| [Objective]1.To explore the role of ER81 transcription factor in the breast carcinogenesis by detecting ER81 expression in malignant and benign breast lesions. 2.To investigate if the three expression systems which siRNAs targeting against human ER81 could inhibit the expression of ER81 and enhance the apoptosis of breast cancer cell line MDA-MB-231,and provide a new approach for breast cancer therapy.[Methods]1.By immunohistochemical EnVision method we detected ER81 protein expression in 20 cases of normal breast tissue,21 cases of usual ductal hyperplasia,20 cases of breast atypical ductal hyperplasia,20 cases of ductal carcinoma in situ and 89 cases of invasive ductal Carcinoma.2.Three siRNA sequences targeting against ER81 inserted into pGenesill expression vector by us before.With scrambled siRNA(group SS) and empty vector(group EV) as control,recombinant plasmid pGenesil-ER81A-siRNA(group A),pGenesil-ER81B-siRNA(group B), pGenesil-ER81C-siRNA(group C) was transferred stably into human breast cancer MDA-MB-231 by lipofectamine2000 respectively.In this part,using above five stably transfected MDA-MB-231 cells and untransfected cells(untransfected group, group UT),we detected the efficiency of transcription of ER81 by real-time Q-PCR; and measured cell proliferation,cell cycle and cell apoptosis by MTT,colony-forming unit assay and flow cytometry.[Results]1.In normal breast tissue,usual ductal hyperplasia,breast atypical ductal hyperplasia,ductal carcinoma in situ and invasive ductal carcinoma ER81 positive expression rates were 0%(0/20),9.5%(2/21),15%(3/20),30%(6/20) and 42.7%(38/89).Statistical analysis showed that there is statistically significant difference between five groups(P＜0.01).The positive expression rate of normal breast tissue is not significantly different from that of breast atypical ductal hyperplasia(P=0.488) but is significantly different from that of ductal carcinoma in situ(P=0.020);usual ductal hyperplasia demonstrated lower positive expression rate of ER81 than invasive ductal carcinoma(P=0.005);breast atypical ductal hyperplasia generally demonstrated slightly lower levels of ER81 expression than invasive ductal carcinoma(P=0.023);ductal carcinoma in situ generally demonstrated slightly lower ER81 expression than that in invasive ductal carcinoma,but the difference is not statistically significant(P=0.326).2.By real-time Q-PCR,group A and C inhibited 72.6%and 65.8%ER81 mRNA expression when compared with group UT,the difference had statistically significant(P＜0.05);group B decreased a 34.0%ER81 mRNA expression,but the difference had no statistical meaning(P＞0.05).3.MTT showed that the absorbance in group A and C was lower than that in group UT,group SS and group EV in 3 days after inoculated(P＜0.01);group B demonstrated lower absorbance than that in control cells in 5days after inoculated(P＜0.05);4 days after inoculated,group A and C showed lower absorbance than that in group B(P＜0.05); these results illustrated that the growth of MDA-MB-231cells was inhibited significantly in groups A,B and C,especially in group A and C.4.The results of the colony-forming assay showed that the colonies in group A and C were less than that in group UT,SS,EV and B when seeded 250 and 500 cells(P＜0.01),the cloning efficiency in group A and C was also lower than that in controls and group B(P＜0.01);in addition,the cloning efficiency of group B was lower than that in controls(P＜0.05).5.Cells-cycle analysis suggested that the percentege of cells in G0/G1 phase was increased but in S and G2/M phase was decreased in group A,C compared with group UT,SS,EV and B(P＜0.01),and the proliferation index was also decreased in group A and C(P＜0.01).6.TUNEL assay showed the numbers of apoptotic cells in group A and C were both greater than that in group UT,SS,EV and B(P＜0.05),also in group B,the TUNEL positive cells was higher than that in controls(P＜0.05).[Conclusion]1,It is suggested in this study that the expression of transcription factor ER81 is involved in the early stages of breast carcinogenesis(both in breast hyperplasia without atypia and atypical breast hyperplasia)and may play an important role in the development of breast cancers.2.The three groups of siRNA targeting ER81 can interfer in gene transcription markedly,inhibit cells growth and proliferation,arrest cells in G0/G1 phase,enhance apoptosis of breast cancer cell line MDA-MB-231,especially in group A and C.3.Our study suggests that strategies designed to ER81 gene silencing may represent promising new strategy for breast cancer therapy. |
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