Effect Of PDCD4 Gene On Proliferation,Migration,Invasion And Metastasis Of Breast Cancer Cell Line MDA-MB-231

OBJECTIVEInvestigate the effects of lentivirus mediated PDCD4 on the proliferation,migration,invasion and metastasis of breast cancer cell line MDA-MB-231.METHODSLentiviral overexpression vectors(LV-PDCD4)was designed and chemically synthesized,The experimental group was subcutaneously injected with MDA-MB-231 cells transfected with Lentiviral overexpression vectors(LV-PDCD4)whlile the negative control was subcutaneously injected with MDA-MB-231 cells transfected with Ubi-MCS-3FLAG-SV40-EGFP-IRES,the blank contol group was subcutaneously injected with MDA-MB-231 cells alone.The expression of PDCD4 was detected by western blot analysis and real-time PCR.The cell growth and proliferation was evaluated by colony formation assays and CCK-8 assays.The migration was evaluated by wound healing assays and Transwell assays.Invasion was evaluated by Transwell assays.The xenograft nude mouse models was established lentiviral vectors transfected into three MDA-MB-231 cell groups.Investigate the growth ofXenograft nude mouse models,HE pathology investigated the visceral metastasis in nude mice.RESULTS1.This experimental showed that lentiviral overexpression vectors was successful designed and chemically constructed,the relative quantification of PDCD4 m RNA expression of breast cancer cell line MDA-MB-231 in experimental group,negative control group and blank control group were~ 6.440±0.218?1.195±0.029?1.000±0.000,respectively;significant difference was observed in the three groups(F=1772.959,P < 0.05),the PDCD4 m RNA expression in experimental group was significantly higher than negative control group(P<0.05)and blank control group(P<0.05),although there was no statistical significance between negative and blank control groups(P=0.109>0.05).In western blotting,the PDCD4 protein expression of breast cancer cell line MDA-MB-231 in experimental group,negative control group and blank control group were~0.499±0.044?0.180±0.021?0.130±0.006,respectively.Significant difference was observed in the three groups(F=152.811,P<0.05),the PDCD4 protein expression was significantly higher in the experimental group compared to that in the negative control(P<0.05)or the blank control group(P<0.05),although there was no statistical significance between negative and blank control groups(P=0.07>0.05).2.The CCK-8 assay showed that the OD value of experimental group at 24 h,48h,72 h,96h piont were~0.345±0.011 ? 0.404±0.007 ? 0.597±0.008 ?0.850±0.015,the OD value of negative control group at 24 h,48h,72 h,96h piont were~ 0.414±0.008?0.595±0.007?0.912±0.013?1.243±0.015,the blank group OD value at 24 h,48h,72 h,96h piont were~0.418±0.007 ? 0.602±0.013 ?0.925±0.011?1.253±0.034,respectively.Significant difference was investigated in the three groups at different time piont(F=60.230?431.098?893.98?291.579,all P<0.05).The experimental group at 24 h,48h,72 h,96h time piont were significantly lower than the negative control(all P<0.05)and the blank control group(all P <0.05),although there was no statistically significance at different time piont between blank control and negative groups(P=0.596?0.200?0.370?0.610 all>0.05).3.The colony formation assay showed that the rate of colony in experimental group,negative control group and blank control group were~30.3±6.0%?57.3±7.4%?57.0±4.4%,respectively.Significant difference was investigated in the three groups(F=19.74,P=0.002<0.05),the the rate of colony was significantly lower in the experimental group compared to that in the blank control group(P=0.002 < 0.05)or the negative control(P=0.002 <0.05),although there was no statistically significance at rate of colony piont between blank control and negative groups(P=0.948>0.05).4.The wound healing assay showed the rate of migration of experimental group at 24 h,48h piont were~5.19±0.59%?10.55±0.93%,the rate of migration of negative control group at 24 h,48h piont were ~14.74±0.98%?27.21±1.60%,thethe rate of migration of blank group at 24 h,48h piont were~15.25±0.90%?29.39±1.69%,respectively.Significant difference was investigated in the three groups at different time piont(F=135.91?152.27,all P<0.05).The experimental group at 24 h,48h time piont were significantly lower than the negative control(P=0.000?0.000 all<0.05)and the blank control group(all P<0.05),although there was no statistically significance at different time piont between blank control and negative groups(P=0.483?0.114 all>0.05).5.The transwell migration assays assay showed that the cell number of migration in blank control group,negative control group and experimental group were~319.0±32.5 ?305.0±27.5?159.7±18.0,respectively.Significant difference was investigated in the three groups(F=32.763,P=0.001<0.05),the cell number of migration were significantly lower in the experimental group compared to that in the blank control group(P<0.05)or the negative control(P=0.001<0.05),although the cell number of migration was no statistically significance between blank control and negative groups(P=0.544>0.05).6.The transwell invasion assays assay showed that the cell number of invasion in blank control group,negative control group and experimental group were~180.7±21.0 ?173.3±20.6?77.0±9.5,respectively.Significant difference was investigated in the three groups(F=F=31.551,P=0.001<0.05),the cell number of invasion were significantly lower in the experimental group compared to that in the blank control group(P=0.001<0.05)or the negative control(P<0.05),although the cell number of invasion was no statistically significance between blank control and negative groups(P=0.633>0.05).7.The tumor rate was 100% after 5 days.The growth rate of nude mice in the experimental group was significantly slower than negative control group and blank control group(all P<0.05).In the experimental group(LV group),negative control group(Con group)and blank control group,the average volume of the tumor were~377.94±130.07mm3 ? 834.70±97.09mm3 ? 837.03±105.64mm-3,respectively;talthoughthere was no statistically significance between blank control and negative groups(F=67.10,P<0.05).The experimental group(LV group)was significantly lower than the negative control group(Con group)(P < 0.05)and the blank control group(P< 0.05),although there was no statisticallysignificant difference between the blank control and the negative groups(P=0.960>0.05).In the experimental group(LV group),negative control group(Con group)and blank control group,the average tumor weight of mice were~0.34±0.11g?0.74±0.10g?0.75±0.10 g,respectively;although there was no statistically significance between blank control and negative groups(F=61.26,P<0.05).The experimental group(LV group)was significantly lower than the negative control group(Con group)(P=0.000 < 0.05)and the blank control group(P < 0.05),although there was no statistically significant difference between the blank control and the negative groups(P=0.897>0.05).8.There was no lung metastasis occurred in the nude mice in the experimental group significantly lower compared with that of three lung metastases negative control group(P<0.05)or three lung metastases blank control group(P<0.05).CONCLUSION1.Stable transfection of LV-PDCD4 can effectively increase the protein expression level and the relative expression level of PDCD4 m RNA in breast cancer MDA-MB-231 cells,represents the successful construction of slow virus expression vector..2.After the stable transfection of LV-PDCD4 into the breast cancer cell line MDA-MB-231,it can obviously inhibitproliferation,invasion and migration of breast cancer cells,PDCD4 gene have the effect of inhibiting the proliferation,invasion and migration of breast cancer cells.3.After the stable transfection of LV-PDCD4 into the breast cancer cell line MDA-MB-231,it can obviously inhibit the growth ? proliferation and metastasis of Xenograft nude mouse,PDCD4 gene have the effect of inhibitingthe growth and metastasis of breast cancer.4.It can be further demonstrated that PDCD4 is a tumor suppressor gene in breast cancer and may be a potential target for breast cancer treatment in vivo and in vitro experiments.