The Effect Of Knockout/ Silencing Of MUC1 On The Proliferation,Invasion And Migration Of Human Triple Negative Breast Cancer Cells

Object:To study the effect of knockout MUC1 by Crispr/Cas9 gene editing technology and silencing MUC1 by short hairpin RNA(sh RNA)technology on the proliferation,invasion and migration of human triple negative breast cancer(TNBC)cells(Hs578T and MDAMB-231).Analyzing the changes in the transcriptome of Hs578T which knocking out the MUC1 gene,and looking for the possible pathways and mechanisms.Method:1.The expression level of MUC1The GEDS and Biogps were used to analyze the expression level of MUC1 in human normal breast tissue.The TCGA database was used to analyze the expression level of MUC1 in breast cancer and its subtypes.2.Knockout and silencing of MUC1 geneUsing Crispr/Cas9 gene editing technology to knock out the MUC1 gene of TNBC cells(Hs578T,MDA-MB-231),construct 578T-MUC1 KO cell line and 231-MUC1 KO cell line.The expression of MUC1 gene in TNBC cells was silenced by sh RNA technology,and 578T-sh MUC1 cell line and 231-sh MUC1 cell line were constructed.Western Blot verifies the knockout and silence effects.3.The effect of knockout and silencing MUC1 on the proliferation of TNBC cellsCCK-8 assay and colony formation assay were used to detect the effect of knockout and silencing MUC1 on the proliferation of TNBC cells.4.The effect of knockout and silencing MUC1 on the invasion and migration of TNBC cells.The effect of knockout and silencing MUC1 on the invasion ability of TNBC cells was detected by the Transwell assay,and the effect on the migration ability was detected by the scratch assay.5.The effect of MUC1 knockout on gene transcriptome of Hs578T cellsHigh-throughput sequencing was used to analyze the differences in transcriptome gene between Hs578T and 578T-MUC1 KO cell lines,GO analysis and KEGG analysis to find the possible pathways and regulatory mechanisms.Result:1.The expression level of MUC1 in human breast duct cells is the highest,and the expression level of breast stromal cells is the lowest.The expression level of MUC1 in human breast cancer tissues was significantly higher than that in normal breast tissues.Among the subtypes of breast cancer,MUC1 expression level of Luminal subtype is the highest,and of TNBC-LAR subtype is the second.2.Western Blot assay proved that after MUC1 was knocked out by Crispr/Cas9,MUC1 protein was not expressed in Hs578T and MDA-MB-231 cells.After sh RNA silenced MUC1,the expression level of MUC1 protein in Hs578T and MDA-MB-231 cells decreased significantly.3.Whether MUC1 was knocked out or MUC1 was silenced,the proliferation rate and colony forming ability of Hs578T and MDA-MB-231 cells did not change significantly.4.In Transwell,both knocking out MUC1 and silencing MUC1 can effectively reduce the number of Hs578T and MDA-MB-231 cells passing through the chamber,and the difference is significant(P<0.05).In the scratch assay,either knocking out MUC1 or silencing MUC1 can effectively reduce the migration distance of Hs578T and MDA-MB-231 cells.5.Hs578T and 578T-MUC1 KO cells were subjected to high-throughput transcriptome sequencing.Comparison of 578T-MUC1 KO cells and Hs578T cells revealed a total of 1,519 differentially expressed genes,932 up-regulated genes,and 587down-regulated genes.The GO enrichment analysis of differential genes showed that the biological processes were mainly enriched in proteolysis and cell adhesion,and each enriched 53 differential genes.KEGG enrichment analysis is mainly enriched in PI3 KAkt signaling pathway and MAPK signaling pathway,and enriched to 43 and 30 related genes,respectively.13 genes about invasion and migration which are related to MUC1 were found by overlapping the differentially expressed genes with the GSEA CELL MIGRATION dataset.Conclusion:Knockout of MUC1 gene and silencing of MUC1 gene can effectively inhibit the invasion and migration ability of Hs578T and MDA-MB-231 cells,but there is no obvious effect on their proliferation ability.MUC1 may mediate the invasion and migration of TNBC through PI3K-Akt signaling pathway and MAPK signaling pathway,and regulate DPYSL5,UNC5 C,AMOT,GDNF,CX3CL1,ANOS1,SAA1,NEXN,PARP9,CXCL8,PARD6 B,ITGB2,VEGFC.