摘要
为了验证转录因子FOXD1(Forkhead D1)在人乳腺癌组织不同时期的表达差异,本实验采用实时荧光定量PCR和Western blotting对浸润性乳腺癌和乳腺癌癌旁组织中FOXD1的表达做了分析。在此基础上,以浸润性乳腺癌为实验材料,应用ChIP技术初步富集了FOXD1作用的目的DNA。
本实验首先对所收集的乳腺癌病例样本进行冰冻切片和HE染色分析,然后按照医学分类学方法对病例组织进行病理组织学分类和临床分期,将病例样本分为浸润性乳腺癌、乳腺原位癌、乳腺癌癌旁组织三类。
在HE染色分析的基础上,我们选取了4例浸润性乳腺癌样本和4例乳腺癌癌旁样本作为实验材料,分别提取了这8例组织的总RNA和核蛋白进行了实时荧光定量PCR实验和Western blotting实验,实验结果利用生物统计学软件进行分析。在Western blotting实验分析的基础上,我们分别选取了两例FOXD1表达量相对较高的浸润性乳腺癌和乳腺癌癌旁组织作为实验材料,利用ChIP技术富集FOXD1作用的目的基因DNA片段,并通过核酸定量测定所得到的DNA片段的浓度。
实验数据分析表明,无论是转录水平还是翻译水平,FOXD1在浸润性乳腺癌组织中的表达量均明显高于乳腺癌癌旁组织,且表达差异显著。核酸定量的结果表明,所富集的DNA能够进行下一步实验。
Breast cancer is a malignant tumor that starts from cells of the breast.A malignant tumor is a group of cancer cells that may invade surrounding tissues or spread to distant areas of the body.Most breast cancers begin in the cells that line the ducts or lobules.According to the modern medical studies,cancer occurs as a result of mutations or abnormal changes.Activation of oncogenes and deactivation of anti-oncogenes cause the loss of control on the cell growth and differentiation,which finally results in cancer.The transcription is a key process for DNA to transfer genetic information to proteins as well as a main period for genes to express and regulate.Most genetic expression of eukaryotic organisms is regulated at the moment of initiating transcription,which is considered as the key point of regulating gene expression.During the occurring and developing processes of cancer, transcription factors interact with oncogenes and anti-oncogenes and mediate their expression.Therefore,it's necessary for us to study transcription factors with priority before we research genes relevant to cancer.Discovering the target genes interacting with transcription factors can be helpful for the further research on cancer.
FOX,as a transcription factor,widely exists in eukaryotic cells.Scientists have identified more than 100 members in 17 subgroups of FOX family.The FOX family, sharing a common DNA binding domain(DBD) and transcriptional regulating domain,recruit reactivators and interact with the factors of transcription complexes to activate transcription.The Functions of the FOX family cover many processes such as embryonic development,cell cycle control,carbohydrate and lipid metabolism,organism aging and immunological regulation.The mutation and abnormal expression of the FOX family are connected with development malformation,metabolic diseases and tumor occurrence.
FOXD1(or BF2,FREAC4) belongs to the member of the FOX family,which is located on the q12-q13 of the No.5 human chromosomal.The studies reported show that FOXD1 plays an important role in the kidney and amphiblestroid development and also regulates the activities of NF-AT and NF-γB.The researches have focused on the kidney differentiation during the process of embryonic development by now, while other functions of FOXD1 are seldom studied.The up-regulation of the FOXD1 expression in breast cancer tissues has been found in analyzing the protein chips,which probably reflects that FOXD1 may take effect in the occurring and developing processes of breast cancer.Accordingly,it is indispensible to test different changes of FOXD1 during occurring periods of breast cancer and then study its transcriptional regulating function further.
The samples of this experiment were breast cancer tissues and normal breast tissues collected from August,2007 to September,2008.Pathohistology classification and clinical stage were carried out with the use of frozen section and HE stain.Then four samples of invasive breast cancer tissues and another four samples of normal breast tissues were examined by Western blotting and qRT-PCR respectively,which testified changes of FOXD1 expression.On such a basis,two specimens of invasive breast cancer tissues were measured by ChIP to obtain the target genes that FOXD1 regulates.
After analyzing all samples with frozen section and HE stain,we classified samples into invasive breast cancer tissue 36,breast tumor in situ tissue 8,normal breast tissue 77 according to standards of pathohistology.In addition,some samples that were not fit for experiments had been dealt with appropriately under the guidance of relevant national regulations.
We examined FOXD1 by qRT-PCR,which suggested that FOXD1 expression increased significantly in invasive breast cancer tissues as compared with that in normal breast tissues.Furthermore,the relative FOXD1 expression of the four samples reached 0.88,10.23,15.50 and 1.40 in invasive breast cancer while the expression of another four samples were 0.1,0.19,0.06 and 0.03 respectively in normal breast tissues.Therefore,the result showed that FOXD1 expression was significantly different(P＜0.01) through the statistic analysis.
The result verified by Western blotting also demonstrated that FOXD1 increased significantly on the translational level.Moreover,FOXD1 expression of four samples in invasive breast cancer tissues were7.63,4.69,6.89 and 11.57 whereas FOXD1 expression reached 2.01,1.06,2.59 and 2.80 in normal breast tissues.Therefore,invasive breast cancer tissues expressed more FOXD1 than normal breast tissues with the significant difference(P＜0.05) through the comparison of the above eight datas.
Based on the above experiments,we performed ChIP by selecting two samples of invasive breast tissues with comparably high FOXD1 expression.As a result, DNA combined by FOXD1 proteins was enriched in the first period of ChIP.
Through this experiment,we verified that FOXD1 expression increased in breast cancer tissues on the transcriptional or translational level.Besides,the results showed that FOXD1in the breast cancer tissues were significantly different from that in the normal breast tissues,which will contribute to studying functions of FOXD1 in the occurring and developing process of breast cancer.Additionally,we enriched four target DNA samples regulated by FOXD1 through ChIP,which can be used in the following experiments.
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