Biotin Deficiency Alters the Expression Profile of Colonic microRNAs: Possible Contribution to the Alterations in Expression of Proteins Involved in the Maintenance of Colonic Physiology and Inflammation

TL;DR

Biotin deficiency changes microRNA levels in the colon, which may affect proteins important for gut health and inflammation.

Contribution

This study identifies specific microRNAs altered by biotin deficiency and links them to key proteins in colonic function and inflammation.

Findings

Biotin deficiency alters expression of 26 colonic miRNAs, with ten validated for roles in gut physiology and inflammation.

Altered miRNAs (e.g., miR-190a-5p and miR-199a-5p) target proteins like ZO1, LGR5, NLRP3, and calprotectin, which are dysregulated in biotin deficiency.

Functional regulation of miRNA targets was confirmed in human colonic cells, showing reduced mRNA levels after miRNA transfection.

Abstract

Background/Objectives: Biotin plays important roles in critical metabolic reactions and also contributes to the regulation of gene expression. While its role in regulating gene expression via transcriptional/epigenetic mechanisms is well established, little is known about its ability to regulate expression at the post-transcriptional level. Methods: To address this, we examined how biotin deficiency affects microRNAs (miRNAs) expression in the colon, a tissue that is impacted by deficiency of this micronutrient. Results: We identified (by miRNA sequencing) 26 miRNAs whose expression was significantly altered in the colon of biotin-deficient mice compared with pair-fed controls. Among these, ten miRNAs with known roles in mucosal physiology and inflammation were selected for direct validation, and their altered expression patterns were confirmed by RT-qPCR. In silico analyses further revealed that important proteins involved in maintaining normal colonic function (the tight junction protein ZO1 and the stem cell marker LGR5) and in mediating inflammation (NLRP3 and calprotectin), all of which are dysregulated in biotin deficiency, possess putative binding sites on 3′-UTR for several of the altered miRNAs. Moreover, transient transfection of miR-190a-5p and miR-199a-5p, whose expression was upregulated during biotin deficiency and predicted to target ZO1 and LGR5, respectively, in human colonic NCM460 cells, led to a significant reduction in the level of the corresponding mRNAs, confirming functional regulation of these targets. IPA further showed that the differentially expressed miRNAs are associated with gastrointestinal and inflammatory disease pathways. Conclusions: Findings of this investigation show that biotin deficiency disrupts colonic miRNA expression, potentially contributing to downstream alterations in important physiological and inflammatory protein expression.

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1. Introduction

Biotin (vitamin B7), a member of the water-soluble family of vitamins, is an indispensable micronutrient for normal human health due to the critical roles it plays in a variety of essential cellular processes [1]. This includes normal functions of mitochondria [2] and the immune system [3,4,5,6], as well as the regulation of gene expression [7,8,9,10] and the level of cellular oxidative stress [11,12]. Humans/mammals cannot synthesize biotin; rather, they obtain micronutrients from exogenous sources via absorption in the intestinal tract. Two sources of biotin are available to the human/mammalian host: dietary (absorbed in the small intestine) and gut microbiota (absorbed in the large intestine; [13,14,15]). In both regions of the gut, absorption of biotin occurs via a sodium-dependent carrier-mediated process that involves the sodium-dependent multi-vitamin transporter (SMVT; product of the SLC5A6 gene; [1,15,16,17,18]).

Deficiency and sub-optimal levels of biotin have been recognized with increased frequency in recent years and occur in a variety of conditions that include inflammatory bowel diseases (IBD) [19,20], chronic alcoholism [21], inborn errors of biotin metabolism and biotin membrane transport [22,23,24,25], as well as severe obesity [26], during pregnancy [27], and following long-term use of certain anticonvulsant drugs [28,29]. Such a deficiency leads to a variety of clinical abnormalities that include immunodeficiency, failure-to-thrive, and cutaneous and neurological features [6,30]. In addition to the above well-described consequences of biotin deficiency, recent preclinical investigations by our group have further expanded its pathological profile. We have shown that biotin deficiency in mice, whether induced by specific deletion of the intestinal SMVT system or by feeding the animals a biotin-deficient diet, leads to the development of severe chronic gut inflammation [31,32,33,34,35,36,37]. This inflammation is accompanied by marked histological abnormalities, particularly in the colon, that closely resemble the pathological changes observed in human ulcerative colitis [31,32,33,34,35,36,37]. The inflammation was associated with several notable molecular and physiological changes that include: (i) An increase in gut permeability accompanied by altered expression of key tight junction (TJ) proteins, including zonula occludens-1 (ZO1) and claudin-1/2 [32,33,34,35,36]; (ii) A decrease in the expression of the colonic stem cell marker LGR5 [35]; (iii) An increase in the level of expression of calprotectin (S100a8/S100a9), a well-established marker of intestinal inflammation and neutrophil infiltration, along with elevated levels of proinflammatory cytokines such as TNF-α, IL-1β, IL-6, and IFN-γ [31,32,36,38]; (iv) Activation of inflammasomes, particularly NLRP3, was evident [32,35]; and (v) An increase in the level of expression of the nuclear factor NF-κB, a major proinflammatory signaling pathway that regulates the transcription of cytokines including TNF-α, IL-6, IFN-γ, and the NLRP3 inflammasome [32,35,36,39,40]. Finally, we found the inflammation to be associated with gut microbial dysbiosis [32,37]. As to the histological abnormalities, they include focal cryptitis/crypt abscesses, focal low-grade adenomatous changes, and extensive submucosal edema [31]. Interestingly, all these abnormalities/changes were found to be significantly attenuated/normalized following oral supplementation of the affected animals with biotin [33,36].

From the above, it becomes clear that biotin plays an important role in regulating the expression of a variety of genes in mammalian cells [3,4,5,6,34,36]. To date, however, most research has focused on delineating biotin’s role in modulating gene expression at the transcriptional and epigenetic levels [3,4,5,6]. Much less attention has been given to its possible role in regulating gene expression at the post-transcriptional level. Such a mode of gene regulation has been recently described in the case of several other micronutrients [41,42,43,44,45,46]. Our aim in this study was to examine the effect of biotin deficiency on the expression profile of miRNAs in the mouse colon.

miRNAs are small non-coding RNAs (around 18–23 nucleotides) that exert significant regulatory effects on gene expression via base-pairing to complementary sequences (mainly) in the 3′-untranslated region (3′-UTR) of their target mRNAs that leads to their degradation and/or suppression in their translation [47,48,49]. Thus, miRNAs can negatively regulate the levels of expression of proteins when their own level of expression is induced and positively regulate when their levels are suppressed [50]. In addition, the expression of miRNAs is tissue-specific [50,51], and a single miRNA can regulate numerous protein-coding target mRNAs. To achieve our aim, we induced biotin deficiency in mice via dietary means, then examined changes in the pattern of expression of miRNAs in their colon compared to pair-fed controls. The results showed that biotin deficiency leads to significant alterations in the miRNA expression profile in the colon compared to that of controls. Further, we found that the 3′-UTR of proteins that play important roles in the maintenance of normal colonic physiology (e.g., ZO1 and LGR5) and those that play roles in mucosal inflammation (e.g., NLRP3 and calprotectin) and whose levels were found in previous studies from our laboratory to be altered in biotin deficiency [32,33,34,35,36] have putative interacting sites with a number of the altered miRNAs. Furthermore, transient transfection of mimic miR-190a-5p and miR-199a-5p, microRNAs that were predicted to target ZO1 and LGR5, respectively, into human colonic epithelial NCM460 cells resulted in a significant reduction in levels of the corresponding mRNAs. These findings demonstrate functional regulation of these targets by altered miRNAs. Finally, the results of the IPA analysis showed that a few of the differentially expressed miRNAs in biotin deficiency were associated with gastrointestinal and inflammatory diseases.

2. Materials and Methods

2.1. Induction of Biotin Deficiency

Dietary-induced biotin deficiency was established in mice as described previously [34,36]. Briefly, 12 pairs of age-matched 4-week-old male C57BL/6J mice (Jackson Laboratories; Ellsworth, ME, USA) were used and were divided into biotin-deficient and pair-fed control groups. Both groups of mice were housed individually in a cage at the animal facility of the University of California-Irvine (UCI), and all Institutional Laboratory Animal Guidelines were followed. The biotin-deficient group was allowed free access to a biotin-deficient diet that contains 30% egg white (TD.81079, Envigo, Indianapolis, IN, USA), while the control group was pair-fed the same diet but with supplemented biotin (0.004 g biotin/kg) (TD.97126, Envigo, IN, USA). For pair-feeding, caloric intake was strictly matched between the biotin-deficient and control groups based on daily food consumption, and body weight was also monitored throughout the experimental period to ensure comparable nutritional status between the two groups. Mice were maintained on these diets for 16 weeks, then used in the described studies. The colon and small intestinal tissue samples were collected from both biotin-deficient and their pair-fed control mice immediately after euthanasia by a standard carbon dioxide inhalation procedure followed by cervical dislocation. All experiments were performed according to the Guideline for Animal Care and Use Committee of the University of California-Irvine (approval number #AUP-23-117).

2.2. Determination of Biotin Status

Biotin levels in mice that were fed the biotin-deficient diet and their pair-fed controls were assessed by measuring the total level of biotinylated proteins in their livers using Western blot analysis, as described previously [34,36]. In brief, mice liver total proteins were run on a 4–12% NuPAGE gel electrophoresis, followed by western blotting using a PVDF membrane that was initially incubated with anti-β-actin antibodies. This was followed by incubation with anti-mouse IR 680 dye (LI-COR) for β-actin and with anti-avidin IR 800 dye (LI-COR) for biotinylated proteins.

2.3. RNA Isolation

Total RNA, including miRNAs, was extracted from intestinal tissues (i.e., colon and small intestine) of biotin-deficient and pair-fed control mice using the Qiagen miRNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The concentration of total RNA was measured and immediately frozen and stored at −80 °C. The extracted RNA was used for miRNA sequencing analysis and subsequent validation via RT-qPCR.

2.4. Preparation of the miRNA Library, Next-Generation Sequencing (NGS), and Data Analysis

The isolated intestinal RNA from both biotin-deficient and control mice was processed by Qiagen Genomic Services (Frederick, MA, USA) for library preparation, NGS, and data analysis. The RNA quality was assessed before and after library preparation using Bioanalyzer 2100 and TapeStation 4200 (Agilent, Santa Clara, CA, USA). The miRNA sequencing libraries were prepared using 100 ng total RNA with the QIAseq miRNA Library Kit from Qiagen (Venlo, The Netherlands). To introduce adapters containing unique molecular indices (UMIs) during the reverse transcription step, the cDNA was amplified using PCR (22 cycles) and then sequenced using the NextSeq 500 (Illumina, San Diego, CA, USA) per the manufacturer’s instructions. The obtained raw data were then converted to FASTQ files using the bcl2fastq software, and the reads with identical UMIs were collapsed into a single read and aligned to miRBase using Bowtie2 to identify known miRNAs in mice and to generate miRNA counts. For data analysis, we used GRCm38 version 98 (mm10) as the reference mouse genome and miRBase_20 as the annotation reference. Differential expression analyses of miRNAs were performed using the EdgeR Bioconductor package [52]. For normalization, the trimmed mean of the M value method based on log-fold and absolute gene-wise changes in expression levels between samples (TMM normalization) was used.

2.5. Validation of the Differentially Expressed miRNA by RT-qPCR

For this, the cDNA was transcribed from 0.2 µg of each total intestinal total RNA isolated from biotin-deficient and paired-fed controls using the miRCURY LNA RT Kit (Qiagen) according to the manufacturer’s instructions and stored at −20 °C in 1:10-diluted aliquots. The relative expression of selective miRNAs was then analyzed following the manufacturer’s instructions using the miRCURY LNA SYBR Green PCR kit [53] and specific miRCURY LNA miRNA PCR assay primers (both QIAGEN, Hilden, Germany). Thermal cycling was performed on a CFX96 Touch Real-Time PCR detection system (BioRad, Munich, Germany). The cycling conditions included an initial heat activation at 95 °C for 2 min, followed by 45 cycles at 95 °C for 10 s and 56 °C for 60 s. RT-qPCR reactions were performed in duplicates of four biological replicates in each of the biotin-deficient and paired-fed control groups. Relative changes of miRNAs were calculated using a 2^−∆ΔCt^ equation using the expression level of RNU1A1 and UniSp6 as an internal control.

2.6. Cell Culture, Transient Transfection with miRNA Mimics, and Validation of the Effects of miRNAs on the Level of Expression of Selected Targets

Human-derived colonic epithelial NCM460 cells were grown in Dulbecco’s modified Eagle medium (DMEM; Gibco, Waltham, MA, USA) with 20% (vol/vol) FBS and supplemented with streptomycin (100 μg/mL) and penicillin (100 U/mL) at 37 °C in 5% CO_2_—95% air environment in T-75 plastic flasks. Transient transfection of NCM460 cells was done using 200 nM miRNA mimic miR-190a-5p (MCE MedChemExpress, Cat# HY-R00361) and miR-199a-5p (MCE MedChemExpress, Cat# HY-R00399) with appropriate negative control miRNA mimic (MCE MedChemExpress, Cat# HY-R04602) for 48 h, using RNAiMax (Cat# 13778150) transfection reagent to achieve maximum transfection efficiency (Invitrogen, Carlsbad, CA, USA). After 48 h of post-transfection, the total RNA was isolated from the cells by using QIAzol reagent (QIAGEN) and RNeasy Kit (QIAGEN) following the manufacturer’s instructions. The total RNA (0.5 μg) was then converted to complementary DNA (cDNA) using the Verso-cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) and then subjected to RT-qPCR analysis in a CFX96 real-time system (Bio-Rad, Hercules, CA, USA) using iQ SYBR Green Supermix (Bio-Rad) using gene-specific primers as follows:

Human ZO1: Forward primer 5′-AACCCAGCATCATCAACC TC-3′; Reverse primer 5′-ATCTACATGCGACAATGATG-3′; Human LGR5: Forward primer 5′-GAGTTACGTCTTGCGGGAAAC-3′; Reverse primer 5′-TGGGTACGTGTCTTAGCTGATTA-3′

Finally, relative mRNA expression was calculated using the 2^−ΔΔCt^ method using the expression level of β-actin as an internal control.

2.7. Statistical Analysis

Statistical analysis of each of the RT-qPCR data was performed by Student’s t-test using the GraphPad Prism 10 statistical software (GraphPad Software, La Jolla, CA, USA). All results are presented as mean ± standard error (SE) of 4–5 individual animals and presented in the figure as percentage relative to controls using GraphPad Prism 10. A p-value < 0.05 was considered as being statistically significant. For miRNA differential expression analysis, all p-values were subjected to multiple corrections using Benjamini and Hochberg’s false discovery rate (FDR) correction method. Expression of the differentially expressed miRNAs was considered significant when the FDR-corrected p-value was less than 0.01, and the fold change was greater than 1.5. The sample size was determined a priori based on feasibility, prior experience, and consistency with previously published studies using similar experimental models. Group sizes were selected to detect biologically meaningful differences while minimizing variability and unnecessary use of samples. No formal a priori power calculation was performed.

3. Results

3.1. Verification of Biotin Deficiency and Associated Phenotypes in Mice

As described in “Methods”, biotin deficiency was induced by feeding mice (for 16 weeks) a biotin-deficient diet [34,36]. Control animals were pair-fed the same diet supplemented with biotin. All biotin-deficient mice developed the hallmark symptoms of biotin deficiency, which include alopecia, perioral dermatitis, and reduced growth rates [34,36]; no such symptoms were observed in the pair-fed control group (Figure 1A). To further confirm biotin status, we measured hepatic biotinylated protein levels (via Western blot analysis) in the group that was fed a biotin-deficient diet and the pair-fed group, with the results showing a significantly (p < 0.01) lower level in the former compared to the latter group (Figure 1B).

3.2. Differential Expression of miRNAs in the Colon of the Biotin-Deficient Mice

In this study, we examined the effect of biotin deficiency on the miRNA expression profile in the mouse colon. Our focus on colonic tissue is based on previous findings from our laboratory showing that this tissue is particularly susceptible to the deleterious effects of biotin deficiency [34,35,36]. For this, we extracted total RNA (including miRNAs) from the colons of biotin-deficient and control pair-fed mice and subjected the samples to miRNA sequencing (see Section 2). The results (based on an FDR-adjusted p-value of <0.01 and a fold change of >1.5) showed a total of 26 miRNAs that were differentially expressed in the colon of the biotin-deficient mice compared to controls (Figure 2 and Table 1), with 19 miRNAs being upregulated and 7 being downregulated.

In a related study, we also assessed the effect of biotin deficiency on miRNA expression profile in the small intestine, a region that is relatively less affected by biotin deficiency [31,32]. The results here showed that expression of only four miRNAs was significantly altered in biotin-deficient mice compared to pair-fed controls (FDR-adjusted p-value < 0.01, fold change > 1.5), with one being upregulated and three being downregulated (Figure 2 and Table 1). [Note: Group-specific miRNA signal intensities, along with associated log2 ratios and fold changes in both intestinal regions, are detailed in Table 1. miRNAs with log2 fold change ≥ 0.5 were categorized as significantly upregulated; those with log2 fold change ≤ 0.5 were considered significantly downregulated.]

3.3. Validating the Observed Changes in Levels of Expression of Selected miRNAs in Biotin Deficiency

In this study, we aimed to validate (by means of RT-qPCR) the changes in levels of expression of selected miRNAs in the colon of biotin-deficient animals that were observed in the above-described miRNA sequencing analysis. We specifically focused on miRNAs that are known to play roles in normal colonic physiology (e.g., regulating expression of tight junction proteins), and in intestinal inflammation [54,55,56,57]. Eight miRNAs from the colon (namely: mmu-miR-126a-5p, mmu-miR-126a-3p, mmu-miR-199a-5p, mmu-miR-199a-3p, mmu-miR-7b-5p, mmu-miR-21a-3p, mmu-miR-21a-5p, and mmu-miR-34a-5p) and two from the small intestine (namely: mmu-miR-146a-5p and mmu-miR-142a-3p) were selected for this purpose. Results of the RT-qPCR analysis confirmed that the levels of mmu-miR-126a-5p, mmu-miR-126a-3p, mmu-miR-199a-5p, mmu-miR-199a-3p, mmu-miR-7b-5p, mmu-miR-21a-3p, and mmu-miR-21a-5p were significantly upregulated, while those of mmu-miR-34a-5p, mmu-miR-146a-5p, and mmu-miR-142a-3p were significantly downregulated in biotin-deficient mice compared to pair-fed controls (Figure 3).

3.4. Identification of Putative miRNA Binding Sites in the 3′-UTR of Selected Colonic Proteins Whose Expressions Are Known to Be Altered in Biotin Deficiency

In this study, we aimed at determining if the 3′-UTR regions of selected colonic proteins possess putative binding sites for the above-described altered miRNAs in biotin deficiency. We focused on proteins that play important roles in colonic physiology (e.g., ZO1 and LGR5) and inflammation (e.g., NLRP3 inflammasomes and calprotectin) and whose levels were previously shown to be significantly altered in biotin deficiency [31,32,33,34,35,36]. For this, we subjected the 3′-UTR regions of the selected proteins to TargetScan analysis, with the results showing that these regions indeed process such putative miRNA-binding sites (Table 2). Specifically, the 3′-UTR of the tight junction protein ZO1 (whose level is suppressed in biotin deficiency [34]) was found to possess multiple putative binding sites for miRNAs that are induced in biotin deficiency (namely, mmu-miR-190a-5p, mmu-miR-802-5p, and mmu-miR-7b-5p) (Table 2). Similarly, the 3′-UTR of the colonic stem cell marker LGR5 (whose level is suppressed in biotin deficiency [35]) was found to have a putative binding site for the induced mmu-miR-199a-5p. In contrast, the 3′-UTR of the colonic inflammatory protein, NLRP3 inflammasome (whose level is induced in biotin deficiency [32,35], was found to have a putative binding site for miRNA mmu-miR-34a-5p that is suppressed in biotin deficiency (Table 2). Similarly, the 3′-UTR of calprotectin (i.e., S100a9) was found to have a putative site for miRNA mmu-miR-211-5p that is also suppressed in biotin deficiency (Table 2).

3.5. Altered Levels of Expression of Colonic miRNAs in Biotin Deficiency Impact the Level of Expression of Their Target mRNAs

In these studies, we examined whether alterations in levels of expression of colonic miRNAs in biotin deficiency impact the levels of expression of their target mRNAs. We focused on examining the effects of miR-190a-5p and miR-199a-5p miRNAs since levels of these miRNAs are induced in the colon of biotin-deficient mice, and since putative binding sites for these miRNAs exist in the 3′-UTR regions of two physiologically important target mRNAs, namely that of the tight junction protein ZO1 and that of the stem cell marker LGR5, respectively. In this study, we transiently transfected a mimic of miR-190a-5p (200 nM) and miR-199a-5p (200 nM) into human colonic epithelial NCM460 cells, followed (48 h later) by examining (by RT-qPCR) the effect on the level of expression of ZO1 and LGR5 mRNAs. The results showed a significant (p < 0.01 for both) reduction in the level of mRNA expression of both ZO1 and LGR5 in cells transfected with the mimic of miR-190a-5p and miR-199a-5p, respectively, compared with cells transfected with a negative control mimic (Figure 4A,B).

3.6. Pathway and Disease-Association Analysis of Differentially Expressed miRNAs

In this study, we aimed at exploring the functional relevance of the differentially expressed miRNAs in the colon of biotin-deficient mice. For this, we performed Ingenuity Pathway Analysis (IPA) on the sequencing data, with the results showing that the altered miRNAs are significantly associated with various diseases and functional categories, including gastrointestinal, inflammatory, immunological, developmental, infectious, and neurological disorders (Table 3 and Figure 5). Additionally, molecular and cellular function analysis identified the cell cycle as a significantly impacted process in colonic tissue in biotin deficiency (p = 0.0476) (Table 3).

4. Discussion

A role for the water-soluble vitamin B7 in regulating gene expression at the transcriptional level has been well established [3,31,32,33,34,35,36,37,58], but its ability to influence expression at the post-transcriptional level is not clear. We examined this issue in this study by investigating the effect of biotin deficiency on the expression profiles of miRNAs in mice. We focused our investigations on the colon since previous studies from our laboratory have shown that biotin deficiency markedly impacts colonic physiology and permeability; it also leads to the development of active mucosal inflammation [3,31,32,33,34,35,36,37,58]. Our findings provide clear evidence that biotin does indeed possess the capability to also affect gene expression at the post-transcriptional level via modulating the levels of expression of miRNAs [7,8,9,10].

Results of the colonic miRNA sequencing showed that biotin efficiency leads to significant alterations in the expression profile of 26 miRNAs compared to the colon of pair-fed controls, with 19 miRNAs being upregulated and 7 being downregulated. Confirmation of these changes in expression of a number of these miRNAs was also done by RT-qPCR, focusing on those that are known to play roles in colonic physiology and inflammation. In analyzing the differentially expressed miRNAs in the colon of biotin-deficient mice, it was found that a number of these miRNAs are involved in post-transcriptional regulation of expression of proteins that are known to play important roles in maintaining epithelial integrity as well as in mucosal inflammation/immune regulation [54,55,56,57]. Examples are miR-126a-5p, miR-21a-3p, and miR-199a-5p, whose levels are induced in the colon of biotin-deficient mice and which have been previously shown to play roles in modulation expression of tight junction proteins and pro-inflammatory cytokines [54,55,56,57]. In contrast to the number of miRNAs whose level of expression is altered in the colon in biotin-deficient mice, expression of only four miRNAs was found to be significantly altered in the small intestine in these animals. This disparity aligns with previous reports from our group and others showing that the colon is more adversely affected by biotin deficiency [31,32,33,34,35,36,37]. The greater sensitivity of the colonic mucosa is likely related to the unique immunology and physiology of the lining mucosa as well as the unique luminal environment in the large intestine [59].

In the study to identify important colonic proteins that are potential targets for action of the altered miRNAs in biotin deficiency, we subjected the 3′-UTRs of selected proteins (those that play important roles in gut physiology and inflammation, namely, the tight junction protein ZO1, stem cell marker LGR5, NLRP3 inflammasome, and calprotectin [32,33,34,35,36,38] to TargetScan analysis to determine if these regions have putative interacting sites with the altered miRNAs. Indeed, the 3′-UTR of ZO1 (a protein whose level is significantly suppressed in biotin deficiency [32,33,34,35,36] was found to have several putative interacting sites with the biotin-deficiency-induced miRNAs mmu-miR-190a-5p, mmu-miR-802-5p, and mmu-miR-7b-5p. Similarly, the 3′-UTR of LGR5 (whose level is also significantly inhibited in biotin deficiency [35]) was found to have a putative binding site for the biotin-deficiency-induced miRNA mmu-miR-199a-5p. In contrast, the 3′-UTR of NLRP3 and calprotectin (whose levels are significantly induced in the colon in biotin deficiency [32,35]) were found to have putative binding sites for miRNAs mmu-miR-34a-5p and mmu-miR-211-5p, respectively, whose levels are significantly suppressed in biotin deficiency. Collectively, these results indicate that biotin deficiency induces a coordinated miRNA-mRNA regulatory network that contributes to epithelial barrier dysfunction and colonic inflammation.

It is also important to note that the biotin deficiency induced alterations in expression of important inflammatory protein markers in the mouse colon and that these alterations are significantly attenuated/fully normalized following supplementing biotin to the affected animals [33,36]. This observation raises an important question for future research: Are the beneficial effects of biotin supplementation on gut inflammation mediated (in part) via modulation in the level of expression of specific miRNAs? Addressing this issue would further strengthen the translational significance of the current findings and clarify whether miRNA-dependent mechanisms contribute to biotin’s protective actions in the colon.

Our studies also demonstrated that the altered levels of expression of colonic miRNAs in biotin deficiency can indeed impact the level of expression of their target mRNAs. This was demonstrated in the case of miR-190a-5p and miR-199a-5p, whose levels are induced in biotin deficiency and which have putative binding sites in the 3′-UTR of two physiologically important colonic proteins, namely ZO1 and LGR5, respectively. The latter was shown via examining the effects of transfecting human colonic epithelial cells with miRNA mimics of miR-190a-5p and miR-199a-5p on the level of expression of ZO1 and LGR5 mRNAs, with the results showing a significant reduction in their levels in the miR-190a-5p and miR-199a-5p mimic-transfected cells compared to those transfected with the negative control. These findings demonstrate that the biotin-responsive miRNAs can indeed exert regulatory effects on key colonic epithelial targets. Future studies on the effects of the other miRNAs whose expression is altered in biotin deficiency are needed, including examining the effects of overexpressing and knocking down these miRNAs as well as performing 3′-UTR-luciferase reporter assays, coupled with analyses of protein levels of their targets.

In addition to the above, results of the IPA further revealed that the differentially expressed miRNAs are associated with the biological processes and disease categories linked to gastrointestinal inflammation, immune dysregulation, and cellular stress responses [54,55,56,57]. Furthermore, the enrichment of pathways related to cell cycle control and inflammatory diseases suggests a greater role of miRNA dysregulation in shaping mucosal vulnerability. These findings raise the possibility that impaired biotin status not only disrupts local nutrient signaling but may also play a role in reprogramming the regulatory networks that drive the onset and/or progression of inflammation.

5. Conclusions

In conclusion, our study shows for the first time that biotin deficiency leads to significant changes in the pattern of the gut miRNA expression profile. Additionally, the study reveals a novel post-transcriptional mechanism through which biotin influences colonic physiology and inflammatory status.