| Objective: Globally,breast cancer is one of the most frequently diagnosed cancer of women.Chemotherapy is the major method for breast cancer patiens.However,the chemotherapy resistance still a major limitation in clinical treatment.As a proto-oncogenes,MTDH is associated with aberrant proliferation,evasion of apoptosis,angiogenesis,invasion and drug resistance.In this study,we aimed to investigate the effect of MTDH on proliferation,apoptosis and taxol sensitive via knockdown of MTDH in MCF-7 breast cancer cell in vitro and in vivo.Methods: A model of MCF-7 breast cancer cells stable silencing MTDH gene was constructed by lentivirus-mediated short hairpin RNA.Real-time PCR and Western blot were used to determine the mRNA and protein expression of MTDH in blank control group(MCF-7),negative control group(MCF-7-control)and experimental group(MCF-7-MTDH/knockdown).CCK-8 assay was used to detect the effect of MTDH silencing on the proliferation and the sensitivity to taxol of MCF-7 cells.Cell cycle and apoptosis were examined by flow cytometry(FCM).The expression leves of P65 and I?B? were measured by real-time PCR and western blot.Tumor growth,tumor volume and weight and the sensitivity of taxol were observed by xenograft modles and FCM.Statistical analysis was performed using SPSS 21.0.P<0.05 was considered statistically significant.Each group at least was three separate experiments.Results:1 A model of MCF-7 breast cancer cells stably knockdown of MTDH was constructed by lentivirus and its identification.A stably knockdown of MTDH(MCF-7-MTDH/knockdown)cells was successfully obtained.Real-time PCR was used to detect the relative mRNA leve of MTDH.The results of MCF-7,MCF-7-control and MCF-7-MTDH/ knockdown were 1.001±0.010,1.014±0.018 and 0.285±0.036.Western Blot results showed that the relative protein leve of MTDH in thses three different cell lines were 0.871±0.126,0.816±0.067 and 0.089±0.011.The expression of MTDH mRNA and protein in MCF-7-MTDH/knockdown cells was lower than that in MCF-7 and MCF-7-control cells,and the difference was statistically significant(P<0.05).2 The effect of MTDH silencing on proliferation,cell cycle and apoptosis.CCK-8 assay was used to exam cell growth on 0h,24 h,48h and 72 h.The results showed that there was no significant difference(P>0.05)between control groups,but cell proliferation was significantly suppressed in MCF-7-MTDH/knockdown(P<0.01),compared with control groups.Flow cytometry was used to detect cell cycle distribution.The proportion of G0 / G1 phase MCF-7,MCF-7-control and MCF-7-MTDH/ knockdown were 41.61±0.53%,40.27±1.22%,and 55.70±1.83%.Compared with control groups,the G0/G1 phase of the MCF-7-MTDH/ knockdown cells was prolonged,while the proportion of S phase and G2/M phase of was decreased(P<0.05).Cells apoptosis were analyzed by flow cytometry.The results of MCF-7,MCF-7-control and MCF-7-MTDH/ knockdown were 4.85±0.64%,5.35±0.56% and 10.21±0.29%.Compared with control groups,cell apoptosis was increased in MCF-7-MTDH/knockdown cells(P<0.05).3 Effects of taxol on cell inhibition rate,cell cycle and apoptosis before and after MTDH gene silencingMCF-7,MCF-7-control and MCF-7-MTDH/knockdown cells were treated with taxol(0.1 and 1?g/ml)for 48 hours.The inhibition rate was detected by CCK-8 assay.The results showed that the inhibition rates of 0.1?g/ml taxol to cells were 40.71±0.08%,40.96±0.16% and 54.25±0.06%.And the inhibition rates of 1?g/ml taxol to cells were 56.54±0.13%,56.49±0.04% and 77.19±0.17%.The inhibition rates of 0.1?g /ml and 1?g /ml in MCF-7-MTDH/knockdown cells were higher than those in MCF-7 and MCF-7-control cells,and the difference was statistically significant(P<0.01).The results of cell cycle in each group showed that G2/M phase arrest increased in three groups of cells after treated with taxol and the 1?g/ml group was significantly higher than the 0.1?g /ml group(P<0.05).The proportion of G2/M phase of MCF-7-MTDH/knockdown cells treated with 1?g/ml taxol was significantly increased,compared with MCF-7 and MCF-7-control cells(P<0.05).The results of apoptosis in each group showed that the apoptosis rate increased dramaticlly in a dose-dependent manner in MCF-7,MCF-7-control and MCF-7-MTDH/knockdown cells.Additionally,Compared with control groups,cell apoptosis was higher in MCF-7-MTDH/knockdown cells(P<0.001).4 The expression level of P65 and I?B? before and after MTDH gene silencingReal-time PCR and Western blot results showed that compared with MCF-7 and MCF-7-control group,the mRNA and protein levels of P65 decreased in MCF-7-MTDH/knockdown cells,while the mRNA and protein levels of I?B? increased(P<0.001).5 The effect of MTDH gene silencing on xenograft tumorKnockdown of MTDH inhibited tumor growth in xenograft model.The tumor volumes of MCF-7,MCF-7-control and MCF-7-MTDH/knockdown tumors were 902.7±26.53mm3,840.79±25.82mm3 and 374.35±16.68mm3.The tumor weights were 1.106±0.095 g,1.103±0.101 g and 0.459±0.051g(P<0.001).Knockdown of MTDH increased the sencitivity of taxol.Compared with control group,the tumor volumes and tumor weights of MCF-7-MTDH/knockdown tumors were much smaller.After treated with taxol,the tumor volumes of MCF-7,MCF-7-control and MCF-7-MTDH/knockdown tumors were 340.21±26.35mm3,322.27±25.82 mm3,91.13±16.68 mm3(P<0.001).The tumor weights were 0.440±0.01 g,0.397±0.03 g,0.113±0.02 g(P<0.001).Flow cytometry analysed the apoptosis of xenograft tumors.In untreated with taxol group,the apoptosis of MCF-7,MCF-7-control and MCF-7-MTDH/knockdown tumors were 2.477±0.055%,2.823±0.770% and 9.527±0.262%(P<0.001).The apoptosis of MCF-7-MTDH/knockdown was higher than that in control group.In taxol treated group,the apoptosis were 14.547±0.625%,13.320±1.529% and 25.613±0.996%(P<0.001).MCF-7-MTDH/knockdown xenograft tumors were more sencitive to taxol.Conclusions:1 A model of stably knockdown of MTDH in MCF-7 cells is successfully constructed by lentivirus.2 Knockdown of MTDH inhibits proliferation,induces G0/G1 phase arrest and promots apoptosis of MCF-7 cells.3 Knockdown of MTDH increases the sensitivity of MCF-7 cells to taxol through NF-?B/I?B pathway. |
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