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

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Construction of murine model of ulcerative colitis-related colorectal cancer and expression profile of mi RNA-200 b in the modelObjective: To construct a murine model of ulcerative colitis-related colorectal cancer(UCRCC),and the expression of mi RNA-200 b in the intestine of each group of mice was detected to investigate whether the expression profile of mi RNA-200 b is related to UCRCC.Methods: A lentiviral vector(LV)labeled with enhanced green fluorescent protein(EGFP)over-expressing mi RNA-200 b was constructed,and a negative control lentivirus(lentivirus-negative control,LV-NC)was established.UCRCC model was induced in specific pathogen free(SPF)C57BL/6J mice weighing 18-20 g by a single injection of azoxymethane(AOM)in combination with 3 cycles of dextran sulfate sodium(DSS).They were randomly divided into five groups as follow: Normal group,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)and Compound Sophorae Decoction group(AOM/DSS+CSD).Expression profile of mi RNA-200 b in mouse colon tissue 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 and NC group compared to the Normal group were discovered,meanwhile the mi RNA-200 b expression levels in mi RNA-200 b group and CSD group were between Model group and Normal group,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 on UCRCC and intervention effect of CSDObjective: To study the effect of over-expresssed mi RNA-200 b and CSD on UCRCC.Methods: The general condition,weight change,and survival rate of each group were recorded and statistically analyzed;The colorectal length of each group of mice was statistically compared;The green fluorescence expression in frozen colonic sections of mi RNA-200 b mice was observed under a fluorescence microscope;The number and diameter of tumors in each group were statistically evaluated and and histopathological analysis of the colorectal in each group of mice was performed by hematoxylin-eosin staining(H&E staining).Results: The green fluorescence expression was observed under the microscope and the virus infection area coincided with the expected transfection area,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 significantly reduced.In line with this,the hair was brighter,and the weight loss along with intestinal shortening of the mice from mi RNA-200 b group were also relatively reduced.Furthermore,HE staining showed that the grade of epithelial dysplasia,inflammatory cell infiltration,and mucosal ulcer in the mi RNA-200 b group were significantly lighter than those in the model group.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 improve the general health and survival of AOM/DSS-treated mice,relieve the occurrence and progression of UCRCC and play as a tumor suppressor in UCRCC.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 overexpressed 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 like changes in colonic cell morphology,cell arrangement,and tight junctions.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.Epithelial characteristics of colonic cell of mi RNA-200 b and CSD groups have not completely disappeared under TEM,and cell adhesion with arrangement exist.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.Conclusion: EMT is involved in the development of UCRCC.Overexpressed 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.