Functional And Mechanistic Study Of MiR-31 In Intestinal Stem Cells,Epithelial Regeneration And Tumorigenesis

The intestinal tract is the main organ for digestion and nutrient absorption in mammals.The rapid tissue turnover in intestine is driven by the intestinal stem cells(ISCs)that reside at the bottom of the crypt.Thus,intestine is one of the ideal models for stem cell research.The rapid turnover and differentiation in intestinal epithelium is required for the absorption of nutrients,but also important for injury repair of the damaged epithelium.Colorectal cancer occurrence could be caused by abnormal activation of ISCs.Understanding the molecular mechanism on self-renewal and proliferation of ISCs helps uncover the basal principles of intestinal development and intestinal disorders resulting in tissue death and regeneration,including colorectal cancer.It has not only scientific significance for basic research,but also potential application value.The ISC compartment includes the active crypt base columnar(CBC)stem cells,which are labeled by Lgr5,and more dormant reserve ISCs referred as+4 cells due to their position at the crypt,which is marked by Hopx.The self-renewal and proliferation of ISCs is regulated by multiple signaling pathways.MiRNAs,a class of 20-24 nucleotide small non-coding RNA,negatively regulate gene expression of their target gene by binding to the 3’untranslated region(3’-UTR)of target mRNAs.Specifically,miRNA-31(miR-31)has been implicated to play important roles in regulating fate determination of adult muscle and mesenchymal stem cells,and tumorigenesis.Particularly,its expression is markedly up-regulated during the progression of inflammation-associated intestinal neoplasia and over-expressed in colorectal cancer.However,little is known about the function of miR-31 in intestinal stem cells and its in vivo role in tumorigcnesis.Therefore,this research aims to investigate the function of miR-31 in ISCs,injury repair of damaged intestinal epithelium,and tumorigenesis,as well as its underlying mechanisms.We isolate Lgr5+ CBCs and+4 reserve stem cells from Lgr5-EGFP-IRES-CreERT2 and Hopx-CreERT2;Rosa26mTmG transgenic mice with flow cytometry and then quantify miR-31 expression level with qRT-PCR analysis.It revealed that miR-31 expression levels were higher in the Lgr5+ CBCs and+4 reserve stem cells relative to their controls.MiR-31 expression was also markedly up-regulated upon 12 Gy γ-IR irradiation and dextran sulfate sodium salt(DSS)treatment as compared to controls.It suggests a potential role of miR-31 in the maintenance of ISCs during homeostasis and the injury repair process during regeneration.To determine the function of miR-31 in the mouse intestine,we generated gain-of-function mouse models using Tet-On system with a targeted,inducible Rosa26-rtTA;TRE-miR-31 mouse model(TRE-miR31)and doxycycline(Dox)-mediated induction of miR-31 in the intestinal epithelium.For the loss-of-function,this study generated constitutive miR-31 null mice using RNA-guided CRISPR/Cas9 nucleases method.This research also generated a Villin-Cre-mediated intestine-specific conditional miR-31 null mice(cKO)using traditional homology-directed gene targeting.MiR-31 induction in response to Dox administration resulted in a significant expanded of crypt with more proliferative cells,promotes Lgr5-G FP+ stem cell expansion.In contrast,loss of miR-31 repressed cell proliferation,reduced Lgr5+ cells,enhanced apoptosis of Lgr5+stem cells in both miR-31 germline knockout(KO)and Vilin-Cre-driven intestinal epithelial conditional KO(cKO)mice.These data strongly indicated that miR-31 promotes proliferative expansion of ISCs and protects ISC against apoptosis during homeostasis.In response to irradiation,miR-31 protects intestinal stem cells against apoptosis at the early stage,and subsequently activates those surviving stem cells,promoting the intestinal epithelial regeneration and the proliferation and regeneration within the Lgr5+ ISCs compartment.To further test the role of miR-31 in intestine epithelial regeneration,this research administered dextran sulfate sodium(DSS)to induce colitis.Loss of miR-31 resulted in more severe weight loss and lower survival rate,and impaired epithelial regeneration in response to DSS-induced colitis.These data highlight the importance of miR-31 in epithelial regeneration under stresses environment.In the HCT116 cell xenograft tumor model,miR-31 promoted the tumor growth.In the AOM-DSS(Azoxymethane-Dextran Sodium Sulfate)model of the inflammation-driven colorectal adenocarcinoma,a marked decrease in both tumor size and number was found in miR-31-/－mice.When miR-31 was deleted in Vil-Cre;APCflox/+ mice,tumor size and number were remarkably reduced in the intestine.These data demonstrate that miR-31 acts as an oncogenic miRNA in intestinal tumorigenesis.Using Dual luciferase reporter and CLIP-qPCR assays,this research demonstrated miR-31 activates Wnt signal pathway by directly targeting to Dkk1,Axinl and Gsk3β and represses the BMP and TGFβ pathways by targeting Smad3,Bmprla,Smad4,resulting in proliferative expansion of ISCs and activation of dormant stem cells and accelerated tumor progression.Meanwhile miR-31 protects ISC against apoptosis by directly targeting p38 and Gsk3β.In the upstream mechanism of miR-31,our findings suggest that γ-IR activated STAT3 mediating miR-31 expression in regenerating foci,while the activities of STAT3 and NF-κB signaling pathways mediated miR-31 expression in colon in response to DSS.In conclusion,the main findings can be summarized as follows:1)MiR-31 functions as a novel intestinal stem cell-intrinsic activating signal that promotes proliferation of intestinal stem cells by activating Wnt and repressing BMP/TGFβ signaling pathways,and protects intestinal stem cells from apoptosis by repressing Gsk3β and p38.2)During epithelial regeneration,our data support a model in which miR-31 protects intestinal stem cells against early-stage apoptosis and subsequently promotes rapid cycling in surviving stem cells through the repression of BMP and potentiation of canonical Wnt signaling.3)We identified miR-31 as a pro-oncogenic miRNA involved in a Stat3-miR-31-Wnt/BMP/TGFβ signaling pathway that promotes intestinal tumorigenesis.4)To the best of our knowledge,our work is the first to reveal critical in vivo role for a specific miRNA in intestinal stem cells.These findings identify miR-31 as a critical modulator of ISCs,and a potential therapeutic target for a broad range of intestinal regenerative disorders and cancers.