Down-Regulation Of Filamin A Expression Impacts Biological Behaviour Of HER2 Positive Breast Cancer Cells

Objective:In the present study,the experiments were performed as following:(1)Construction of stably transfected cell lines with knockdown of FLNa.(2)Examining the biological behavior of transfected cell lines,including proliferation,migration and invasion by MTS,Wound healing assay and Transwell cell invasion assay.(3)Western blot was used to detect levels of HER2 phosphorylation and a variety of its downstream signal molecules.Aim to analyze the effects of FLNa on biological behaviour of the cell line MDA-MB-453 and explore the mechanisms of HER2 phosphorylation and its downstream signaling molecules regulated by FLNa.It will be a promissing target for the treatment of HER2 positive breast cancer.Methods:1 Construction of stably transfected cell linesMDA-MB-453 cells were stably transfected either with plasmids expressing FLNa shRNA or control shRNA and selected with puromycin,resulting in MDA-MB-453/KD and MDA-MB-453/Ctrl cell lines.The knockdown efficiency of FLNa was measured by Western blot assay.2 Proliferation assayMDA-MB-453/KD and MDA-MB-453/Ctrl cells were seeded in 96-well plates,respectively.After stimulating with different concentrations of EGF(0nM,4nM,20 nM,100nM)for 48 hours,MTS assay was used to measure the OD values and examine the proliferation ability of stably transfected cell lines.3 Migration assayWound healing assay was used to examine the abilities of stably transfected cell lines.MDA-MB-453/KD and MDA-MB-453/Ctrl cells were seeded in 24-well plates,respectively.Cells were serum-starved for 4 hours,then scratched with a 10?l pipette tip and washed with serum-free medium(SFM)to remove floating cells.Cells were divided into 2 groups: EGF stimulated group and no EGF group.Images of cells at the same field were taken until closure of the scratch.4 Invasion assayThe invasion assays were carried out using Transwell chamber with 10 mm diameter and 8mm pore size polycarbonate membrane coated with matrigel.After starved with serum-free 1640 medium for 4 hours,MDA-MB-453/KD and MDA-MB-453/Ctrl cells were seeded into upper chamber with serum-free 1640 medium,respectively.Lower chamber were added into 10% serum 1640 medium.After incubated for 6 days,the invaded cells were fixed with 95% ice-cold ethanol and then stained with hematoxylin and eosin(HE).5 Western blottingMDA-MB-453/KD and MDA-MB-453/Ctrl cells were seeded in 35 mm dishes,starved with serum-free 1640 medium for 4 hours.After stimulating by EGF for different time(0min,5min,10 min,30min),total proteins were extracted from cells.Western blot was used to examine the levels of FLNa,p-HER2,HER2,p-Akt,Akt,p-ERK,ERK and ?-actin.Results:1 The knockdown efficiency of FLNa in stably transfected cell linesThe relative levels of FLNa protein in MDA-MB-453/KD cells(0.60±0.05)were significantly lower than MDA-MB-453/Ctrl cells(1.05±0.01)(P<0.01).2 Proliferation,migration and invasion abilities in stably transfected cell lines2.1 Proliferation abilities of stably transfected cell linesThe OD values of MDA-MB-453/KD cells stimulated with different concentrations of EGF(0nM?4nM?20nM?100nM)(1.26±0.03,1.39±0.01,1.55±0.02,1.82±0.04)were significantly higher than MDA-MB-453/Ctrl cells(0.88±0.02,0.98±0.03,1.07±0.14,1.38±0.09)(P<0.01,respectively).2.2 Migration abilities of stably transfected cell linesMigration rates of MDA-MB-453/KD cells without EGF stimulating for 4h,8h,12 h,and 16h(23.06±0.14%,23.26±0.14%,39.62±0.04%,47.16±0.16%)were significantly higher than MDA-MB-453/Ctrl cells(7.85±1.08%,7.85±1.87%,12.15±0.53%,20.93±1.39%)(P<0.01,respectively).Migration rates of MDA-MB-453/KD cells stimulated by EGF(20nM)for 4h,8h,12 h,and 16h(30.16±0.62%,36.59±0.77%,42.87±1.53%,50.00±0.00%)were significantly higher than MDA-MB-453/Ctrl cells(13.34±0.51%,14.38±1.18%,15.80±1.56%,25.65±0.43%)(P<0.01,respectively).2.3 Invasion abilities of stably transfected cell linesInvasion abilities of stably transfected cells were detected by Transwell assay.The average invasion cell numbers of MDA-MB-453/KD group(3±0.23)were significantly more than MDA-MB-453/Ctrl cells(14±0.44)(P<0.01).3 HER2 phosphorylation levels and activities of downstream signal moleculesThe expression of different proteins in stably transfected cells were detected by Western blot.After stimulating by EGF(20nM)for 0min,5min,10 min and 30 min,the levels of FLNa expression in MDA-MB-453/KD group(0.22±0.02,0.25±0.03,0.22±0.02,0.23±0.04)were significantly lower than MDA-MB-453/Ctrl cells(0.82±0.03,0.83±0.03,0.82±0.03,0.84±0.0)(P<0.01,respectively).The levels of p-HER2 expression in MDA-MB-453/KD group after stimulating by EGF(20nM)for 5min,10 min,30min(0.83±0.02,0.71±0.05,0.59±0.03)were significantly higher than MDA-MB-453/Ctrl cells(0.42±0.05,0.17±0.01,0.07±0.03)(P<0.01,respectively).The levels of p-Akt expression in MDA-MB-453/KD group after stimulating by 20 nM EGF for 5min,10 min,30min(0.84±0.05,0.63±0.04,0.46±0.03)were significantly higher than MDA-MB-453/Ctrl cells(0.44±0.03,0.24±0.03,0.06±0.02)(P<0.01,respectively).The levels of p-ERK expression in MDA-MB-453/KD group after stimulating by 20 nM EGF for 5min,10 min,30min(0.83±0.02,0.86±0.02,0.79±0.02)were significantly higher than MDA-MB-453/Ctrl cells(0.44±0.04,0.27±0.04,0.17±0.03)(P<0.05,respectively).Conclusions:1 FLNa lower expression can promote proliferation,migration and invasion abilities of MDA-MB-453 cells.2 FLNa may affect biological behaviour of MDA-MB-453 cells through regulating HER2 activation and its downstream MAPK/ERK and PI3K/Akt signal pathways.