Expression Of RUNX3 And β-catenin And Its Roles In Carcinogenesis Of Sporadic Colorectal Tubular Adenoma

Objective:More than 80% of colorectal cancers are developed from the adenomas.Colorectal tubular adenomas are very common,account for about 75% to 80% of colorectal adenomas.In clinical,colorectal cancers changed from sporadic tubular adenomas are not uncommon,but its mechanism of carcinogenesis is not clear.In recent years, the relationship between the epigenetics and tumorigenesis become to be one of the hot spots in medical field, especially DNA methylation in colorectal cancers has also received extensive attention. RUNX3 is a newly discovered gene which plays fundamental roles in the evolution of the mammalian RUNX family[1]. Recently,several lines of evidence have demonstrated that RUNX3 is a downstream effector of the TGF-βpathway, it can direct activated TGF-β/smad complex from the cytoplasm into the nucleus to the specific target sites,and enhance the strength of combination between the complex and target sites, and then participate in cell proliferation ,differentiation,apoptosis and other biological behaviors[2].At the same time, RUNX3 has a close relationship with many tumors.In many tumors ,its expression is downregulated,but the mechanism is not completely understand.It has also been reported that it was silenced mainly by DNA methylation [3].The role of Wnt pathway in colorectal carcinogenesis has been recognized by majority of relevant scholars.β-catenin is an important component of Wnt pathway.β-catenin is a multifunctional protein with cell adhesion and signal transduction function [4].Firstly it can link with E-cadherin to form E-Cadherin-β-catenin complex,mediate cell adhesion,and then act as a signaling molecule involved in Wnt signaling pathway,participate in embryogenesis and tumorigenesis[5].Only a little ofβ-catenin can be detected in normal cells.When the APC,β-catenin or axin gene bears mutations,β-catenin will accumulate in the cytoplasm,translocate to the nucleus,bind with the transcription factors and induce the expression of target genes[6] .A recent discovery by Kosei Ito [7] indicated that RUNX3 form a ternary complex withβ-catenin/TCF4,weaken the affinity with DNA and attenuates Wnt signaling activity in initiating the development of colorectal cancer (CRC).Colorectal adenoma with dysplasia and carcinomatous change often showed focal distribution.Laser capture microdissection (LCM) is a technology can fast, accuratly cut and separate the target cells, effectively overcome the bias caused by cell heterogeneity. LCM used in the research of sporadic colorectal tubular adenoma carcinomatous change and the expression of RUNX3 andβ-catenin has never been reported.To make the machanisms of sporadic colorectal tubular adenoma carcinomatous change more clearly,we used LCM combined with pathological morphology, immunohistochemistry, Western blot ,RT-PCR and methylation-specific PCR,analyzed the expression of RUNX3 andβ-catenin in the normal colorectal mucosa,sporadic colorectal adenomas with varying degrees of dysplasia and carcinomatous changed tissues.We also discussed the correlation between RUNX3 andβ-catenin and the possible role in the process of colorectal tubular adenoma carcinomatous change.This research aim at building the basis of diagnosis and treatment of colorectal tubular adenoma carcinomatous change.Method:1 ImmunohistochemistryWe used immunohistochemical ElivisionTMplus two-step method to study the expression of RUNX3 andβ-catenin in 23 cases of normal colorectal mucosa, 81 cases of sporadic colorectal tubular adenoma with different dysplasia and 20 cases of colorectal tubular adenoma with carcinomatous changes. 2 Western blotTotal protein was extracted from 17 colorectal tubular adenoma carcinomatous changed tissues and corresponding normal mucosa, separated by 15% SDS-PAGE electrophoresis and transferred to PVDF membranes.PVDF membranes were probed with primary monoclone antibodies (RUNX3 1:200;β-catenin 1:400)and secondary monoclone antibodies . Immunoreactive bands were visualized using ECL kit.3 RT-PCRTotal RNA was extracted from 17 colorectal tubular adenoma carcinomatous changed tissues and corresponding normal mucosa using Trizol reagent according to manufacturer instructions (Invitrogen, USA). The integrity of total RNA was identfied at 90V on 1% agarose gels containing EB (0.5μg/ml). The UV Spectrophotometer was used for the quantitation of the total RNA.The cDNA reverse transcripted from 500ng of total RNA was used as the template for PCR reaction.RUNX3 mRNA extracted from the cancerous tissue and corresponding normal mucosa was detected, and quantitied by gel analysis software (BIO-LD).The ratio of target gene and internal reference geneβ-actin represented the relative amount of the target gene.4 Methylation specific PCR(MSP)4.1 Laser capture microdissection17 colorectal tubular adenoma carcinomatous changed tissues and corresponding normal mucosa and 41 cases of tubular adenoma tissues which formalin-fixed and paraffin-embedded were cut in 10μm.The sections were mounted to the LCM slides (Leica).Frozen tissue sections stained with 2% methyl-green and paraffin tissue sections stained with hematoxylin were microdissected on Leica 6000 LMD microdissection system. The microdissected tissue was then transferred to an eppendorf cap. DNA extracted from the microdissected tissue was used in MSP.4.2 Methylation specific PCR(MSP)DNA was extracted from microdissected tissue according to the QIAamp DNA Micro Kit instructions. DNA was bisulfite modified with sodium metabisulfite and hydroquinone.The treated DNA was stored in -20℃.We designed RUNX3 gene methylatied primers and unmethylated primers for MSP.5 Statistical analysisThe experimental data was analyzed with Chi-square test, wilcoxon rank sum test and spearman correlation analysis using SPSS13.0 software.Result:1 The expression of RUNX3 in the process of colorectal tubular adenoma carcinomatous change1.1 The expression of RUNX3 protein1.1.1 Immunohistochemical resultIn the normal colorectal mucosa, tubular adenoma with varying degrees dysplasia and the colorectal carcinomatous changed from tubular adenoma, the positive rate is 91.30% (21/23), 54.32% (44/81) and 15.00% (3/20).The expression of RUNX3 in cancerous tissue and tubular adenoma is significantly lower than that in the nomal mucosa (P<0.05). The expression of RUNX3 in cancerous tissue is significantly lower than that in the tubular adenoma (P<0.05). The positive expression rate of RUNX3 in the cases of colorectal tubular adenoma with different dysplasia was 75.76%(25/33)(grade I), 60.87%(14/23)(grade II) and 20.00%(5/25)(grade III)respectively. The expression of RUNX3 in sporadic colorectal tubular adenoma with sever dysplasia is significantly lower than in the adenoma with mild/moderate dysplasia (P<0.05). The expression of RUNX3 in sporadic colorectal tubular adenoma with moderate dysplasia is lower than in the adenoma with mild dysplasia, but there is no statistical significance (P>0.05).1.1.2 Western blot resultRUNX3 is about 45~46 KD.We used GAPDH as the internal reference.Statisical analysis showed that the expression of RUNX3 in the cancerous tissue was significantly lower than that in nomal mucosa (P<0.05).1.2 The expression of RUNX3 mRNA in the adenoma carcinomatous changed tissues and corresponding normal mucosa We could detect the expression of RUNX3 mRNA in all of 17 pair cases, regardless of the normal mucosa or the cancerous tissues. The statistical analysis hint that the expression of RUNX3 mRNA in carcinomous tissues is significantly lower than that in normal mucosa (P<0.05).1.3 Methylation special PCR (MSP) result1.3.1 Laser capture microdissectionThe cancer nests in 17 cases of cancerous tissues and the glands in corresponding normal mucosa and 41 cases of formalin-fixed and paraffin-embedded adenomas were completely separated from the matrix.After cutting, the target cells were all transferred to the cap from the slices with no hyrid cells.1.3.2 Methylation special PCR (MSP) resultThe hypermethylation of the RUNX3 has been found in 41.18% (7/17) of colonrectal tubular adenoma carcinogenesis, 17.07% (7/41) of colorectal tubular adenomas and 5.89% (1/17) of the corresponding normal mucosa. The methylation of RUNX3 in cancerous tissue is significantly more frequently than that in the nomal mucosa and tubular adenoma (P<0.05). The methylation of RUNX3 in tubular adenoma is more frequently than that in the nomal mucosa, but there is no statistical significance (P>0.05).The hypermethylation of the RUNX3 has been found in 13.33%(2/15) (grade I), 16.67%(2/12) (grade II), 21.43%(3/14) (grade III).The rate of methylation increased with the grade of dysplasia,but there is no statistical significance(P>0.05).1.4 The relationship between the methylation of RUNX3 and its expressionIn 17 cases of adenomas carcinomatous changed, the protein and mRNA is much lower in the RUNX3 methylated cases than in the unmethylated cases.It has statistical significance (P>0.05).2 The expression ofβ-catenin in the process of sporadic colorectal tubular adenoma carcinomatous change2.1 Immunohistochemical resultIn the normal colorectal mucosa, tubular adenoma with varying degrees of epithelial dysplasia,and the colorectal caicinoma changed from tubular adenoma,the positive rate ofβ-catenin is 13.04%(5/23),60.49%(49/81) and 90.00%(18/20). The expression ofβ-catenin in cancerous tissue and tubular adenoma is significantly higher than that in the normal mucosa (P<0.05). The expression ofβ-catenin in cancerous tissue is higher than that in the tubular adenoma(P<0.05).The positive expression rate ofβ-catenin in the cases of colorectal tubular adenoma with different dysplasia was 39.40%(13/33)(grade I), 65.21%(15/23)(gradeII) and 84.00%(21/25)(grade III)respectively. The expression ofβ-catenin in sporadic colorectal tubular adenoma with sever dysplasia is significantly higher than in the adenoma with mild dysplasia (P<0.05). The expression ofβ-catenin in sporadic colorectal tubular adenoma sever dysplasia is higher than in the adenoma with moderate dysplasia, the expression ofβ-catenin in sporadic colorectal tubular adenoma with moderate dysplasia is higher than in the adenoma with mild dysplasia,but there is no statistical significance (P>0.05).2.2 Western blot resultβ-catenin is about 92 KD.We used GAPDH as the internal reference. Statisical analysis showed that the expression ofβ-catenin in the cancerous tissue was significantly higher than that in nomal mucosa (P<0.05).3 The relationship between RUNX3 andβ-catenin expression in the carcinogenesis of sporadic colorectal tubular adenomaCorrelation analysis showed: in the carcinogenesis of sporadic colorectal tubular adenoma, there is no significant correlation between RUNX3 andβ-catenin (P>0.05).Conclusion:1 RUNX3 protein and mRNA expression decreased in the process of colorectal tubular adenoma carcinomatous change.2 The RUNX3 methylation is more and more frequent in the process of colorectal tubular adenoma carcinomatous change.RUNX3 protein and mRNA expression is lower in the cases which have methylation than that in the cases not. 3β-catenin protein expression was gradually increased in the process of colorectal tubular adenoma carcinomatous change.With the dysplasia increased,β-catenin translocated from the cytoplasm to the nucleus.4 No relationship was found between RUNX3 andβ-catenin in the cacinomatous change of colorectal tubular adenoma.