Study On Subtype Transformation Of Breast Cancer By Multi-Gene Editing System

Breast cancer is divided into different subtypes due to its high heterogeneity,marking the entry of breast cancer treatment into the era of precision medicine.For most breast cancer subtypes,there are clinically relevant treatments.For example,Luminal subtype breast cancer is usually treated with endocrine therapy,and HER2 overexpression subtype breast cancer adopts targeted drug therapy.However,Its lack of effective targets leads to limited treatment for triple-negative breast cancer.Patients are prone to recurrence and have a poor prognosis.At present,scholars have tried to treat the triple-negative breast by new treatment methods,but the results have been minimal.Therefore,this study proposes a treatment strategy for triplenegative breast cancer subtype transformation by means of somatic cell reprogramming concept,and achieves a treatment strategy by reducing the malignant degree of triple-negative breast cancer and changing the tumor-associated treatment of cells.Bioinformatics analysis and literature support have locked in key genes affecting breast cancer subtype transformation.The study using the self-constructed CRISPR-dCas9 multi-gene editing system for multi-gene regulation,and has verified from in vitro functional studies,drug response and mechanism exploration.The results laid the foundation for the development of precision medicine for triplenegative breast cancer.The main results are as follows:(1)Using the characteristics of self-shearing of endogenous tRNA,tRNA and sgRNA were combined in series to realize the construction of CRISPR-dCas9 multi-gene editing system.In the breast cancer MCF7 cells,the four transcription factors of OCT4,KLF4,MYC and SOX2 were successfully expressed and the stable cell line OKMS was obtained.In the breast cancer MDAMB231 cells,both ANLN and KDR genes were simultaneously expressed to obtain stable cell line AdKu.(2)Cell migration ability,proliferation ability and stem cell proportion in OKMS and AdKu cell lines before and after gene regulation was detected.In the OKMS cell line,the invasive ability was enhanced,the proliferation rate was increased,and the proportion of stem cells was significantly increased.In the AdKu cell line,the invasive ability,proliferative ability,and stem cell ratio were all decreased,and the degree of malignancy was decreased.(3)The expression levels of the typing markers of the OKMS and AdKu cell lines was quantified In the OKMS cell line,ER was lowly expressed,HER2 was highly expressed,and cell subtype was changed from Luminal A to HER2 overexpression.In AdKu cell line,ER was highly expressed,HER2 was not significantly changed,and cell subtype was changed from triple negative breast cancer to Luminal A(4)The response of two cell lines,OKMS and AdKu,upon drugs treatment was measured.In the OKMS cell line,this cell line is as sensitive to the targeted drug Herceptin as the HER2 overexpressing breast cancer cell SKBR3.It is found that the gene regulation affects the MAPK and NFκB signaling pathways through phosphorylation of the signaling pathway.Subtypes of breast cancer cells have changed due to the AdKu cell line is as sensitive to the drug tamoxifen as the cell line MCF7.