Exploration Of Application And Mechanisms Of Non-coding RNAs In Disease Activity Evaluation And Malignancy Surveillance In Ulcerative Colitis

Background and ObjectivesUlcerative colitis related colorectal cancer(UCRCC)is one of the major complications of ulcerative colitis(UC).Currently there is no ideal serum biomarker for the evaluation of disease activity and malignancy surveillance in UC.Our research is intended to explore the application and potential mechanisms of microRNA(miRNA)and long non-coding RNA(lncRNA)in disease activity evaluation and malignancy surveillance in UC patients and mice models.Part I.Screening of Serum Non-coding RNAs for Evaluation of Disease Activity and Malignancy Surveillance in UC Methods:1.We used qRT-PCR to screen 5 candidates non-coding RNAs(miR-223,miR-21,miR-126,GAS5 and SLC25A25-AS1)in serum samples of Control(n=35),Mild UC group(n=21)and Severe UC group(n=33).Receiver operator characteristic curve is used for assessment of the evaluation efficacy of non-coding RNAs for disease activity.Then we analyze the correlation of non-coding RNAs expression with ESR,hsCRP and Mayo endoscopic subscore.2.We screened candidate miRNAs in serum samples of Colorectal cancer group(n=30)and Control(n=35)with qRT-PCR.Results:1.Disease activity related:miR-223 is 2.40 times(P=0.0018)and 6.97 times(P<0.0001)upregulated in Severe UC group compared with Mild UC group and Control;miR-223 is 2.90 times(P=0.0024)upregulated in the Mild UC group compared with Control.GAS5 is downregulated to 81.2%(P=0.0063)and 79.3%(P=0.0197)in Severe UC group compared with Mild UC group and Control.The sensitivity and specificity to evaluate the disease activity with combination of miR-223 and GAS5 is 75.8%and 85.7%,respectively.miR-223 expression has a positive correlation with ESR,hsCRP and Mayo endoscopic subscore(P<0.05),and GAS5 expression has a negative correlation with hsCRP and Mayo endoscopic subscore(P<0.05).miR-223 demonstrated a higher Spearman r value than ESR and hsCRP with Mayo score.2.Malignancy surveillance related:Serum miR-21 expression increased 2.42 times(P=0.0030),5.66 times(P<0.0001)and 2.40 times(P<0.0001)in Colorectal group compared with Control,Mild UC group and Severe UC group.Conclusions:1.Serum expression of miR-223 and GAS5 are correlated with UC disease activity.2.The sensitivity and specificity to evaluate the disease activity with combination of miR-223 and GAS5 are relatively high,indicating that the combination of miR-223 and GAS5 is a potential candidate for UC activity evaluation.3.The expression of miR-223 and GAS5 are correlated with commonly used clinical measures for activity evaluation,and miR-223 expression is more correlated with Mayo score than ESR and hsCRP.4.miR-21 is a potential serum biomarker for colorectal cancer.Part ?.Exploration of Expression and Mechanisms of Non-coding RNAs and Related mRNAs in Colon Tissues of Mouse Models and UC PatientsMethods:We used qRT-PCR to evaluate the expression levels of 5 candidate non-coding RNAs and miR-223 related mRNAs in colon tissues of colitis mouse model,colitis-associated colorectal cancer(CAC)mouse model and UC patients.We set up DSS7 group,DSS9 group and DSS12 group which were sacrificed on day 7,day 9 and day 12 after DSS drinking in colitis mouse model.Results:1.The mucosal inflammation levels in colitis mouse model:DSS7 group>DSS9 group>DSS 12 group.2.Non-coding RNAs:miR-223,miR-21 and miR-126 are significantly upregulated in the colon tissues of DSS7 group compared with Control,DSS9 group and DSS 12 group.GAS5 is significantly downregulated in the colon tissues of DSS7 group compared with Control,DSS9 group and DSS 12 group(P<0.05).miR-223 expression is significantly increased in CAC group(P=0.0420).miR-223 and miR-21 are significantly upregulated in the colon tissues of Severe UC patients group compared with Mild UC group and Control.GAS5 and SLC25A25-AS1 are significantly downregulated in the colon tissues of Severe UC patients group compared with Mild UC group and Control(P<0.05).3-Related mRNAs:(1)Tissue expression of miR-223 targeted CLDN8 mRNA:significantly decrease in DSS7 group compared with Control,DSS9 group and DSS 12 group(P<0.05);significantly decrease in CAC mouse group compared with Control(P=0.0259);significantly decrease in Severe UC patients group compared with Mild UC group and Control(P<0.05).(2)Tissue expression of miR-223 targeted RhoB mRNA:decrease in DSS7 group compared with Control,DSS9 group and DSS 12 group(P=0.0019,P=0.0846 and P<0.0001);decrease in CAC mouse group compared with Control but not significantly different;not significantly different in UC patients.(3)Tissue expression of miR-223 related inflammatory cytokine IL-1?mRNA:significantly increase in DSS7 group compared with Control,DSS9 group and DSS 12 group(P<0.05);significantly increase in CAC mouse group compared with Control(P=0.0097);significantly increase in Severe UC patients group compared with Mild UC group and Control(P<0.05).(4)Tissue expression of miR-223 related inflammatory cytokine IL-6 mRNA:increase in DSS7 group compared with Control,DSS9 group and DSS 12 group(P=0.0142,P=0.1209 and P=0.1987 respectively);significantly increase in CAC mouse group compared with Control(P=0.0341);significantly increase in Severe UC patients group compared with Mild UC group and Control(P<0.05).(5)Tissue expression of miR-223 related inflammatory cytokine TNF-a mRNA:increase in DSS7 group compared with Control,DSS9 group and DSS12 group(P=0.0095,P=0.0998 and P=0.4081 respectively);significantly increase in CAC mouse group compared with Control(P=0.0148);increase in Severe UC patients group compared with Mild UC group and Control(P=0.3148 and P=0.0289 respectively).Conclusions:1.Tissue expression of miR-223,miR-21 and GAS5 are correlated with mucosal inflammation in colitis mouse model and UC patients.In addition,tissue expression pattern of miR-223 and GAS5 are the same as serum expression pattern.2.miR-126 tissue expression is correlated with mucosal inflammation in mouse models,and SLC25A25-AS1 tissue expression is correlated with mucosal inflammation in UC patients.3.The results indicate that miR-223 may play a role in UC and UCRCC by downregulating CLDN8 expression and upregulating IL-1? and IL-6 expression.miR-223 may downregulate RhoB expression to mediate the intestinal inflammation in mouse models,and may increase TNF-? expression to mediate the intestinal inflammation and malignancy in mouse models.