The Role And Mechanism Of PPAPDC1A In The Occurrence And Development Of Breast Carcinoma

BackgroundBreast carcinoma is ranked as the first incidence in women malignant tumors,the metastasis is the main cause of death in patients with breast carcinoma.Phosphatidic acid phosphatase type 2 domain containing 1A(PPAPDC1A)is one of the important members in phosphatidic acid phosphatase family,which main function is able to dephosphorylate substrate and involved in cell proliferation,movement,and may be related with tumor occurrence and development.The previous research has found that PPAPDC1 A was a differentially expressed gene between normal mammary tissues and breast carcinoma tissues by gene expression profiling,the further study has not been reported.In this research group,the above result is verified by gene expression omnibus(GEO)public database.But whether PPAPDC1 A is related to the occurrence and development of breast carcinoma,and its regulation mechanism remains unclear.ObjectivesThe expression of PPAPDC1 A in normal mammary and breast carcinoma was investigated from the level of cell and histology.The role of PPAPDC1 A in breast carcinoma cells proliferation,metastasis,invasion and its related molecular mechanism were investigated in vitro experiments.In order to clarify the role and mechanism of PPAPDC1 A in the occurrence and development of breast carcinoma.Methods1.The expression of PPAPDC1 A in normal mammary epithelial HBL-100 cells,low metastatic breast carcinoma MCF-7 cells,high metastatic breast carcinoma MDA-MB-231 cells and MDA-MB-435 S cells were detected by real-time quantitative polymerase chainreaction(qPCR)and Western blotting assay.2.The expression of PPAPDC1 A in normal mammary,breast fibroadenoma,breast carcinoma tissues were detected by immunohistochemistry(IHC).3.The expression of PPAPDC1 A in breast carcinoma cells which were transfected PPAPDC1 A recombinant expression plasmid and target siRNA were detected by Western blotting assay;CCK-8 assay was used to detect the breast carcinoma cells proliferation;Scarification assay and Transwell assays were used to detect the migrative and invasive ability of breast carcinoma cells.4.The signal pathway in breast carcinoma with high expression of PPAPDC1 A were analysised by GEO public database and gene set enrichment analysis(GSEA).5.The relationship between PPAPDC1 A and FAK was detected by Western blotting assay,immunofluorescence co-localization and Co-Immunoprecipitation.Results1.The qPCR and Western blotting assay results both showed that: the mRNA and protein expressions of PPAPDC1 A in three different metastatic ability of breast carcinoma cells were significantly increased than the normal mammary epithelial cells(P<0.01).Furthermore the mRNA and protein expressions of PPAPDC1 A in high metastatic breast carcinoma MDA-MB-231 cells and MDA-MB-435 S cells were significantly higher than the low metastatic breast carcinoma MCF-7 cells(P<0.01).2.The IHC results showed that: the positive expression rate of PPAPDC1 A in normal mammary,breast fibroadenoma,breast carcinoma tissues were 11.36%,48.10%,77.08%,respectively.The positive expression rate of PPAPDC1 A gene in breast fibroadenoma tissues(?2=16.774,P<0.01)and breast carcinoma tissues(?2=53.001,P<0.01)were both significantly increased than normal mammary tissues.And PPAPDC1 A positive expression rate in breast carcinoma tissues was significantly higher than breast fibroadenoma tissues(?2=15.799,P<0.01).3.Western blotting assay results showed that: the protein expression of PPAPDC1 A inMCF-7-PPAPDC1 A cells was significantly higher than MCF-7-vector cells(P<0.01).And in MDA-MB-231 and MDA-MB-435 S cells with transfected PPAPDC1A-siRNA were respectively lower than the negative control group cells(P<0.01).Among them,MDA-MB-231-siRNA2 and MDA-MB-435S-siRNA2 cells silencing effect is the best(P<0.01).4.CCK-8 assay results showed that: the growth rate of MCF-7-PPAPDC1 A cells was much more significantly faster than MCF-7-vector cells(P<0.01).And the growth rate of MDA-MB-231-siRNA2,MDA-MB-231-siRNA3 and MDA-MB-435S-siRNA1,MDA-MB-435S-siRNA2 were significantly lower than the negative control group MDA–MB-231-NC and MDA-MB-435S-NC cells(P<0.01).5.Scarification assay results showed that: the migration velocity of MCF-7-PPAPDC1-A cells was obviously faster than MCF-7-vector cells(P<0.01).The migration velocity of MDA-MB-231-siRNA2,MDA-MB-231-siRNA3 and MDA-MB-435S-siRNA1,MDAMB-435S-siRNA2 cells were significantly lower than the negative control group MDA–MB-231-NC and MDA-MB-435S-NC cells(P<0.01).6.Transwell migration and invasion assay results both showed that: the migrative and invasive cell number of MCF-7-PPAPDC1 A were obviously more than the MCF-7-vector cells(P<0.01).The migrative and invasive cell number of MDA-MB-231-siRNA2,MDA-MB-231-siRNA3 and MDA-MB-435S-siRNA1,MDA-MB-435S-si RNA2 were significantly less than the negative control group MDA–MB-231-NC and MDA-MB-435S-NC cells(P< 0.01).7.The GSEA analysis result showed that: Focal adhesion signal pathway was up-regulated in breast carcinoma with high expression of PPAPDC1 A,and the pathway related genes were significantly enriched(P<0.05).8.Western blotting assay results showed that: there was a positive correlation between the protein expression of FAK and PPAPDC1 A in breast carcinoma cells which were transfected PPAPDC1 A recombinant expression plasmid and target PPAPDC1A-si RNA.9.Immunofluorescence co-localization results showed that: FAK and PPAPDC1 A were both located in the breast carcinomar MCF-7?MDA-MB-231 and MDA-MB-435 S cells,which were both located in the cell membrane and cytoplasm.10.Co-IP results showed that: there was exsisting interaction between PPAPDC1 A and FAK protein.Conclusions1.PPAPDC1 A gene is highly expressed in breast carcinoma,which can promote the ability of breast carcinoma cells proliferation,migration and invasion,and it play an important role in the development and evolution of breast carcinoma.2.FAK is a vital molecule target in the above mentioned effects of PPAPDC1 A in breast carcinoma.