Analysis On Changes And Functions Of FOXD1 In Different Periods Of Human Breast Cancer Tissue

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.