EnChIP Identified Pcl2 Regulating Cap Cell Phenotypes In Mammary Glands

Background The mammary gland is a unique glandular organ that distinguishes mammals from other animals.Mammary glands develop in the following stages: embryo,preadolescence,adolescence,pregnancy,lactation and degeneration.In each stage,mammary cells are stimulated by hormone to proliferate,differentiate,or apoptose,causing significant remodeling of the glandular tissue architecture.Therefore,in the study of mammary gland development,it is significant to explore the the mechanism,prevention and treatment of breast cancer.Par3L is expressed at low levels in tissues,and restricted in few cells.In previous studies Par3 L is found to express in cap cells of the terminal end buds.Par3 L does not interact with a PKC.Instead,it binds to the tumor suppressor protein Lkb1 and inhibits its kinase activity.Moreover,the loss of Par3 L in mouse mammary cells led to apoptosis of stem cells.Therefore,it is of great significance to find how Par3 L is regulated.Aims: Optimize the en Ch IP technology,and identify fate determinants of cap cells of the terminal end bud.Methods: We constructed p LVTHM-2A-d Cas9-sg Par3 L cell line and used immunofluorescence staining and Ch IP assay to test the specificity of pard3 b guide RNA;we then captured proteins by d Cas9 Ch IP and analyzed to find the protein molecules associated to the promoter of pard3 b gene.Immunofluorescence staining of mammary tissues and Ch IP assay was carried out to verify that candidate proteins associate to the pard3 b gene promoter region in mammary cells.We overexpressed pcl2 in mammary cell lines Comma-1D and analyzed the corresponding phenotypes.We used luciferase assay to detecte the regulation of pcl2 on the transcription level of pard3 b gene promoter.The CCK8 assay,MTS assay,and Ki67 immunofluorescence staining assay was conducted to detect the regulation of pcl2 overexpression on cell proliferation.Results:(1)The p LVTHM-2A-dcas9-Sg Par3 L cell lines were successfully constructed;Immunofluorescence staining on these cells showed that d Cas9 was recruited in a few focal points in the nucleus,compared to the difuse staining in control cells.Ch IP assay on d Cas9 showed significant enrichment of pard3 b gene promoter region;(2)Mass Spectrometry showed that pcl2 was co-precipitated with pard3 b gene promoter;(3)Immunofluorescence of mammary tissues,found that pcl2 expressed in both myoepithelial cells and luminal epithelial cells,pcl2 Ch IP assay showed significant enrichment of pard3 b gene promoter region,compared to the control;(4)pcl2 overexpression increased the ratio of CK5 and decreased the ratio of CK8 positive cells;(5)CCK8 assay and MTS assay showed that pcl2 inhibit cell proliferation;(6)statistical analysis used t test,and p< 0.05 with statistical significant.Conclusion:(1)pcl2 binds to the pard3 b promoter region;(2)pcl2 promote myoepithelial cell phenotypes and supresss luminal cell phenotypes;(3)pcl2 inhibit cell proliferation.