| BackgroundUlcerative colitis(UC) is a chronic non-specific colonic inflammatory disease with uncertain etiologies. Recently, the incidence of IBD in China has increased rapidly. One of the most important complications of UC is Ulcerative colitis related colorectal cancer(UCRCC). Timely and accurate evaluation of disease activity and malignancy surveillance in ulcerative colitis is mandatory to improve patient outcome. So far, the evaluation of disease activity and malignancy surveillance has mainly depended on endoscopic examinations, lacking noninvasive ideal laboratory tests. Our research is intended to explore the application of MicroRNA(miRNA) for the evaluation of disease activity and malignancy surveillance in UC patients and mice models with UC and UCRCC.The first part The preliminary study of serum miRNAs for evaluation of disease activity and malignancy surveillance in UCMethodsGroups 1. Disease activity related:Training set-Severe group(30 severe patients) vs Control(30 healthy people).Validated set-Severe validated group(64 severe UC patients) vs Mild validated group (44 mild UC patients) vs Control(60 healthy patients).2.Malignancy surveillance related:Long course group(39 UC patients lasting longer than five years) vs Short course group(69 patients less than five years) according to course; Subgroup based on disease activity-Severe long course group(n=24) vs Severe short course group(n=40), Mild long course group(n=15) vs Mild short course group(n=29).Candidated miRNAs miR-21,126,31,192,19a,224,155,150.miRNAs screening 1.Disease activity related:We used qRT-PCR to screen 8 candidated miRNAs in the training set, followed by validation in those with differential expression. Then risk score analysis is used for assessment of the evaluation efficacy of post-screening miRNAs for disease activity in the validated set.2.Malignancy surveillance related:With qRT-PCR as well, Statistical analysis of expression change of candadated miRNAs based on course grouping; Subgroup analysis for severe and mild UC respectively; We then used correlation analysis to study the relation of age with miR-21 and miR-126, respectively.Result1. Disease activity related:(DmiR-21 is over expressed in Severe validated group compared to Mild validated group and Control in the validated set with 2.19 and 3.14 times, respectively(P<0.001); miR-21 shows a elevation trend in the Mild validated group compared to Control with 1.44 times(P=0.47). ②miR-126 is over expressed in Severe validated group compared to Mild validated group and Control in the validated set with 2.63 and 2.55 times, respectively(P<0.001); miR-21 shows no significant differentiation between Mild validated group and Control(P=0.52). ③The specificity and positive predictive value in the differentiation between Severe validated group and Control with combination of miR-21 and miR-126 is 90.0% and 84.6%, respectively; The specificity and positive predictive value in the evaluation efficacy between severe validated UC and mild validated UC with combination of miR-21 and miR-126 is 82.1% and 85.7% respectively.2. Malignancy surveillance related:①miR-21 is two times over expressed in Long course group compared to Short course group(P<0.05); Further analysis of disease course in severe UC and mild UC subgroups shows that miR-21 increases to 2.14 times in terms of diseae course in the severe UC(P<0.05) while miR-21 increases to 1.82 times(P=0.12) in terms of disease in the mild UC;The serum expression of miR-21 has no significant correlation with age(P=0.266).②miR-126 is over expressed in Long course group compared to Short course group with 1.68 times(P=0.16); Further subgroup analysis of disease course in severe UC and mild UC subgroups shows:miR-126 increases to 2.23 times in terms of disease course in the severe UC(P<0.05) while there is no significant differential expression of miR-126 in terms of disease in the mild UC (P=0.86); The serum expression of miR-126 has no significant correlation with age(P=0.233).Conclusion1. The serum expression of miR-21 and mir-126 is correlated to disease activity of UC with expression of miR-21 paralled to disease activity.2. The evaluation efficacy for Severe validated group vs Control and Severe validated gourp vs Mild validated group with combination of miR-21 and miR-126 shows relatively high specificity and positive predictive value, suggesting that combination of serum miRNAs is a potential candidate for UC activity evaluation.3. The expression of miR-21 and miR-126 is correlated to UC disease course, but they have no correlation with age, suggesting that the serum expression of miR-21 and miR-126 may be correlated to the risk of UCRCC; Subgroup analysis reveals that the expression change of the two miRNAs with disease course is more evident in the severe UC than mild UC, which further supports this possibility.The second part The preliminary study of expression correlation between serum miRNAs and targeted mRNA in the mouse model with UC and UCRCCMethodsGroups 1.miRNA related:Control(9 normal mice) vs UC group(10 DSS-inducing UC mice) vs UCRCC group(10 DSS/AOM-inducing UCRCC mice).2.mRNA related: Control(6 normal mice) vs UC group(7 DSS-inducing UC mice) vs UCRCC group(7 DSS/AOM-inducing UCRCC mice).Candidated miRNAs and mRNA 1.miRNAs:we choose miRNAs with differential expression in the first part, namely, miR-21,126.2.Targeted mRNA:Rhob and PDCD4(miR-21), NFKBIA (miR-126)Expression quantity of miRNAs and mRNA we measure expression change of candidated miRNAs and targeted mRNA in the colon epithelium of Control, UC group group and UCRCC group, discussing the correlation between miRNA and Targeted mRNA and possible mechanism in the development of UC and UCRCC.Result1. miRNA related:①miR-21 is overexpressed in the colon epithelium of UC group and UCRCC group compared to control with 9.24 times(P<0.05) and 7.24 times(P<0.01) respectively while there is no significant differential expression between UC group and UCRCC group(P=0.75). ②miR-126 is overexpressed in the colon epithelium of UC group and UCRCC group compared to control with 6.08 times(P<0.05) and 6.91 times(P<0.05) respectively while there is no significant differential expression between UC group and UCRCC group(P=0.88). ③Tissue pathological pattern reveals that the UCRCC model is successful and there is less inflammation in UCRCC than UC.2. mRNA 相关:①miR-21 targeted PDCD4 is lower in Control compared to UC group with 0.60(P=0.14) and is overexpressed when compared to UCRCC group with 2.3 times(P<0.01). It is also overexpressed in UC group compared to UCRCC group with 3.83 times(P<0.01). ②miR-21 targeted Rhob is overexpressed in Control compared with UC group and UCRCC group with 1.43 times(P=0.17) and 1.51 times(P=0.06) respectively while there is no significant differential expression between UCRCC and UC(P=0.77). ③miR-126 targeted NFKBIA is overexpressed in the colon epithelium of control compared to UC group and UCRCC group with 17.35 times(P<0.001) and 19.02 times(P<0.001) respectively while there is no significant differential expression between UCRCC group and UC group(P=0.85).Conclusion1. The expression of miR-21 and miR-126 is correlated with mucosal inflammation in mouce with UC, presenting overexpression and consistence with serum change in UC patients.2. There is no significant differential expression of miR-21 or miR-126 in the colon epithelium between UC group and UCRCC group. By combination with tissue pathological pattern, it suggests that miR-21 and miR-126 may be correlated to the onset and progression of UCRCC with overexpression.3. PDCD4 is reversely expressed in the colon epithelium of UCRCC group compared with miR-21. Given its biological effect, our fingdings hint that miR-21 influences the onset and development of UCRCC by down-regulation of PDCD4 which plays a role in cell apotosis. As for the result that PDCD4 is over expressed in the colon epithelium of UC, further study is needed.4. Rhob is reversely expressed in the colon epithelium of UCRCC group and UC group compared with miR-126. Given its biological effect, our fingdings hinted that miR-21 mediates the onset and progression of UC by down-regulation of Rhob which may deal damage on the bowel epithelium tight junction and promote UCRCC by affecting cell migaration.5. NFKBIA is reversely expressed in the colon epithelium of UCRCC group and UC group compared with miR-126. By combiniation of its biological effect, it preliminarily suggests miR-126 mediates the onset and development of UC and UCRCC by down-regulation of NFKBIA which induces NF-κB pathway.Final conclusion1. Serum and tissure expression changes of miR-21 and miR-126 may be predictive on the disease activity and carcinogenesis risk of UC.2. The combination testing of miRNAs may be potential ideal laboratory tests in the evaluation of diseae activity in UC.3. miR-21 and miR-126 mediate onset and development of UC and UCRCC by suppressing the expression of Rhob、PDCD4(miR-21), NFKBIA(miR-126). |
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