The intestinal epithelium undergoes a regenerative process after injury, the destruction of which can lead to the development of inflammatory bowel disease and tumors. Intestinal stem cells (ISCs) are essential for maintaining intestinal epithelial homeostasis and promoting post-injury regeneration. However, the parenchymal function of dGCR8 in intestinal regeneration, as a key component of microRNA (miRNA) biogenesis, is poorly understood.
Figure 1Schematic diagram of miRNA-200-LNPs promoting intestinal regeneration in mice.
In this paper, the authors demonstrate through a series of experimental demonstrations that epithelial miRNAs play an indispensable role in intestinal regeneration in mice (damaged by 5-FU or irradiation). Through a comprehensive pooled screen of miRNA function in DGCR8-deficient organoids, the authors observed that deletion of the miR-200 family leads to overactivation of the p53 pathway, which hinders the proliferation of ISCs as well as the regeneration of epithelial cells (Figure 1).
Notably, downregulation of the Mir-200 family and overactivation of the P53 pathway were confirmed in colon tissue of patients with active ulcerative colitis (UC). Most importantly, after acute injury in mice, by oral administration of Mir-200-LNPS, temporary** Mir-200 restores ISCS and promotes intestinal regeneration. Therefore, the Mir-200 p53 pathway is a promising target for patients with active UC with reduced levels of the Mir-200 family. The author's experiment can be divided into 6 parts, which will be briefly described below
1.dgcr8Deficiency impairs intestinal regeneration after 5-fu treatment
Figure 2Deletion of dGCR8 disrupts the biogenesis of miRNA in the intestinal epithelium.
dGCR8 is an important factor in miRNA biogenesisThe biogenesis of miRNAs was significantly inhibited after dGCR8 deletion (Figure 2).
Figure 3DGCR8 is essential for intestinal regeneration after irradiation Figure 3DGCR8 is essential for intestinal regeneration after irradiation.
The authors compared the weight changes, post-injury survival time, and changes in the levels of the pro-inflammatory cytokines IFN and PNF-B between DGCR8 KO mice and WT mice through multiple sets of experiments (Fig. 3).dGCR8 is essential for intestinal epithelial regeneration.
2. dgcr8It is required for intestinal organoid formation and ISC maintenance
Figure 4DGCR8 is required for intestinal organoid formation and ISC maintenance.
The authors used intestinal organoid models of DGCR8-deficient mice and control mice to investigate the role of DGCR8 in ISCS.
The expressions of ISC marker genes LGR5, OLFM4, ASCL2 and SLC12A2 were significantly down-regulated after DGCr8-induced deletion. In contrast, several pri-miRNAs, including MIR17HG, lncppara, and lncpint, are upregulated in DGCR8-deficient organoids. Gene set enrichment analysis (GSEA) showed that deletion of DGCR8 reduced the expression of ISC and proliferation signature genes (Figure 4D), which was consistent with the downregulation of ISC and proliferation markers (Figure 4E, F). Flow cytometry analysis confirmed a decrease in the number of ISCs of LGR5-GFPHIGH in intestinal organoids due to DGCR8 deficiency (Figure 4G). Combined with more findings to show thatLack of DGCR8 in organoids leads to a decrease in the number of ISCs, a decrease in cell proliferation, and activation of the p53 pathway.
3.dgcr8Overactivation of the p53 pathway in the missing intestine
Figure 5Lack of DGCR8 induces overactivation of the p53 pathway.
Protein levels of P53 and P21 were elevated in the dGCR8-deficient epithelium by immunoblotting (Figure 5H), in combination with data from other experimental groups (Figure 5).It supports the inference that the p53 pathway is involved in the regulation of ISC proliferation and epithelial regeneration
4.p53Attenuation of the pathway promotes intestinal regeneration of the dGCR8-deficient epithelium
Figure 6Inhibition of the p53 pathway promotes epithelial regeneration in 5-fu-induced mice with dGCR8-deficient mouse.
Next, the effect of inhibition of the p53 pathway on intestinal regeneration was studied: treatment of DGCR8-deficient organoids with the P53 protein inhibitor polyvinyl fluoride-hydrobromide (PFT) could rescue the downregulation of genes related to ISCS and proliferation, increase the number of buds, and improve organoid survival (Fig. 6). This provides evidence that overactivation of the p53 pathway is associated with the pathogenesis of colitis.
5.mir-200The absence of family members explains the activation of p53 in DGCR8-deficient cells
Figure 7The absence of the Mir-200 family leads to overactivation of the p53 pathway in the intestinal epithelium.
Transfection of a chemically modified miR-200B-3P agonist (AgoMiR-200B) facilitated the germination process of organoids, while transfection of a miR200B-3P antagonist (AntagagoMiR-200B) inhibited the budding process and reduced the survival of organoids (Figure 7E). Transfection of Agomir200B decreased p53 and p21 protein levels in DGCR8-deficient organoids, whereas transfection of AntagomR-200b increased p53 and p21 protein levels (Figure 7H). Combined with other experimental findings, it is shown that:Dysregulation of miR200 family members leads to overactivation of the p53 pathway, impaired epithelial regeneration, and increased inflammation.
Intestinal regeneration in injured mice by lipid nanoparticle delivery of miR-200
After acute injury, LNPs carrying MIR-200 are delivered orallyThe transient recovery of miR-200 levels promotes the process of intestinal regeneration in mice。The authors' findings reveal a regulatory mechanism, the DGCR8 mir-200 p53 pathway, which plays a crucial role in intestinal regeneration.
Summary
The authors focused on revealing the key role of the regulatory mechanism of DGCR8 miR-200 p53 in intestinal regeneration, and expected that miRNA-200-LNPs could provide better ** for UC patients, and believed that nucleic acids** with LNPs as carriers would play a broader application space in the near future.
References:
1] wei x, yu s, zhang t, liu l, wang x, wang x, chan ys, wang y, meng s, chen yg. microrna-200 loaded lipid nanoparticles promote intestinal epithelium regeneration in canonical microrna-deficient mice. acs nano. 2023 nov 28;17(22):22901-22915. doi: 10.1021/acsnano.3c08030. epub 2023 nov 8. pmid: 37939210; pmcid: pmc10690841.
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