The Research Of The MicroRNA-21is Involved In The Regulation Of Intestinal Barrier Function And Colitis-associated Colon Cancer

Objective:To determine the role of miR-21in colitis and damage progression ofintestine in a genetically modified murine model and investigated the role ofmiR-21in colitis-associated colon cancer (CAC).Methods:(1) In the present study, colon tissue samples were obtained frompatients with histologically confirmed chronic UC and colon cancer andnormal control tissue. The mucosal and tumor tissue levels of miR-21weredetermined by using quantitative real-time polymerase chain reaction(QRT-PCR) analysis. The expressions of proteins were evaluated byimmunohistochemistry (IHC) and Western Blot analyses.(2) The miR-21KO mice (miR-21flox/flox, E2α-Cre homozygous mice)construction methods as follows: the miR-21flox/flox mice (neo) wereintercourse with EIIa-Cre mice (Strain Name: B6.FVB-Tg (EIIa-cre) C5379Lmgd/J, The Jackson Laboratory, USA).(3)Experimental colitis was induced in miR-21KO and wild-type (WT)mice by3.5%dextran sulphate sodium (DSS) administration for7days.Disease activity index(DAI), blood parameters, intestinal permeability,histopathologic injury, cytokine and chemokine production, and epithelialcells apoptosis were examined in colons of miR-21KO and WT mice. The mouse model of colitis-associated colon cancer (CAC), which is induced byadministration of azoxymethane (AOM) followed by repeated oraladministration of dextran sulfate sodium (DSS). The serum level of TNF-α,IL-6, IL-17A, and IL-21were measured in patients with colon cancer andCAC mice as manufacturer’s instructions.Results:(1) The miR-21is overexpression in human colorectal cancer and mouseCAC. There were significantly correlated expression between miR-21andmRNA and protein level of TNF-α, IL-6, IL-21in CRC samples.(2) miR-21was overexpressed in intestine of inflammatory boweldiseases (IBD) and acute intestinal obstruction (AIO) patients whencompared with normal intestinal tissues. Likewise, miR-21was up-regulatedin colon of IL-10KO mice when compared with control mice. WT micerapidly lost weight and were moribund5days after treatment with3.5%DSS,while miR-21KO mice survived for at least6days. Elevated leukocytes andmore severe histopathology were observed in WT mice when compared withmiR-21KO mice. Elevated levels of TNF-α and macrophage inflammatoryprotein-2(MIP-2) in colon culture supernatants from WT mice exhibitedsignificant higher than miR-21KO mice. Furthermore, CD3and CD68positive cells, intestinal permeability and apoptosis of epithelial cells weresignificantly increased in WT mice when compared with miR-21KO mice.Finally, we found that miR-21regulated the intestinal barrier functionthrough modulating the expression of RhoB and CDC42.(3) The expression of miR-21was significantly increased in the tumortissues of37patients with colorectal cancer, and in mice with CAC induced by azoxymethane (AOM) and dextran sulfate sodium (DSS). AfterAOM+DSS treatment, miR-21KO mice showed reduced infiltration ofinflammatory cells, diminished production of pro-inflammatory andpro-carcinogenic cytokines (TNF-α, IL-6, IL-17a, and IL-21), and developedfewer and smaller tumors compared with control wild-type mice. Absence ofmiR-21reduced expression of Ki-67result attenuated proliferation of tumorcell, while increased E-cadherin and decreased β-catenin and stem cellmarker SRYbox containing gene9(Sox9) expressions in tumor tissue ofCAC mice. Furthermore, absence of miR-21reduced NF-κB and signaltransducer and activator of transcription3(STAT3) activation and decreasedexpression of Bcl-2in tumor and stromal cells result increased apoptosis oftumor cells in CAC.Conclusion:(1) The miR-21is overexpressed in intestinal inflammation and injurytissue, while knockout of miR-21in mice improve the survival rate inDSS-induced fatal colitis through protecting against inflammation and tissueinjury. Therefore, attenuated expression of miR-21in gut may prevent theonset or progression of inflammatory bowel disease in patients.(2) The overexpression of miR-21amplifies an inflammatory milieu thatpromotes CAC, and suggests that miR-21blockade may be useful in reducingthe risk of UC-associated colon cancer.