mRNA in two separate clones of sphingosine kinase 1 (SphK1) stable cells, Clone 1 (A) and Clone 2 (B). Aa and Ba: levels of c-Myc mRNA as measured by quantitative (Q)-PCR analysis in SphK1 stable cells that were treated as described. Data were normalized to GAPDH mRNA levels, and values are shown as the means ± SE from six samples. *P < 0.05 compared with results for cells infected with Adnull. Ab and Bb: the half-life of c-Myc mRNA expressed from overexpression (O/E) miR-542-5p in SphK1 stable cell-1 and SphK1 stable cell-2. After the administration of actinomycin D, total RNA was isolated at the indicated times and the remaining levels of c-Myc mRNA were measure by Q-PCR analysis. Data were normalized to GAPDH mRNA levels from three separate experiments.

To determine if this observed reduction was due to decreased stability of the c-myc mRNA, we measured the half-life of the mRNA in control conditions and with miR-542-5p expression and found no difference in stability of the c-myc mRNA in either condition (Fig. 3, Ab and Bb).

miR-542-5p Interacts with c-Myc mRNA

To further elucidate the effect of overexpression of miR-542-5p on c-Myc mRNA translation, we examined the relative distributions of c-Myc mRNA on individual fractions from polyribosome gradients in Sphk1 stable cells. Polyribosome distribution profiles were examined in control Sphk1 clone cells and Sphk1 clone cells exposed to mi542-5p as described previously (6). In this study, fractions 1–4 included mRNAs that were not associated with components of the translation machinery or co-sedimented with ribosome subunits (monosomes); hence, they were not considered to be translated. Fractions 5–7 included mRNAs that bound to single ribosomes or formed polysomes of low molecular weight, and they were considered to be translated at low-to-moderate levels. Fractions 8–10 included the mRNAs that were associated with polysomes of high molecular weight, and they were thus considered to be actively translated. We found that overexpression of miR-542-5p had no effect on housekeeping GAPDH mRNA (Fig. 4A, top) but did affect the distribution on c-myc mRNA (less abundant) in the high-translating fractions (Fig. 4A, bottom).

Figure 4.

miR-542-5p interacts with and inhibits c-myc mRNA translation. A: polysome distributions of c-Myc mRNA. Distribution of c-Myc mRNA and housekeeping GAPDH mRNA in each gradient fraction prepared from three groups. Total RNA was isolated from the different fractions, and the level of c-Myc and GAPDH mRNA were measured by RT- quantitative (q)PCR analysis and plotted as a percentage of the total c-Myc or GAPDH mRNA levels in that sample. The translational activity associated with each fraction is indicated as untranslated (NB, not bound to polysomes; NT, not translated), moderately translated (LMW, low-molecular-weight polysomes), and actively translated (HMW, high-molecular-weight polysomes). Data represent the average of three independent experiments yielding similar results. Levels of biotinylated miR-542-5p after transfection for 48 h. B: schematic representation of biotinylated miR-542-5p. C: total mRNA levels of c-jun, Cdk2, and JunD with biotinylated miR-542-5p as measured by Q-PCR analysis. Values are represented as means ± SE from five separate experiments. **P < 0.05 compared with cells transfected with control scrambled RNA. D: binding of biotinylated miR-542-5p to c-Myc, c-Jun, Cdk2 mRNA in pull-down materials from 5 separate experiments.

To determine the role of miR-542-5p in the regulation of c-myc, we examined the association of miR-542-5p with c-myc mRNA using biotin-labeled miR-542-5p to perform an RNA pull-down assay (Fig. 4B). As shown in Fig. 4, C and D, c-myc mRNA was enriched in the materials pulled down by biotin-miR-542-5p, but not in materials from cells transfected with control scramble RNA. Thus, the association of miR-542-5p with c-myc mRNA was specific since increasing the levels of biotin miR-542-5p did not increase its interaction with c-jun and cdk2 mRNA.

In Vivo Studies Demonstrate S1P Reverses Effects of MIR

To determine if S1P would affect mucosal barrier function in vivo, we examined for mucosal injury and repair after exposure to mesenteric ischemia reperfusion (MIR). We utilized a MIR model in mice. To further understand the role of miR-542-5p in this process of ischemia reperfusion, we examined levels of this microRNA in the tissues and found that miR-542-5p was significantly expressed in the more damaged intestinal tissue. S1P analog FTY720 not only preserved the intestinal villi but demonstrated reduced levels of miR-542 simultaneously (Fig. 5A). To grade the degree of intestinal injury for this we used Chui scores that grades morphological changes to the villus. We found on histopathological analysis that MIR resulted in significant mucosal injury compared with sham surgery with swollen villi with copious cell sloughing. As shown in Fig. 5B, this process was attenuated with the administration of FTY720, an S1P agonist, with better preservation of villi architecture and less sloughing, demonstrative of a lower Chui score. Finally, in Fig. 5C, we demonstrate that gut permeability to FITC-dextran increased in all littermates exposed to MIR, but that the degree of permeability was lessened with concomitant administration of S1P analog FTY720, which paralleled both the levels of miR-542-5p and the observed histological changes.

Figure 5.

Tissue levels of microRNA-542-5p taken tissue in mice undergoing mesenteric ischemia-reperfusion (MIR), results are from seven separate experiments. A: representative sections for Sham (left) surgery, MIR surgery, and MIR with a pretreatment with sphingosine kinase 1 (S1P) agonist FTY720 (all taken 2 h after reperfusion). **,***P < 0.05 vs. Sham and column 3. B: representative H&E staining of mid-jejunal portions of intestine taken after sham surgery, and 2 h of reperfusion. Mesenteric ischemia-reperfusion (MIR), center, demonstrates significant mucosal sloughing compared with sham surgery (left) that improves with pretreatment with S1P agonist FTY720. Scale bar depicts 50 µm. C: changes in gut permeability to FITC dextran; this was given orally and collected in blood after conclusion of the experiment for seven separate experiments.

DISCUSSION

S1P has been shown to regulate cell proliferation, paracellular permeability, and susceptibility to apoptosis (4, 5). Our previous studies (2, 6) demonstrated that increased S1P, seen in intestinal epithelial cells overexpressing its rate-limiting enzyme sphingosine kinase 1, showed increased proliferation and increased levels of c-myc. However, little was known about the exact mechanism that led to the c-myc increase.

The present study demonstrates that IECs overexpressing sphingosine kinase 1 or S1P itself shows decreased expression of miR-542-5p expression, which in turn is a direct regulator of c-myc expression. This regulation occurs at the translational level, as miR-542-5p directly binds to the c-myc mRNA. Further experiments showed that S1P analog FTY720 was able to successfully mitigate damage to in vivo intestinal epithelia induced by mesenteric ischemia. Intestine demonstrating greater damage showed increased expression of miR-542-5p than those that recovered. These findings provide a novel understanding of miR-542-5p and its interaction with normal physiological function of intestinal mucosal renewal.

S1P is a bioactive entity that is ubiquitous, as it is created from the catabolism of eukaryotic cell membranes (21, 22) and also created from dietary sphingomyelin (23). S1P has been shown to be a potent regulator of cell proliferation and survival, and S1P itself is under tight regulation, as are the enzymes that help regulate its concentration within the cytoplasm (4). S1P enacts its effects in part through several G-protein cell surface receptors (5) and also through direct intracellular signaling pathways that are not as well characterized (24). S1P has been shown in several non-intestinal organ systems to govern its proliferative effects through regulation of growth-related genes (5). c-myc is one such growth-related gene and is known to have trophic effects on proliferation, differentiation, and resistance to apoptosis among other effects (25, 26) and our prior studies showed that intestinal epithelial cells with increased S1P would demonstrate increased c-myc expression (6).

A significant finding from this study was the regulation of cellular proliferation through growth-related genes in intestinal epithelial cells through expression of miR-542-5p. MiR-542-5p has been shown in recent studies to play an integral role as a tumor suppressor in lung cancer cells (10), where downregulation of miR-542-5p was associated with vascular invasion and lymphatic metastases, and conversely higher expression carried a better prognosis. In another study, miR-542-5p decreased cell invasiveness without affecting proliferation (11), although a precise mechanism was not known. The results reported here are the first to demonstrate that miR-542-5p functions as a biological repressor of normal physiologic growth, and from this study the intestinal mucosa.

Another significant finding is that miR-542-5p is dependent on expression of S1P. The studies on miR-542-5p in other tumor lines did not offer a clear mechanism for its regulation. It is noteworthy that S1P has been shown to be involved in the mechanism of miR-542-3p regulation of breast cancer cells (27). Nevertheless, this study in intestinal epithelial cells demonstrates the relation of miR-542-5p to S1P activity and a direct interaction with growth-related genes.

Our prior studies reported on S1P-related effects on intestinal mucosa have been through in vitro studies in cultured IEC-6 cells. Here, we present evidence of S1P also promoting intestinal epithelial barrier function in vivo. This study clearly demonstrates the dramatic effect on the murine intestinal barrier due to mesenteric ischemia-reperfusion, and that therapeutic provision of S1P analog FTY720 ameliorates this effect. Though this analog does not completely prevent damage from MIR, the effect is significant and a major finding of this study. Though the mechanism is not completely understood, and further studies are ongoing to evaluate this, it is notable as it parallels the effects of previous studies in vitro (6). FTY720 has been studied as a model of S1P upregulation and has been shown as a promising model of therapeutic benefit through its upregulation of S1P activity, and is in advanced clinical studies for the treatment of multiple sclerosis, myasthenia gravis, and rheumatoid arthritis (28, 29). Several studies have shown that upregulation of S1P can demonstrate improved survival from ischemia-reperfusion insults, most notably in cardiac tissue through antagonism to sphingosine lyase, which eliminates S1P (29). It is important to note that FTY720 is not an exact replication of S1P and that many of its described effects are via agonism at the S1P receptors, generally all five except S1P2 (29). S1P itself has demonstrated effects both intracellularly and agonistically against the S1P receptors (6). Nevertheless, we found that FTY720 demonstrated protective effects in parallel to those we have seen with S1P.

An important finding of the in vivo studies is that the damage to the intestinal villi correlated with the miR-542-5p levels, which the work described herein would attribute to effects from the S1P analog that was provided. The effects of miR-542-5p have been shown to be associated with damage and decreased growth and proliferation, albeit prior studies reported have primarily been in cancers. This study shows that miR-542-5p is involved in normal intestinal growth and is associated with physiological stress via mesenteric ischemia reperfusion. Its parallel to S1P activity provides some hope for a therapeutic action for recovery from mesenteric ischemia.

DATA AVAILABILITY

Data will be made available upon reasonable request.

GRANTS

This work was supported by Merit Review Awards from the US Department of Veterans Affairs (to D. J. Turner, J. N. Rao, and J. -Y. Wang) and from the National Institutes of Health under Grants DK057819, DK061972, and DK068491 (to J. -Y. Wang).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.N.R., J.-Y.W., and D.J.T. conceived and designed research; R.L., A.D.S., and H.K.C. performed experiments; R.L., J.N.R., A.D.S., L.X., J.-Y.W., and D.J.T. analyzed data; J.N.R., A.D.S., H.K.C., L.X., and D.J.T. interpreted results of experiments; R.L. and D.J.T. prepared figures; D.J.T. drafted manuscript; J.N.R., J.-Y.W., and D.J.T. edited and revised manuscript; J.N.R., J.-Y.W., and D.J.T. approved final version of manuscript.