| Breast cancer is one of the most common malignant tumors in women and a major killer of women’s health.The occurrence of breast cancer is closely related to life habits.In European and American countries,the incidence of breast cancer ranks first among female malignant tumors.In China,with the continuous improvement of people’s living standard and the change of life style,the incidence of breast cancer is increasing year by year.It is estimated that the number of breast cancer patients in China will reach 2.5 million by 2020,and the patients will gradually become younger.At present,the treatment of breast cancer mainly includes surgery,radiotherapy,chemotherapy,endocrine therapy,drug therapy and molecular targeted therapy.Despite the continuous improvement of medical level in recent years,the research on the pathogenesis of breast cancer has made great progress,but its characteristics of high incidence and low survival rate have not been improved.Postoperative recurrence and distal metastasis are the main causes of death.Epithelial-mesenchymal transition(EMT)is an important factor in the ability of tumor cells to acquire invasion and metastasis.It refers to the process in which epithelial cells lose their epithelial phenotype and transform into mesenchymal cells through specific procedures.Epithelial-mesenchymal transformation is involved in many biological processes:embryonic development,organogenesis,wound repair,tissue regeneration,organ fibrosis and invasion and migration of tumors.Epithelial-mesenchymal transformation promotes the occurrence of malignant phenotypic transformation of tumors.It is an important reason that induces breast cancer cells to break through extracellular matrix and vascular basement membrane into circulatory system,and then invade and migrate,forming distant secondary metastases.Therefore,looking for the molecules involved in regulating Epithelial-mesenchymal transition in breast cancer and elucidating its regulatory mechanism will provide new ideas and targets for future research and treatment of breast cancer.Stem cells(SC)are self-replicating multipotent cells that can differentiate into multiple functional cells under specific conditions.Studies have shown that abnormal regulation of normal stem cells can lead to abnormal expansion,which increases the susceptibility of cancer,and is considered to be one of the sources of cancer stem cells.Cancer stem cells(CSC)were proposed by Mackillop in 1983.He believed that there might be some cancer stem cells with self-renewal ability that could differentiate into other types of tumors and initiate the formation of tumors.More and more studies have confirmed that cancer stem cells play a decisive role in maintaining malignant proliferation,invasion,drug resistance,metastasis and recurrence of tumors.SIRT4 is an important member of the Sirtuins protein family.Sirtuins protein family is a highly conserved nicotinamide adenie diueleotide(NAD)-dependent deacetylase(HDAC).The family includes seven members,SIRT1-SIRT7,which regulates many biological processes including apoptosis,aging and carcinogenesis,and is associated with many diseases such as cancer,cardiovascular disease and diabetes mellitus.Occurrence and development are closely related.SIRT4,unlike other members of the family,has been shown to contain conserved deacetylase domains,but not deacetylase activity.It has ADP-ribosyltransferase activity and is involved in regulating the catabolism of various nutrients in cells,such as glutamine metabolism,glucose metabolism,lipid metabolism and so on.SIRT4 can regulate intracellular energy metabolism by regulating glutamine catabolism.Studies have shown that S1RT4 can inhibit the occurrence and development of various cancers,including hepatocellular carcinoma,gastric cancer,lung cancer,prostate cancer,etc.However,whether SIRT4 plays a regulatory role in the occurrence and development of breast cancer,has not been systematically reported.In this study,we mainly discussed the regulation of SIRT4 on EMT and invasion and metastasis of breast cancer cells,and found that SIRT4 also plays an important role in regulating the stem of breast cancer cells,the research content is mainly divided into the following three parts:1.Clinical significance of SIRT4 in breast cancer tissues:Bioinformatics analysis technology was used to analyze the correlation between SIRT4 expression level and malignant degree of breast cancer patients in TCGA database and GEO database,and use Kaplan-Meier Analytical method in the TCGA database to analyze whether the expression level of SIRT4 is related to the survival prognosis of patients,and initially confirm that SIRT4 has a regulatory effect on the occurrence and development of breast cancer.The expression of SIRT4 in breast tissue microarray(including normal breast tissue,in situ cancer tissue and breast cancer tissue with metastatic characteristics)was detected by immunohistochemical staining to further verify the correlation between the expression of SIRT4 and breast cancer metastasis.2.Inhibitory effect of SIRT4 on EMT and invasion and migration of breast cancer cells:In four breast cancer cell lines(MDA-MB-468,MDA-MB-231,MCF7,BT549)and a normal breast epithelial cell line(MCF10A),the basic expression of SIRT4 was detected by qRT-PCR and Western blot.According to the detection results,the SIRT4 overexpressing breast cancer cell line MDA-MB-231 and the SIRT4 knockdown breast cancer cell line MCF7,and their corresponding control cell lines were constructed by using lentiviral transfection technology.The effects of SIRT4 on proliferation,invasion,migration and EMT of breast cancer cells were detected by MTT,clonogenesis assay,Western blot,immunohistochemical staining,Transwell,Matrigel,cell scratch and tumorigenesis in nude mice.3.Studies on the characteristics of stem cells regulated by SIRT4 in breast cancer cells:RNA-seq assay confirmed that the decrease of SIRT4 expression can lead to the change of expression of marker genes related to breast cancer stem cells and the change of signal pathway related to breast cancer stem cells.Western Blot and qRT-PCR results showed that the expression of SIRT4 in SP cells and breast cancer stem cells was significantly lower than that in non-SP cells and normal breast cancer cells.In addition,flow cytometry and Sphere Formation experiments further confirmed that the reduction of SIRT4 expression could promote the formation of breast cancer stem cells.The successful progress of this project is expected to reveal the new mechanism of recurrence and metastasis of breast cancer,clarify the specific role of SIRT4 in the occurrence and metastasis of breast cancer and its clinical significance,and provide new ideas and targets for the diagnosis and treatment of breast cancer. |
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