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MiR-1-3p and MiR-124-3p Synergistically Damage the Intestinal Barrier in the Ageing Colon

Abstract Background and Aims Disruption of the intestinal barrier of the digestive tract is a common pathophysiological change in the elderly, which may partly contribute to gut dysfunction and inflammatory bowel disease [IBD]. This study aimed to discover new interactive epigenetic regulation patterns involved in intestinal barrier dysfunction and colitis in elderly populations. Methods Intestinal barrier function and structure were evaluated in naturally ageing mice and elderly people. High-throughput analysis was performed on colonic tissues from humans and mice. The synergistic roles of miR-1-3p and miR-124-3p were identified using microRNA mimic/agomirs. Related genes were examined in biopsies of old IBD patients. Results A defective mucus barrier was observed before mucosal microstructural damage during ageing. Elevated miR-1-3p expression in the colons of older individuals impaired the mucus barrier by directly targeting T-synthase, similarly to the mechanism of miR-124-3p, which we reported previously. Importantly, the synergistic effect of a half dose of each microRNA supplement on T-synthase and CDK4/6 was stronger than that of a full dose of miR-1-3p or miR-124-3p alone, and mice co-treated with two microRNAs showed greater susceptibility to chemical-induced colitis than mice treated with either microRNA alone. These two microRNAs were up-expressed in old IBD patients. Conclusions The slight increases in miR-1-3p and miR-124-3p expression with ageing may be important contributors to the breakdown of intestinal homeostasis by targeting divergent genes in different cells. These data reveal the potential ability of multiple microRNAs to exert synergistic effects to damage the intestinal barrier and promote inflammatory bowel disease development in elderly populations.


Introduction
Inflammatory bowel disease [IBD], which includes ulcerative colitis [UC] and Crohn's disease [CD], is a group of chronic, recurrent, incurable intestinal diseases that cause severe inflammation and ulceration of the gastrointestinal [GI] tract. As the global prevalence of IBD has continued to increase in recent years, IBD has imposed a significant financial and resource burden on health care systems. 1,2 With the rapid increase in the ageing population throughout the world [according to the United Nations 2019 Revision of World Population Prospects], the elderly population is the fastest-growing subpopulation of people with IBD, due to both the ageing of previously diagnosed patients and the diagnosis of new cases in this subpopulation. 3,4 Elucidating the precise aetiology of GI inflammation, particularly in IBD, in elderly populations is important for addressing the challenge of treating the increased number of ageing patients and to help achieve gradual healthy ageing.
IBD is a multifactorial disease involving host genetic susceptibility, environmental triggers, immune dysregulation, and altered intestinal microbiota homeostasis, the last of which is partly influenced by dynamic cross-talk between the gut microbiota and intestinal barrier. 5,6 Generally, the normal intestinal barrier consists of multiple layers, including: [1] the micro-ecological barrier; [2] the mucus gel layer; [3] intestinal epithelial cells [IECs] that form a single-cell layer of physical protection; and [4] the immune barrier. 7 Accumulating experimental evidence indicates a crucial role of intestinal barrier dysfunction in the onset of IBD, suggesting that such dysfunction is a potential predisposing factor for IBD. 8,9 As one of the hallmarks of health, 10 the intestinal barrier is progressively damaged during senescence. 11 Our recent work 12 demonstrated that age-related changes in mucus properties increase the susceptibility to colitis in elderly populations. However, research on the underlying regulatory mechanism is far from comprehensive.
Broadly, microRNAs [miRNAs] are believed to be among the key molecular regulators of gut homeostasis. Abnormal miRNA expression and function appear to be pervasive features of almost all related diseases. 13,14 Evidence regarding the mechanisms by which specific miRNAs control the progression of digestive tract inflammation and tumour has emerged from many studies, showing that the concerted targeting of multiple genes by a single miRNA is responsible for dramatic effects. However, the interaction and mode of action of different miRNAs in GI diseases remain unclear. Here, we identified a set of miRNAs, miR-1-3p and miR-124-3p, whose expression was mildly upregulated in the ageing distal colonic mucosa. These miRNAs convergently target T-synthase and cyclin-dependent kinase [CDK] 4 and CDK6, and comprise a cooperative miRNA network that impairs intestinal barrier integrity. Our study may provide new research targets for both delaying GI senescence and ameliorating IBD symptoms.

Animals
Male C57BL/6 mice were purchased from Capital Medical University and divided into three groups of 10 each according to age, i.e., 2, 16, and 24 months [mo], to simulate the life phases at approximately 20, 50, and 70 years of age in humans. 15 An additional 30 male mice [aged 2 mo] were used to establish the miR-1-3p agomir treatment model [n = 5 per group] and the dextran sulphate sodium [DSS]-induced colitis model [n = 5 per group]. All mice were housed in a controlled environment under a 12-h light/dark cycle at 22 ± 2 ℃ and 55 ± 5% humidity, with ad libitum access to food and water, and were sacrificed by appropriate anaesthesia and/or cervical dislocation before sample collection. Colon and liver tissues were surgically and aseptically removed from the mice.

DSS-induced colitis model
The procedure used to generate the DSS colitis model is shown in Supplementary Figure 1A. Our published data 12 demonstrated that miR-1-3p expression in the distal colon mucosa of 24-mo-old mice was 1.6 times higher than that of miR-124-3p. These previous data provide a reference for the miRNA agomir dose required in the animal model. Four groups of young mice [aged 2 mo, n = 5 per group] received tail vein injections of: [1]

Statistical analysis
All quantitative data are presented as the mean ± standard deviation [SD]. We analysed the normality of the data distribution and homogeneity of variance using Shapiro-Wilk's and Levene's test, respectively. Statistical differences were analysed using Student's t test [

Distal colons show hyperpermeability and impaired epithelial barrier integrity with age
Our previous data showed that the mouse colon exhibits age-related microbiota dysbiosis and a breached inner mucus layer. 12 In this study, we measured mouse gut permeability in vivo to further assess intestinal barrier function during ageing. The guts of mice aged 16 and 24 mo showed greater permeability to fluorescein isothiocyanate [FITC]-dextran [ Figure 1A]. Considering that the epithelial barrier acts as a physical barrier, we compared the transepithelial resistance [TER] of ex vivo distal colons and paracellular permeability based on the luminal-to-serosal flux of FITC-dextran across distal colon segments from mice in different age groups. Mice aged 24 mo showed distinctly decreased TER [ Figure 1B] and markedly increased permeability [ Figure 1C]. Then, the architecture of mouse and human distal colons was examined by measuring the expression of intercellular junction proteins, such as claudin-1, claudin-2, claudin-3, occludin, zonula occludens-1 [ZO-1], and E-cadherin, by western blot analysis [ Figure 1D; Supplementary Figure 1B, C] and/or immunofluorescence [ Figure 1E, F, G; Supplementary Figure 1D, E, F]. Impaired mucosal microstructure was clearly observed in mice aged 24 mo. We also found reduced numbers of BrdU-and Ki67-positive cells within the crypts in aged mice [ Figure 1H; Supplementary Figure 1G, H, I], indicating that intestinal hyperpermeability in the colons of 24-mo old mice might partly be induced by mucosal microstructural damage and reduced epithelial self-renewal. Intriguingly, changes in intestinal epithelial microstructure appeared at 24 mo, but changes in mucus properties were observed beginning at 16 mo in the distal colon, 12 suggesting that changes in colonic mucus properties occur before those in intestinal microstructure.

MiR-1-3p expression is upregulated in the distal colon with age
Given that miRNA molecules are dysregulated in many diseases, miRNA expression in the normal distal colon mucosa from young and old humans and mice was determined by miRNA microarray analysis and miRNA sequencing, respectively.  Figure 2A. Five miRNAs [miR-29c-3p, miR-1-3p, miR-378d, miR-133a-3p, and miR-490-3p] had similar expression patterns in both humans and mice. Our previous study 12 found that T-synthase, the rate-limiting enzyme in mucin-type O-glycosylation, displays an age-related decline in expression. We integrated these five miRNAs and eight miRNAs predicted to regulate the T-synthase gene C1GALT1 in the public databases PicTar, TargetScan, and miRanda 12 and obtained one specific miRNA, miR-1-3p. We further showed that miR-1-3p expression in the distal colonic mucosa was significantly increased in old mice and elderly persons, but no noticeable differences were observed in the proximal colonic mucosa [ Figure 2B, C; Supplementary Figure 2, available as Supplementary data at ECCO-JCC online], indicative of a distinct regional expression pattern and trend opposite to that of T-synthase in the distal colon. 12 Thus, further experiments in this study focused on miR-1-3p.  12 We further demonstrated that these two miRNAs had a synergistic inhibitory effect on T-synthase expression [ Figure 4I; Supplementary Figure 4J]. HT-29 and HCT116 cells were transiently transfected with full-dose miR-1-3p mimic, full-dose miR-124-3p mimic, or half-dose miR-1-3p mimic combined with half-dose miR-124-3p mimic. The results showed that compared with the control group, the single transfected groups and the group cotransfected with miR-1-3p and miR-124-3p exhibited significantly decreased T-synthase expression levels at 72 h, but only the cotransfected cells displayed reduced T-synthase expression at 48 h, illustrating that the T-synthase level decreased earlier and more sharply in the cotransfection group than in the single transfection groups.

MiR-1-3p and miR-124-3p have synergistic inhibitory effects on epithelial cell proliferation
In addition to mucus barrier breakdown, slow epithelial cell renewal was observed in old mice [ Figure 1H]  and miR-124-3p in enterocytes by analysing the pan-proliferation marker Ki67 and BrdU by immunohistology and immunofluorescence, respectively, in the colons of agomir-treated mice. Consistent with the increased expression of miR-1-3p [ Figure 4A] and miR-124-3p, 12 reduced numbers of Ki67-and BrdU-positive cells were observed within the crypts of the distal colon of agomir-treated young mice [ Figure 5A; Supplementary Figure 5A, B, available as Supplementary data at ECCO-JCC online]. Additionally, in accordance with the diminished proliferative ability, the miR-1-3p/miR-124-3p agomir-treated population showed attenuated CDK4/6 expression [ Figure 5B; Supplementary Figure 5C, D], and these results were consistent with those in mucosal tissues from older individuals [ Figure 5C; Supplementary Figure 5E, F]. Given that miR-1-3p and miR-124-3p downregulate CDK4/6 expression, we also examined the synergistic inhibitory effect of these two miRNAs on proliferation. RKO and HCT116 cells were transfected with miR-1-3p and miR-124-3p mimic alone or together. Compared with the control, the cotransfection group showed a sharp decrease in CDK4/6 expression level [ Figure 5D; Supplementary Figure 5G, H]. Similarly, in vitro treatment with miR-1-3p and/or miR-124-3p mimic resulted in suppression of RKO [ Figure 5E, F] and HCT116 [Supplementary Figure  5I, J] cell proliferation, especially in the cotransfection group, as determined by CCK-8 assay and real-time cell analyser [RTCA] experiments. Therefore, the data provide evidence that the cotransfection of miR-1-3p and miR-124-3p downregulates CDK4/6 expression and synergistically inhibits epithelial cell proliferation.

Mice overexpressing miR-1-3p and miR-124-3p
show more severe DSS-induced colitis Our previous study indicated that the miR-124-3p induced defects in the mucus barrier increase the susceptibility to colitis. 12 Here, we observed strong collaboration between miR-1-3p and miR-124-3p in enteritis. Young mice [2 mo] were injected with miR-1-3p and miR-124-3p agomir alone or together [each at a half-dose] and then treated with DSS to induce colitis [ Supplementary Figures 1A, 6A, available as Supplementary data at ECCO-JCC online]. Compared with the mice injected with a single miRNA agomir, those injected with two miRNA agomirs were more susceptible to acute DSS-induced colitis and had much worse colonic injury. This injury was characterised by greater body weight loss [ Figure 6A], a higher disease activity index [DAI] score [ Figure 6B], greater gut permeability to FITC-dextran [ Figure 6C], and a heavier spleen [Supplementary Figure 6B] in cotreated mice than in single miRNA-treated mice, but there was no significant difference among groups in colon length [ Figure 6D; Supplementary Figure 6C]. We further found that co-injected mice developed severe colitis with higher colonic histopathology damage scores [ Figure 6E; Supplementary Figure 6D], confirming superficial inflammation. Consistent with this finding, the proliferative ability of epithelial cells in the co-injected mice was markedly decreased [ Figure 6F; Supplementary Figure 6E]. In addition, the distal colonic mucosa from the co-injection group exhibited a tendency toward lower T-synthase expression and more Tn/sTn accumulation, indicating miRNA-induced aberrant O-glycosylation [ Figure 6G; Supplementary Figure 6F]. These results were associated with reduced CDK4, CDK6, and T-synthase protein levels and elevated Tn and sTn protein levels [ Figure 6H; Supplementary Figure 6G, H, I].
These data suggested that the combination of miR-1-3p and miR-124-3p in enterocytes markedly exacerbated DSS-induced colitis. Furthermore, miR-1-3p and miR-124-3p that increased in the elderly had shown higher expression in elderly IBD patients compared with young IBD samples. Combined with decreased C1GALT1 and CDK4/6 in elderly IBD patients, these results supported the contribution of two miRNAs in IBD progression during ageing and partly explained why elder people often suffer from a relatively high risk of developing IBD [ Figure 6I; Supplementary Figure 6J-M].

Discussion
IBD is an idiopathic disease and a focus of GI research. A variety of factors have been implicated in the complex pathogenesis of IBD. It is generally thought that the intestinal barrier changes with ageing, becoming an important individual contributing factor for IBD in the elderly population. 11,21 Our study showed that intestinal permeability increases with age, adding to the evidence for the 'leaky gut' during ageing. 11 An elegant study showed that intestinal barrier dysfunction is correlated with the lifespan of Drosophila and is a more accurate predictor of age-onset mortality than chronological age, 22 which makes research on the ageing intestinal barrier more meaningful. Substantial research has shown that the intestinal epithelial apical junctional complex, which includes tight junctions and adherens junctions, contributes to intestinal barrier function via its role in regulating paracellular permeability. 23,24 In this study, intercellular junction proteins, such as claudin-1, claudin-3, ZO-1, occludin, and E-cadherin, were downregulated in the epithelium of the distal colon in 24-mo old mice, whereas claudin-2 expression was upregulated, indicating that the hyperpermeability in aged mice was at least partially due to abnormal epithelial microstructure.
Furthermore, increased IEC proliferation contributes to enhanced intestinal epithelial barrier function. 25,26 We discovered that colon epithelial cells from older individuals exhibited decreased growth compared with those from young individuals; this decreased growth will prolong the exposure to bacteria and other harmful metabolites, partially contributing to the disrupted intestinal homeostasis. In fact, changes in IEC proliferation in elderly individuals are somewhat controversial. Moorefield et al. 27 showed increased intestinal epithelial stem cell [IESC] proliferation with age, which might lead to increased DNA damage, resulting in p53-activated IESC apoptosis and decreased IESC function. However, Powell et al. 28 stated that a decline in colonic epithelial cell proliferation is not evident in geriatric mice. Another study found that p16 restraint of CDK activation is sufficient to inhibit IEC proliferation, resulting in the characteristics of ageing in somatic tissues of mammals. 29 Regardless, the intestinal epithelium is central to maintaining the peaceful coexistence of dietary components and commensals, controlling mucosal immunological homeostasis and defending against potentially harmful pathogens, and these cells are continuously replenished by multipotent IESCs residing in intestinal crypts. 30,31 Deficient IEC proliferation is obviously detrimental for the long-term maintenance of GI microstructure and function.
Previous studies have elucidated that CDK4 and CDK6, the proven target mRNAs of miR-206, 17,18 promote cell cycle entry via formation of the CDK4/6-cyclin D complex. 32 MiR-206 shares identical seed sequences with miR-1-3p, 33 allowing us to focus on miR-1-3p, which was identified in the high-throughput analysis. In our study, miR-1-3p exhibited the same regional expression pattern and expression trend as miR-124-3p, which we have reported on previously. 12 Coincidentally, CDK4/6 expression can also be regulated by miR-124-3p. 19,20 Practically, our in vitro and in vivo findings did indeed point to an miRNA-induced decrease in the proliferative ability of cells that expressed high levels of miR-1-3p and miR-124-3p due to the downregulation of CDK4/6 expression, and these phenomena closely resemble the observations made in colons of older individuals. Furthermore, the combination of miR-1-3p and miR-124-3p had a synergistic effect on CDK4/6. RKO and HCT116 cells were transfected with miR-1-3p and miR-124-3p mimics, alone or in combination. Compared with the group treated with miR-1-3p or miR-124-3p alone, the cotransfection group exhibited lower CDK4/6 protein expression, revealing that the combination of these miRNAs has the potential to synergistically suppress epithelial cell proliferation. Most extraordinarily, elevated miR-1-3p and miR-124-3p levels were observed in the mice at 16 mo, whereas a profound decrease in the proliferative capacity of epithelial cells was observed at 24 mo but not at 16 mo. We hypothesised that, as an important factor for maintaining epithelial function and intestinal homeostasis, the renewal of the epithelium is controlled by diverse genes and multistep processes and that miR-1-3p/miR-124-3p may only play a partial role in regulating proliferation. Many issues remain to be studied in the future.
Although a substantial number of studies have shown that intestinal tight junction barrier dysfunction can accelerate the onset and severity of colitis, 34 and this dysfunction has been recognised as a major aetiological factor in IBD, the roles of intestinal epithelial glycosylation in homeostasis and gut microbiota interactions in IBD have received increasing attention. 35 We observed a deficiency in mucin-type O-glycosylation in the distal colon beginning at 16 mo, before the disruption in epithelial microstructure and decrease in proliferation. Our data raised the intriguing possibility that damage to the mucus barrier, rather than disruption of the mucosal architecture, might be a contributing factor that results in epithelial hyperpermeability at the beginning of ageing [at approximately 50 years]. The normally impenetrable inner colon mucus layer is formed by sheets of mucin 2 that separate bacteria from the epithelium. The O-glycosylation of mucin 2 is critical and relies on the rate-limiting glycosyltransferase T-synthase. 36, 37 The development of spontaneous colitis in mucin 2-deficient mice 38,39 and IL-10 deficient mice 40 confirmed the protective properties of the inner colonic mucus layer, suggesting that a decline in mucus barrier function might occur before colitis onset. Our results 12 showed that the loss of T-synthase and reduced mucin glycosylation appeared beginning at 16 mo and became prominent at 24 mo. The changes in mucus properties and the subsequently increased microbiota-epithelial contact in the distal colon led to disturbances in the mucosal immune system and inflammation, which can trigger the development of intestinal inflammatory diseases. Hence, improving the properties of colonic mucus would be an effective strategy to delay GI ageing and ameliorate early age-related inflammatory symptoms in the clinic. In this study, miR-1-3p was confirmed to directly downregulate C1GALT1 expression and impair the intestinal mucus barrier function via decreased T-synthase-related mucin O-glycosylation. Intriguingly, all the age-related changes in miR-1-3p expression were observed exclusively in the distal colon, which was consistent with the distinct regional expression pattern of miR-124-3p/T-synthase we previously reported. 12 Furthermore, we found that miR-1-3p and miR-124-3p exerted a synergistic inhibitory effect on T-synthase, indicating that these two miRNAs are endogenous cooperative factors that regulate dynamic changes in gut mucus by modulating O-glycosylation in ageing people.
Normally, ageing is universal and results in mild or moderate changes in the expression of a range of miRNAs throughout the body rather than the severe abnormal expression of a single miRNA. Given the elevated levels of miR-1-3p and miR-124-3p during ageing and their robust functional roles in inhibiting T-synthase and CDK4/6 expression, we expanded our study by inducing DSS colitis in miRNA agomir-pretreated mice. Compared with high-dose agomir treatment targeting a single miRNA, the co-administration of low doses of two miRNAs exacerbated colitis after DSS treatment, indicating that slightly elevated miR-1-3p and miR-124-3p expression during ageing significantly increases the susceptibility to inflammation. Combined with our results that miR-1-3p and miR-124-3p were upregulated in colonic mucosa biopsies of old IBD patients, these findings support miR-1-3p and miR-124-3p as attractive targets to achieve multitarget and high-efficiency regulatory effects for the management of IBD in the geriatric population. Interestingly, miRNAs that belong to the same miRNA cluster and family are often thought to have close functional relationships and to co-regulate or coordinately regulate multiple biological processes. For instance, Zhao et al. 41 reported that miR-524-3p and miR-524-5p exerted a synergistic inhibitory effect on glioma cells. Pencheva et al. 42 revealed a convergent and cooperative multi-miRNA network including miR-1908, miR-199a-5p, and miR-199a-3p, which drove melanoma metastasis. The miR-23∼27∼24 clusters regulate multiple aspects of T cell biology. 43 However, miR-1-3p and miR-124-3p are not members of the same gene cluster/family, and we suspect that these molecules are unsaturated in our body and exert synergistic effects by binding to different sites of C1GALT1, CDK4, and CDK6 in the same cell or different cells. The exact mechanism underlying this synergistic effect has yet to be investigated.
In Drosophila, gut dysfunction appears to be a primary driver of ageing and mortality, and the intestine represents a potentially important target for interventions that promote longevity. [44][45][46] The identification of the determinants that delay ageing in the human body has proven to be more challenging. In the present study, miR-1-3p and miR-124-3p, which exhibit slightly elevated expression in the ageing colon, work together in both goblet cells and IECs to disrupt the intestinal barrier by changing mucus properties and suppressing epithelial cell proliferation, supporting their crucial synergistic role in accelerating GI ageing and enhancing susceptibility to colitis. The cooperation between these miRNAs is a new feature of key regulators involved in the onset and progression of colitis in elderly populations. Although the manifestations of ageing are a feature of later life, their underlying molecular bases certainly begin earlier. Our discovery provides a possible effective or optimal time window for intervention and a new research direction for postponing the course of GI ageing in the future.