A Study Of The Mechanism Of SIX Family In Regulating Tumorigenesis And Metastasis Of Breast Cancer

Objectives: The SIX family,a transcription factor containing the homeodomain domain,plays an important role in the development of the spine.The SIX gene family has a total of six members: SIX1,SIX2,SIX3,SIX4,SIX5,and SIX6.The SIX family is a homolog of So,Optix and Dsix4 genes in Drosophila,which correspond to SIX1/SIX2(So),SIX3/SIX6(Optix),SIX4/SIX5(Dsix4).The Retinal Developmental Gene Network(RDGN)was first discovered in the ocular development of Drosophila.RDGN is an important regulator of tissue development.The retinal developmental gene network is mainly composed of the following members: dac/Dach;eya/Eya;so/ Six and PAX6-like two homeodomain proteins(ey and toy),which are highly conserved in evolution.The regulation of the development of multiple organ systems is achieved by coding a set of transcription factors or transcriptional cofactors.In this regulatory network,transcription factors are not achieved by simple sequence activation or inhibition of downstream signaling molecules,but rather through sophisticated feedback loops to precisely regulate development.In tumors,the RDGN regulatory network is abnormally regulated,in which the expression of Eya and Six genes is up-regulated,the expression of Dach gene is down-regulated,and the direct balance of self-renewal and differentiation of cancer stem cells(CSC)is also regulated by some RDGNs.In the development of the eye in Drosophila,the loss of So leads to the elimination of the compound eye,Optix expression in the non-retinal tissue induces the formation of the eye,and during the development of the mesoderm,Dsix4 plays an important role,such as muscle,gonad With adipose tissue.Recent reports have found that members of the SIX family are closely related to the development of cancer,but some members of the SIX family(SIX1 SIX2 SIX4)are still unclear about the regulation of breast cancer cell processes.Methods:(1)A eukaryotic expression vector for expressing the SIX1,SIX2,and SIX4 genes was constructed.(2)The protein related to the SIX1 protein was identified by mass spectrometry,and the mass spectrometry analysis results were detected by immunoprecipitation.(3)The GST pull-down method was used to analyze whether SIX1 interacts with MTA1/MTA2 in vitro.(4)SIX1,MTA1 common target gene BRCA1 was screened by reviewing the literature and analyzing the RNA-seq database(GSE93925),and the target gene was verified by luciferase reporter gene assay and chromatin immunoprecipitation assay.(5)A series of phenotypic experiments were performed in cell lines to verify the effect of SIX1 SIX2 SIX4 on proliferation and invasion of breast cancer cells.Results:(1)SIX1 specifically interacts with Nu RD(MTA1/2),but not Nu RD(MTA3)..(2)SXI1 has a direct interaction with MTA1/MTA2.(3)Both SIX1 and MTA1 can bind to the promoter region of BRCA1 and can function as a transcriptional repression to inhibit the expression of the BRCA1 gene.(4)SIX1 SIX2 SIX4 can promote the proliferation and invasion of breast cancer cells.Conclusion: SIX1 and Nu RD/(MTA1/2)form a transcriptional repressor complex,and SXI1 and MTA1 act together on the promoter region of BRCA1,causing transcriptional inhibition of BRCA1,thereby promoting the proliferation and metastasis of breast cancer cells.SIX2 and SIX4 may promote the proliferation and metastasis of breast cancer cells by promoting the EMT process of breast cancer cells.