Effects Of BRWD3 On Microfilament Cytoskeleton And Migration Of Breast Cancer Cells

Previous studies have revealed that mutations of BRWD3(Bromodomain and WD repeat domain containing 3)are associated with several types of tumors and mental diseases.However,the causal link between the gene mutation,protein expression level changes and related diseases has not been established yet,because of insufficient research on the molecular function of BRWD3.In the Drosophila,it is found that d BRWD3 regulates biological rhythms and affects cell morphology.Cell morphology is affected when BRWD3 is knocked down with the method of RNAi in the cell lines of S2R~+,He La,MCF-7 and MDA-MB-231,which includes our previous studies in the last two cell lines.It means that molecular function of BRWD3 is highly conserved in the regulation of cell morphology.We wondered that if increasing BRWD3 expression levels would affect cell migration and cell morphology,and which may by through regulating the microfilament cytoskeleton.This study included two parts.Part 1:Effect of increasing transcript level of BRWD3 on cell motility and morphology:CRISPR/Cas9 technology was used to activate the endogenous expression of BRWD3 in the two cell lines of MCF-7 and MDA-MB-231.Effect of over-expression of BRWD3 on cell motility was explored with cell scratch assay.Further,cell morphology and the formation of microfilament cytoskeleton were observed with cell immunofluorescence.The result showed that ability of cell motility was improved,and cell morphology and microfilament cytoskeleton were changed,when BRWD3 was endogenous activated in both cell lines.Part 2:Preliminary studies on the mechanism of BRWD3 affecting the microfilament cytoskeleton:For its molecular function on cytoskeleton remains obscure,first,we conducted bioinformatics analysis on the physicochemical properties of BRWD3,and its role in regulation on microfilament.Since BRWD3 has been suggested as a chromatin modifier and a transcriptional regulator,the methods of Ch IP-sequence combining quantitative Real-time PCR were used to screen the genes related to microfilament,and explored the role of BRWD3 in regulation on microfilament.All of the results showed that microfilament was affected,and expression levels of Rho C、ARPC3、ARPC1A and ENAH were increased,when BRWD3 was endogenous activated in both cell lines.Conclusion:BRWD3 were endogenous activated in this study,in contrast to previous studies.We found that alteration of the expression level of BRWD3 would affect the microfilament cytoskeleton,through promoting the formation of stress fibers,causing microfilaments to aggregate at the leading edge of cells,which may explain the alteration of BRWD3 level resulted in cell morphological change and to promote cell movement.It may be helpful for understanding the mechanism of diseases related to BRWD3 abnormality,and provides a reference for the diagnosis and intervention of these diseases.