| Objective: Colorectal adenomas are benign lesions with malignantpotential. More than80%colorectal carcinoma are developed fromcolorectal adenoma. Tubular adenomas are the most common histologicaltypes of colorectal adenomas. The mechanism of carcinogenesis is not clear.Currently, TGF-β/SMAD signaling pathway become the hot spots of themedical field in the study of tumor development.TGF-β/SMAD signalingpathway is involved in physiological processes such as cell growth,proliferation,differentiation.The biological signals binding with TGF-β/SMADfamily ligands and cell membrane receptors are passed to target genes bySmads. The abnormality of some factors of this pathway may promote tumorprogression.c-Ski is identified as an oncogene which could interact with Smad3andinhibit TGF-β/SMAD signaling pathyway.c-Ski prevents the phosphorylationof Smads proteins.There is closly relationgship between c-Ski and tumor.High expression of c-Ski has been reported in many human cancer celllines,including melanoma,breast cancer,prostate cancer, pancreatic cancer, andso on.Smad3is the signal transduction molecules of theTGF-β/SMADpathways downstream,and it has the transcriptionally activation andparticipation in the function of nuclear translocation. Smads complex cancombine with the target gene, and then regulate the transcription ofdownstream related factors, such as c-myc. The study find that highexpression of Smad3in some tumors,such as in choriocarcinoma organizationand glioblastoma.But in liver cancer,stomach cancer and skin squamous cellcarcinoma,the Smad3expression decrease.And some study find the Smad3gene mutations in colon cancer cell lines SNU-769A and SNU-769B. p15is an important tumor suppressor gene, which is one of the TGF-β/SMAD signaling pathways downstream targets by majority of relevantscholars. p15protein is the cell cycle inhibitor. It participates the regulation ofcell growth and proliferation programs. The study find that the deletions andmutations of p15gene exsited in many tumors such as bladder cancer,esophageal cancer, liver cancer and stomach cancer.Currently, there is a small number of research related to the expression ofthe c-Ski,Smad3and p15, TGF-β/SMAD signaling pathways downstreamfactors, in carcinogenesis of colorectal adenoma.So we analyze the expressionof c-Ski,Smad3and p15in the normal colorectal mucosa,sporadic colorectaladenomas with varying degrees of dysplasia and carcinomatous changedtissues by pathological morphology,immunohistochemistry, Western blot, Realtime-PCR and culture of cells,and also combined with clinic pathologicparameters about patients. We also explore the possible role in sporadiccolorectal carcinogenesis significance of intestinal adenomas.This researchaims to providing theoretical basis for colorectal adenoma prevention andclinical treatment of cancer.Method:1ImmunohistochemistryWe use immunohistochemical ElivisionTMplus two-step method to studythe expression of c-Ski,Smad3and p15in20cases of normal colorectalmucosa,81cases of sporadic corectal tubular adenoma with different dysplasiaand32cases of colorectal tubular adenoma with carcinomatous changes.2Western blotTotal protein is extracted from the tissues and cells, separated by12%SDS-PAGE electrophoresis, and transferred to PVDF membranes.PVDFMembranes are probed with primary monoclone antibodies (c-Ski1:800;Smad31:5000;p151:500)and secondary monoclone antibodies.Immunoreactive bands are visualized by Odyssey and Bio-LD software.3Real time-PCRTotal RNA is extracted from the tissues and cells using Trizol reagent according to manufacturer instructions (Invitrogen,USA). The ratio of targetgene is calculated by the method of Comparative Ct (ΔΔCt).4Cell culture and Cell treatmentHIEC are grown in RPMI-1640medium supplemented with10%new-born calf serum (NBCS), streptomycin (100μg/ml), penicillin (100U/ml),insulin (0.625%)in an atmosphere of5%CO2,95%air at37℃.HT-29cells are grown in RPMI-1640medium supplemented with10%new-born calfserum (NBCS), streptomycin (100μg/ml), penicillin (100U/ml) in anatmosphere of5%CO2,95%air at37℃.The cells in logarithmic growth phaseare digested with0.25%trypsin+0.02%EDTA, then collected and detectedrelated indicators.5Statistical analysisSPSS17.0software is applicated. Measurement data are presented as(x±s).The experimental data are analyzed by T-test,Chi-square test,wilcoxon test,spearman correlation analysis.Results:1The expression of c-Ski in the process of colorectal adenoma carcinomatous1.1The expression of c-Ski1.1.1Immunohistochemical resultThe positive expression rate gradually incressed in in the normalcolorectal mucosa, tubular adenoma with varying degrees dysplasia, and thecolorectal caicinoma changed from tubular adenoma.The positive rate of c-Skiprotein is10.00%(2/20),55.56%(45/81),81.25%(26/32) repectively.Theexpression of c-Ski in cancerous tissue and tubular adenoma is significantlyhigher than that in the other groups (P<0.05). the positive expression rate ofc-Ski was28.13%(9/32)(grade I),60.00%(15/25),(gradeII)and87.50%(21/24)(grade III)respectively. The expression of c-Ski in sporadiccolorectal tubular adenoma with moderate/sever dysplasia is significantlyhigher than in the adenoma with mild dysplasia (P<0.05).1.1.2Relationship between the expression of c-Ski and clinical pathologicalcharacteristics The positive rate73.91%(34/46) of c-Ski in size≥2cm is higher than that55.22%(37/67)in size＜2cm (P<0.05), but there is no statistical significancebetween the expression of c-Ski and age,sex and location (P>0.05).1.1.3Western blot resultIn18fresh colorectal adenoma carcinomatous changed tissues andcorresponding normal colorectal rectal mucosa,we use β-actin(43KD) as theinternal reference. Statisical analysis show that the expression of c-Ski (85KD)in the cancerous tissue is significantly higher than that in nomalmucosa(P<0.05).1.2Real time-PCR resultThe expression of c-Ski mRNA in the adenoma carcinomatous changedtissues and corresponding normal mucosaReal-time PCR test results show that c-Ski mRNA expression in tumortissues44.44%(8/18) is significantly higher than their matched normalintestinal mucosa. Further analysis find that the average expression level ofc-Ski mRNA in tumor tissue is significantly higher than that in normalmucosa(P<0.05).2The expression of Smad3in the process of colorectal adenomacarcinomatous2.1The expression of Smad32.1.1Immunohistochemical resultThe positive rate of Smad3is60.00%(12/20),54.32%(44/81),84.38%(27/32)in the normal colorectal mucosa, tubular adenoma with varyingdegrees dysplasia, and the colorectal caicinoma changed from tubularadenoma. The expression of Smad3in cancerous tissue is higher than that inthe normal tissue and the tubular adenoma group (P<0.05). There is nostatistical significance in sporadic colorectal tubular adenoma withmild/moderate/sever dysplasia (P>0.05).2.1.2Relationship between the expression of Smad3and clinical pathologicalcharacteristicsThere is no statistical significance between the expression of Smad3and age,sex, location and size (P>0.05).2.1.3The expression of Smad3protein in the tissuesIn18fresh colorectal adenoma carcinomatous changed tissues andcorresponding normal colorectal rectal mucosa,we use β-actin(43KD) as theinternal reference. Statisical analysis show that the expression of Smad3(58KD)in the cancerous tissue is significantly higher than that in nomalmucosa(P<0.05).2.2Real time-PCR resultThe expression of Smad3mRNA in the adenoma carcinomatous changedtissues and corresponding normal mucosaReal-time PCR test results show that Smad3mRNA expression in tumortissues50.00%(9/18) is significantly higher than their matched normalintestinal mucosa. Further analysis find that the average expression level ofSmad3mRNA in tumor tissue is significantly higher than that in normalmucosa(P<0.05).3The expression of p15in the process of colorectal adenoma carcinomatous3.1Immunohistochemical resultThe positive rate of p15is85.00%(17/20),56.80%(46/81),31.25%(10/32),in the normal colorectal mucosa, tubular adenoma with varyingdegrees dysplasia, and the colorectal caicinoma changed from tubularadenoma.The expression of p15in cancerous tissue and tubular adenoma issignificantly lower than that in the other groups (P<0.05). In the cases ofcolorectal tubular adenoma with different dysplasia,the positive expressionrate of p15was81.25%(26/32)(grade I),52.00%(13/25)(gradeII)and29.17%(7/24)(grade III)respectively. The expression of p15in sporadiccolorectal tubular adenoma with moderate/sever dysplasia is significantlylower than in the adenoma with mild dysplasia (P<0.05). The expression ofp15in sporadic colorectal tubular adenoma with sever dysplasia is lower thanin the adenoma with moderate dysplasia, but there is no statistical significance(P>0.05).3.2Relationship between the expression of p15and clinical pathological characteristicsThe positive rate(32.61%)of p15in size≥2cm is lower than that(61.19%)in size＜2cm (P<0.05), there is no statistical significance betweenthe expression of p15and age,sex and location (P>0.05).3.3The expression of p15protein in the tissuesWe used β-actin(43KD) as the internal reference. Statisical analysis showthat the expression of p15(15KD) in the cancerous tissue is significantlylower than that in nomal mucosa(P<0.05).4The relationship between c-Ski,Smad3and p15protein expression in thecacinogenesis of sporadic colorectal tubular adenomaResults show that there is a positive correlation between c-Ski and partialSmad3in the process of tubular adenoma dysplasia (p <0.05).There is anegative correlation between c-Ski and p15in carcinogenesis of sporadiccolorectal tubular adenoma(P<0.05), But there is no obvious correlationbetween Smad3and p15.5The expression of c-Ski,Smad3and p15protein in the cellsStatisical analysis show that the expression of c-Ski protein and mRNAin HT-29is significantly higher than that in HIEC (P<0.05). The expression ofSmad3protein and mRNA in HT-29is significantly higher than that in HIEC(P<0.05).The expression p15protein in the HT-29is significantly lower thanthat in HIEC (P<0.05).Conclusion:1In the process of colorectal tubular adenoma carcinomotous change, theexpression of c-Ski protein is gradually increased, and the expression of p15protein is gradually decreased. The expression of Smad3in cancerous tissue ishigher than that in the normal tissue and the tubular adenoma group. Theresults show that c-Ski,Smad3and p15may play an important role in sporadiccolorectal carcinogenesis significance of intestinal adenomas.2Immunohistochemistry results suggest that positive correlation existsbetween the c-Ski and Smad3and the negative correlation between c-Ski and p15. There is no significant correlation between Smad3and p15. The resultsshow that the synergistic relationship may exist between c-Ski and Smad3.3In HT-29, the expression of c-Ski protein and mRNA is higher than that inHIEC. The expression of Smad3mRNA and protein in the HT-29is higherthan that in HIEC.The expression of p15protein in HT-29is lower than that inHIEC.The results show that the three factors may play some role in theincidence of colorectal cancer. |
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