| ObjectivesBreast cancer is one of the most common malignant tumors in women all over the world. With the in-depth understanding of breast cancer, people have widely accepted that breast cancer is a systemic disease. On the basis of this understanding, the treatment of breast cancer has developed into a set of comprehensive treatment system including surgery, chemotherapy, radiotherapy, endocrine therapy, biological target therapy and traditional Chinese medicine (TCM) treatment.As a part of breast cancer adjuvant systemic treatment, TCM has attracted more and more attention. Each component of TCM usually has a number of functions, and there also has complex interaction between each component. Therefore TCM usually has multi-target effects. TCM has dialectical and holistic features. So it has unique role and broad application prospects in the clinical application.Huaier is a kind of TCM with anti-cancer effect. It was one of the adjuvant systemetic treatment of primary liver cancer when it came out. In the clinical application, Huaier showed its anti-live cancer effects. However the mechanisms of Huaier still remains unclear. Subsequently, Huaier started to be used in the adjuvant therapy of breast cancer and also showed its anti-cancer effects. As a typical example of TCM, Huaier also has its dialectical and holistic features. Therefore, the study of its mechanisms of action should also start from the comprehensive and integrated perspective. It can not be confined to the traditional research methods.Microarray is a kind of new technology which has been developed rapidly in recent decades. By using microarray technology we can detect thousands or even tens of thousands of genes expression information. The genetic information detected by microarray could be further analyzed and biological functions and signal pathways could be studied. There results will provide guidance to further in-depth studies.The purpose of this paper is to analyze the mechanisms of Huaier in treatment of breast cancer with the help of microarray technology. We will select the biological functions and signal pathways induced by Huaier in breast cancer cells and reveal the mechanisms of Huaier. Our study will provide guidance for the following studies. At the same time, we will locate the key genes in multi-effects of Huaier in breast cancer and research its functions and mechanisms to provide new targets for drug research of breast cancer.MethodsIn order to study the mechanisms of Huaier, we first determined the inhibitory effect of Huaier by using MTT assay. According to the results of MTT, we chose suitable drug concentration and action time to stimulate breast cancer cells. Then RNA was extracted from breast cancer cells which were treated by Huaier to complete microarray assay.Fold-change method was used to identify differentially expressed genes (DEGs) and real-time quantitative PCR (RT-qPCR) method was applied to validate the veracity of microarray results. Then Gene Ontology (GO) analysis was conducted to research the biological functions and Pathway analysis was applied to study the molecular signal transduction pathways of DEGs. Finally, the intersection of DEGs from GO and Pathway analysis were screened out. Under the guidance of Kyoto Encyclopedia of Genes and Genomes (KEGG) database, gene-gene interaction network was conducted and could intuitively reflect the multi-effects of Huaier.With the prompt of microarray results, we used Western blot and RT-qPCR methods to detect the expression changes of potential tumor-suppressor gene, P53-bingding protein 1 (53BP1). To extend function rsearch of 53BP1, we established the breast cancer cells in which 53BP1 was over-expressed or knocked-down for future study by plasmid construction and transfection. Cell morphological observation, Western blot, RT-qPCR and cell immunofluorescence chemistry methods were applied to detect the expressions of epithelial-mesenchymal transition (EMT) markers. Then Transwell assays were applied to research influence of 53BP1 on the ability of migration and invasion of breast cancer cells.In order to clarify the detailed mechanisms of 53BP1, we used Western blot and RT-qPCR methods to detect the expression levels of possible signal pathways. Then we transferred the miRNA mimics into the breast cancer cells with low expression of miRNAs and transferred miRNA inhibitors into the cells with high expression of miRNAs. After that Western blot and Transwell assays were conducted to make sure the roles of miRNA in the regulation of 53BP1 to EMT. In vivo tumorigenesis assay was established and immunohistochemistry assay was conducted to study the expression of EMT markers in the tissue sections to verify the results in vitro. Finally RT-qPCR was applied to confirm our results from the clininal perspective.ResultsMTT assay results showed that the viability of breast cancer cells was suppressed more obviously with the higher concentration and longer reaction time of Huaier. Huaier aqueous extract showed significant dose-and time-dependent effects. According to the results of MTT assay, we chose the concentration of 8 mg/ml Huaier aqueous extract and reaction time of 72 h to treat breast cancer cells. After treatment, cells were collected and DEGs were screened by microarray assay.After treatment of Huaier, the expression of 1655 genes (907 upregulated and 748 downregulated) was changed statistically in MDA-MB-231 cells and 1320 genes (600 upregulated and 720 downregulated) was changed in MCF-7 cells. There were 172 genes which were regulated in both breast cancer cells, including 98 genes up-regulated and 74 genes down-regulated. RT-qPCR method was used to verify the results of microarray assay. And the results showed that the RT-qPCR results were consistent with the microarray.Then GO analysis was applied to identify the altered biological functions of breast cancer cells after treatment of Huaier. The results showed that in MDA-MB-231 cells, 562 GO functions (353 up-regulated and 209 down-regulated) were regulated obviously, and 695 GO functions (307 up-regulated and 388 down-regulated) in MCF-7 cells.129 GO functions (77 up-regulated and 52 down-regulated) were regulated by Huaier in both breast cancer cells.Pathway analysis was conducted to identify the altered signal pathways of breast cancer cells after treatment of Huaier. The results showed that in MDA-MB-231 cells, 104 Pathways (59 up-regulated and 45 down-regulated) were regulated obviously, and 77 Pathways (39 up-regulated and 38 down-regulated) in MCF-7 cells.36 Pathways (8 up-regulated and 28 down-regulated) were regulated by Huaier in both breast cancer cells.At last, we constructed the signal network of MDA-MB-231 and MCF-7 after treatment of Huaier. And we also constructed the common signal network in both breast cancer cells which indicated the multi-target roles of Huaier.With the prompt of microarray results, we found that after treatment of Huaier, the expression of 53BP1, which is a key gene in DNA repair, was up-regulated obviously in breast cancer. This finding was different with other DNA repair genes. According to our former researches, we speculated that 53BP1 functioned as a tumor-supprressor gene in this process. Therefore we expend the studies of 53BP1.During the culture of breast cancer cells which were established to over-expresss or knock-down the expression of 53BP1, we observed that the morphology of cells changed obviously. This indicated that 53BP1 might inhibit the EMT process of breast cancer. In order to clarify this assumption, we detected the expression of EMT markers and found that 53BP1 indeed inhibit the EMT. At the same time, we found that 53BP1 could also restrained the migration and invasion of breast cancer cells. Then we detected the change of EMT transcription factors (EMT-TFs) and found that the expression of ZEB1 was regulated most significantly.53BP1 might inhibit the EMT by regulating ZEB1. micro-RNAs (miRNAs) play important roles in regulation of EMT. And ZEB1 is target of miR-200 family. As a result we detected the expression of miR-200 family. We found that miR-200b and miR-429 were regulated most. Then we interfered miRNA in cells with high expression of miRNA and over-expressed them in cells with low expression of miRNAs and found that the changes induced by 53BP1 were all reversed. All these in vitro results confirmed that 53BP1 inhibited the EMT and migration and invasion in breast cancer by regulating miR-200b-429/ZEB1/E-cadherin pathway. Finally we confirmed our results in vivo and in clinical level.Conclusions1. Huaier has obvious anti-breast cancer effects.2. Inhibitory of Huaier on breast cancer are multi-target effets and covers many important biological functions and signal pathways during the breast cancer progression.3. Huaier up-regulate the expression of 53BP1 which functions as a tumor-suppressor gene.4.53BP1 suppresses epithelial-mesenchymal transition, migration and invasion by regulating miR-200b-429/ZEB1/E-cadherin pathway in breast cancer.Significances1. We revealed the multi-target effects of Huaier in treatment of breast cancer by using microarray assay and provided guidance for future reserches of Huaier.2. We screened out the tumor-suppressor gene 53BP1 from microarray results and provided new target for breast cancer treatment.3. We confirmed the tumor-suppressor gene functions of 53BP1 and found that 53BP1 suppressed epithelial-mesenchymal transition, migration and invasion by regulating miR-200b-429/ZEB1/E-cadherin pathway in breast cancer. |
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