Role And Mechanism Of MiRNA-200b/AKT2 In Ulcerative Colitis-associated Colorectal Cancer And Regulation Of Compound Sophorae Decoction

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Construction of murine ulcerative colitis-related colorectal cancer model and expression profile of mi RNA-200 b in the modelObjective: To construct a murine model of ulcerative colitis-related colorectal cancer(UCRCC)and detect the expression of mi RNA-200 b to investigate whether the expression profile of mi RNA-200 b is related to UCRCC.Methods: The lentiviral vectors(LV)labeled with enhanced green fluorescent protein(EGFP)overexpressing mi RNA-200 b or acting as negative control(LV-NC)were constructed.UCRCC model was induced in C57BL/6J mice weighing 18-20 g by administration of azoxymethane(AOM)in combination with dextran sulfate sodium(DSS).They were randomly divided into five groups as follow: Model group(AOM/DSS),mi RNA-200 b group(AOM/DSS + overexpressed mi RNA-200 b lentivirus),Negative Control group(AOM/DSS + lentivirus negative control),Normal group and Compound Sophorae Decoction group(AOM/DSS+CSD).Expression profile of mi RNA-200 b in colons were illustrated by PCR and ISH.Dual luciferase assay was adopted to verify whether mi RNA-200 b can target AKT2 on the basis of results from mi RNA target prediction softwares such as Target Scan,Pic Tar and mi RDB.Results: The lentiviral vectors and UCRCC model were successfully constructed.Expression of mi RNA-200 b was detected by ISH in the colonic tissue chip.Lower-grade mi RNA-200 b expression in Model group compared to the Normal group was discovered and the result was validated through PCR.As based upon the results from those softwares listed above,mi RNA-200 b was observed to have a putative binding site at 3?-UTR(3' untranslated region)of AKT2.Moreover,luciferase reporter assay displayed that mi RNA-200 b overexpression reduced luciferase activity from the wildtype(WT)AKT2 3?-UTR compared to mutant(MUT)AKT2 3?-UTR,confirming that mi RNA-200 b can inversely target AKT2.Conclusion: mi RNA-200 b is down-regulated in the murine UCRCC model and AKT2 is a target of mi RNA-200 b.Part ? Regulation of over-expresssed mi RNA-200 b and CSD on UCRCC Objective: To investigate the effect of over-expresssed mi RNA-200 b and CSD on UCRCC.Methods: Body weight,food and water intake were monitored daily over the duration of the experiment.Body weight loss,stool consistency,survival rate and hemafecia ratio were under surveillance and statistically analyzed.After making a survey of length of the colons,the number and diameter of tumors were statistically compared and the histopathological analysis was performed by hematoxylin-eosin staining(H&E staining).Frozen colonic sections of mi RNA-200 b group were observed under microscope to identify the location of LV which was labeled with fluorescence.Results: The green fluorescence was detected under the microscope,suggesting that the lentiviruses were successfully colonized in epithelial cells or submucosa.Compared with Model group,the mortality,tumor number and hematochezia rate of mi RNA-200 b group decreased significantly.In line with this,a tiny increase in intestinal length of mi RNA-200 b group was manifested.Furthermore,higher grade of epithelial dysplasia along with inflammatory cell infiltration and mucosal ulcer in Model group was observed by H&E staining.The results of the Compound Sophorae Decoction group(CSD group)were similar to those in the mi RNA-200 b group,while the negative control group(NC group)is equivalent to the Model group.Conclusion: Over-expressed mi RNA-200 b and CSD not merely improve the general health and survival of AOM/DSS-treated mice but mitigate the AOM/DSS-induced malignancy.Part ? Ectopic mi RNA-200 b inhibits AKT2-mediated inflammatory responses in AOM/DSS-induced mice and CSD exerts anti-inflammatory effect on UCRCCObjective: To investigate whether CSD and over-expressed mi RNA-200 b alleviate or even inhibit UCRCC by inhibiting inflammatory response.Methods: Western blot and immunohistochemistry were used to inspect the levels of inflammatory markers such as tumor necrosis factor ?(TNF-?),nuclear factor-kappa B(NF-?B)and interleukin-6(IL-6),transforming growth factor-?(TGF-?),signal transducer and activator of transcription 3(STAT3)in each group.Results: Compared with Model group,the levels of inflammatory markers in the mi RNA-200 b group and the CSD group were significantly down-regulated.Meanwhile the results of NC group were consistent with those of Model group,with no statistical difference between them.Conclusion: Mi RNA-200 b can inhibit the inflammatory response in UCRCC through AKT2/NF-?B/IL-6/STAT3 signaling pathway and CSD also exhibits obvious anti-inflammatory effect.Part ? Effect of mi RNA-200 b and CSD on epithelial-mesenchymal transition of UCRCCObjective: To investigate whether epithelial-mesenchymal transition(EMT)is involved in the development of UCRCC and further probe whether mi RNA-200 b and CSD can inhibit this process.Methods: Western Blot and immunohistochemistry were used to figure out the expression of EMT-related proteins like E-cadherin and N-cadherin along with ?-catenin,colorectal cancer stem cell marker CD133.Transmission electron microscope(TEM)was used to examine cellular morphological variance.Results: Compared with Model group,E-cadherin was up-regulated in mi RNA-200 b group and CSD group.In contrast,N-cadherin,?-catenin,and CD133 were down-regulated.TEM images of Model group revealed typical tumorigenesis characterized by variform cellular morphology coupled with abnormal and large and deeply stained nuclei,and displayed typical EMT as demonstrated by disappearance of cellular polarity and cell adhesion together with disordered cell arrangement.However,treatment with mi RNA-200 b overexpression or CSD was sufficient to partially reverse the effect.Conclusion: EMT is involved in the development of UCRCC.Mi RNA-200 b can regulate the changes of cell polarity and repress AOM/DSS-induced EMT via AKT2 and also inhibit cancer stem cell phenotype.At the same time,CSD can antagonise EMT,thus mitigating UCRCC.