Expression And Significance Of Wnt/β-catenin In Myelodysplastic Syndrome

Background and ObjectiveWnt signaling, as a fundamental role in embryogensis in all metazoans, is actively regulating adult tissue renewal and organ homeostasis. The accumulated data suggest, the abnormal sign of Wnt is the source of many different kinds of tumors (Rectal cancer, breast cancer, Prostate cancer, pancreatic cancer, and leukemia and so on),arthritis, nephritis and other diseases. As regards to the clearest one that has been studied most, it is the classic pathway, that is, Wnt/β-catenin pathway. At present, many kinds of studies have proved the classic access plays an important part in the development of the embryos and tumors. Various kinds of study results have proved the expression level of the β-catenin in tumors has been obviously improved. The transferring ability of the mouse tumor in which β-catenin has been knocked has been reduced, and by the disturbing technique it is found that SiRNA aimed at the β-catenin can restrain the growth of the tumors. All of these show that targeted therapy aiming at Wnt/β-catenin may become a new way to treat the tumors.MDS (Myelodysplastic Syndromes) is a kind of disease owning a group of candidate stem cells with heterogeneity and clonality, whose biological feature is the abnormal growth and ineffective hamatopoiesis of the medullary system or many systems, along with the increasing of the origin cells. The high-risk group of MDS is often changed into the leukemia. It is common that the hot Wnt/β-catenin access is studied in the leukemia, but in MDS the study of Wnt/B-catenin access is also rare. In the overseas this kind of research is rare, too, while the study of WNT/β-catenin signaling access is mainly focused on the family of the antagonist in China, without any report directly on the study of WNT/β-catenin signaling access. This experimental study applies the ways of RT-PCR and Western blotting to examine the expression level of β-catenin of the single nucleus cell in the MDS patients, and analyze the meaning the β-catenin expressed in different risk groups and the relationship between β-catenin and clinical features. Its aim exists in exploring the possible effects Wnt/β-catenin signaling access has during the production and development of MDS and whether it can help to judge and forecast.Material and Methods1Study ObjectsAll of objects are the inpatient or outpatient cases of the hematology apartment in the First Affiliated Hospital of Zhengzhou University from April,2011to December,2011. MDS patients are up to the diagnostic standards of MDS after a series of examinations, including blood tests, analysis of bone marrow cells, immunology, cytogenetics and molecular biology. Immunology keeps an record about the clinical data, like every patient’s age, gender, karyotype and whether to change into the leukemia.The group with the integral of IPSS less than1.5is called low-risk group, while the group with the integral of IPSS more than1.5is called high-risk group, that is, in the classification of the risk degree of IPSS integral, the low-risk and middle-risk Ⅰ are grouped into the low-risk group, and middle-risk Ⅱ and high-risk are grouped into the high-risk group.Karytope defined as Good:Normal,-Y, del(5q), del(20q); Poor:chromosome7anomalies, complex (3or more abnormalities) and Intermediate:all others).(1) The low-risk group of the patients with MDS includes18cases, with the6male cases and the12female cases. The median age is50, and there are2cases of the prognosis karyotype.(2) The high-risk group with MDS includes21cases, with the7male cases and14female cases. The median age is54, and there are11cases of the prognosis karyotype.(3) The group of leukemia developing from MDS includes3cases, with1male case and2female cases. All of these cases have been changed into AML-M2, and there are3cases of the prognosis karyotype.(4)The controlled group includes14cases, including the8male cases and the6female cases. The median age is42. The bone marrow samples are from non-malignant hematologic diseses or HSCT donors.2Experimental Methods:(1)The way of RT-PCR examines the B-catenin mRNA level of the single nuclear cell in the bone marrow. Select the study object---marrow sample, separate the single nuclear cell, extract the total RNA of the cell by the way of TRIZOL and transcribe them into cDNA, and finally the last PCR expands to30circulations. After the electrophoresis of the ago-gel and the photo of UV lamp, all the expression results are analyzed by grey analysis and calculate theratio between the B-catenin and B-actin.(2)The way of Western blotting is to detect the protein level of B-catenin in the marrow. Select the marrow aspirate of the study object, centrifuge it, and add the lysis buffer of PIPA and proteinase inhibitor. Select some protein, and use BAC determination protein. Select25μg protein, and this protein will be separated by the electrophoresis of100g/L SDS. MaSiliang blue inspection checks the electrophoresis effect of protein, and electricity has been transferred to the film of PVDF. The film of PVDF is kept for10hours at4degree of the refrigerator. Put the film into the container with the confining liquid(defatted milk powder at5%), and seal it up for one hour in the table concentrator. Add the monoclonal antibody of B-catenin that is anti-mice and anti-human, dilute it at the proportion of1:2000, and sway it slowly for incubation for two hours in the table concentrator at room temperature. Use the detection of rat of protein marked with HRP to express. Use the imaging system to do the scanning of optical density, and deal with the scanning results statistically.(3)Data Analysis:the application of SPSS17.0to analyze the experimental data. The qualitative materials are analyzed through the Chi-square test(including Fisher’s test of accurate probability).The quantitative materials are expressed by±SD: analysis of variance among the means of many samples, and t-test applied between two independent samples. The correlation analysis uses the Spearman at the testing standard of a=0.05.Result1RT-PCR is to examineβ-catenin mRNAThe comparison of expression rates among56sample cases:the expression rate of the control group is21.4%, the expression rate of the low-risk group is61.1%, the expression rate of the high-risk group is95.2%, and the expression rate of the blanching group is100%. All of these have statistically significants(expression rates compareχ2=22.154, P<0.05). From the comparison between the two, there is statistically significant shown through the difference between the control group and low-risk group (Fisher=O.036, P=0.028<0.05), there is no statistically significant between the high-risk group and blanching group (Fisher=1.000, P=0.875>0.05), and there is statistically significant between the low-risk group and high-risk group (Fisher=0.015,P=0.012<0.05). As regards to the comparison of expression level, do the independent t-test respectively to compare the normal control group and low-risk group, and the low-risk group and high-risk group. In the comparison of the normal control group and low-risk group,t=0.982, P=0.346>0.05, showing statistically significant, while in the comparison of low-risk group and high-risk group, t=4.349, P<0.05, showing statistically significant.2Western blotting is to examine the expression of B-cateninThe results show that among the sample of56people, the expression rate of the Control group, which includes14people, is zero, while that of the patient sample is54.8%. And the expression rates of the low-risk group and the high-risk group are respectively11.1%and85.7%. The case of turning into leukemia is rate. The3patients are all expressed. The expression rates of the high-risk group and the group of turning into leukemia are obviously higher than those of the low-risk and the control groups, and the expression rate of low-risk group is a little higher than that of the control group.The comparision between expression rates:χ2=38.03, P<0.05. The expression differences is of statistical significance. The comparison between the low-risk and the high-risk group has significant significance in statistics (Fisher=21.038, P<0.05), while neither that between the control group and the low-risk group (Fisher=1.607, P=0.308) nor that between the high-risk group and the group of turning into leukemia has (Fisher=0.469, P=0.657).As regards to the comparison of expression level, do the independent t-test respectively to compare the normal control group and low-risk group, and the low-risk group and high-risk group. In the comparison of the normal control group and low-risk group, showing statistically significant(t=7.920, P<0.05). And the difference got from the independent test between the samples of the low-risk and the high-risk groups has significance in statistics (t=12.890,P<0.05)。3The correlate analysisThe correlate analysis between p-catenin and karyotype shows that β-catenin mRNA and the protein level of β-catenin are both related to the karyotype which is poor.Conclusions1.If the β-catenin and mRNA in MDS and the the protein level rise, which shows β-catenin perhaps has contributed to the cause of MDS and played an important role in the development from low-risk to high risk. The Target treatment for β-catenin is likely to be the new treatment for MDS.2.β-catenin is related to karyotype which is poor, which means it perhaps also related to MDS prognosis.