Function Research Of RBMS1 Gene In Migration And Invasion Process Of Triple Negative Breast Cancer

Objective:Triple negative breast cancer(TNBC)is a type of breast cancer in which estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER2)are all negative.TNBC is more malignant than other molecular typecast breast cancers,which is characterized by high recurrence and metastasis rate,strong aggressiveness,rapid progression and poor prognosis.We expect to find the gene specifically expressed in TNBC and reveal its function,providing clues for the development of TNBC specific targeted drugs.Methods:1.Through bioinformatics technology,the expression of RBMS1 gene in different molecular types of breast cancer in TCGA database was analyzed,and survival analysis and GSEA analysis were performed on RBMS1;2.Knockdown of RBMS1 in two TNBC cell lines(MDA-MB-231 and HCC1937)by lentivirus transfection system,and detect the knockdown efficiency by reverse fluorescence quantitative PCR(qRT-PCR)and Western blotting;3.Upon RBMS1 knockdown in TNBC cells(HCC1937 and MDA-MB-231),the Transwell experiment was conducted to detect the migration and invasion ability;4.The expression level of EMT-related markers were detected by qRT-PCR and Western blotting;5.The situation of cell apoptosis and cell cycle was detected by flow cytometry,and cell proliferation was detected by MTS;6.Overexpression of RBMS1 in non-TNBC cell line MCF7,and Transwell cell migration and invasion experiments were conducted after the overexpressed efficiency was detected by qRT-PCR;7.MDA-MB-231 cell lines with knockdown of RBMS1 were sent to the company for transcriptome sequencing,and correlation analysis was conducted after the data was returned:confirming knock down efficiency,performing gene function enrichment analysis,and analyzing downstream differentially expressed genes;8.QRT-PCR assay verified the changes of RBMS1 downstream differentially expressed genes in MDA-MB-231 cell lines.Results:1.Bioinformatics analysis and laboratory results all suggested that RBMS1 gene was specifically highly expressed in TNBC and poorly expressed in non-TNBC cell lines.Survival analysis suggested that RBMS1 expression was associated with poor prognosis.GSEA analysis indicates that RBMS1 is related to EMT process;2.The qRT-PCR assay confirmed that RBMS1-shRNA could significantly inhibit the expression of RBMS1 mRNA in MDA-MB-231 and HCC1937 cell lines(P<0.001).Western blotting experiments indicated that the protein expression level of RBMS1 in MDA-MB-231 cell line was significantly decreased;3.Knockdown of RBMS1 can inhibit the migration and invasion abilities of MDA-MB-231 cells(P<0.001)and HCC1937 cells(P<0.05);4.Upon RBMS1 knockdown in MDA-MB-231 cells,the expression levels of ZEB1(P<0.001)and Fibronectin(P<0.01)were decreased in the qRT-PCR experiment,while the expression levels of E-Cadherin were increased and the expression levels of N-Cadherin,ZEB1 and Vimentin were decreased in the Western blotting experiment.Upon RBMS1 knockdown in HCC1937 cells,the expression level of E-Cadherin was increased in the qRT-PCR assay(P<0.05),and the expression level of N-Cadherin was decreased in the cell immunofluorescence assay;5.The results of flow cytometry analysis indicated that the apoptosis rate of MDA-MB-231 cells in the RBMS1 knockdown group and the ratio of G1/S/G2 in each period of cell cycle were not significantly different from those in the control group.The results of MTS assay showed that knockdown of RBMS1 had no effect on cell proliferation(P>0.05);6.The qRT-PCR assay confirmed that RBMS1 was effectively overexpressed in MCF7(P<0.001),and the migration and invasion abilities of MCF7 cells were significantly enhanced after overexpression of RBMS1(P<0.001);7.Transcriptome data of MDA-MB-231 cell lines with knockdown of RBMS1 confirmed that the expression of RBMS1 gene was inhibited(P<0.01),and gene function enrichment analysis suggested that RBMS1 was related to EMT process;8.Transcriptome data were analyzed to obtain downstream differentially expressed genes CST1,CST2,CST4,IL-1B,ITGA10,and SPDEF,etc.qRT-PCR confirmed that mRNA expression levels of CST1,CST2,CST4,SPDEF,and ITGA10 decreased after knockdown of RBMS1.Conclusion:1.RBMS1 was specifically highly expressed in triple negative breast cancer,and high expression of RBMS1 was associated with poor prognosis;2.RBMS1 promotes the migration and invasion of TNBC cells;3.RBMS1 regulates the migration and invasion process of TNBC through the EMT signal pathway.The expression of RBMS1 was negatively correlated with the expression of E-Cadherin,and positively correlated with the expression of N-Cadherin,Fibronectin,ZEB1 and Vimentin;4.RBMS1 is correlated with downstream differentially expressed genes such as CST1,CST2,CST4,IL-1B,ITGA10,and SPDEF.