| PART ?The expression of FEAT protein in breast cancer tissues and its clinicopathological significanceObjectiveFEAT protein is uniformly overexpressed in a variety of human cancers but weakly expressed in normal tissue.FEAT has antiapoptotic activity and plays a role in carcinogenesis;however,the correlation between FEAT and clinicopathologic characteristics in cancer has not been reported.Our study explores the expression of FEAT protein and its clinicopathologic significance in breast cancer.MethodsWe examined the expression of FEAT in tissues from 131 cases of breast cancer by immunohistochemistry and analyzed the correlation between FEAT expression and clinicopathologic parameters.The difference in FEAT expression between normal breast tissues and breast cancer tissues was also investigated.Finally,we analyzed the association between FEAT expression and disease-free survival or overall survival.ResultsOur data showed that FEAT was expressed in the cytoplasm.The expression of FEAT protein was significantly higher in breast cancer tissues than in normal breast tissues.Moreover,the expression of FEAT protein was higher in breast cancer with a larger tumor size(>2 cm),negative PR,positive HER2,or higher Ki67 index(?14%)than in breast cancer with a smaller tumor size(?2 cm),positive PR,negative HER2,or lower Ki67 index(<14%)(P<0.05).In addition,the expression of FEAT protein was associated with tumor size,PR status,HER2 expression,Ki67 index,and molecular subtype.Survival analysis showed that disease-free survival and overall survival were significantly shorter in breast cancer patients with high FEAT expression than in those with low expression of FEAT(P<0.05).COX regression analysis showed that FEAT was an independent prognostic factor for recurrence in breast cancer,but not for survival.ConclusionFEAT is highly expressed in breast cancer tissues,and correlated to the clinicopathological characteristics of breast cancer.In addition,FEAT is an independent risk factors for recurrence in breast cancer.In conclusion,FEAT may be a potential biomarker for recurrence of breast cancer.PART ?Effecs of FEAT protein on biological behavior of cell line MCF-7ObjectiveThe expression of FEAT protein was detected in breast cancer cell line MCF-7.After silencing the expression of METTL13 gene in MCF-7 cells,the changes of proliferation,migration,cell cycle and apoptosis were observed in MCF-7 cells.Methods3 strands of siRNA were designed to silence the expression of METTL13 gene in MCF-7 cells,then the experiments were divided into 5 groups:group NC,group MOCK,group FEATsiRNA1,group FEATsiRNA2 and group FEATsiRNA3.Gene silencing effect was verified by fluorescence quantitative PCR(qPCR)and Western-blot.The strand of siRNA with best silencing effect was selected to proceed to further experiments,then the experiments were divided into 3 groups:group NC,group MOCK and group siRNAFEAT.MTT assay was used to detect the cell proliferation ability in 3 groups of MCF-7 cells.After cells were cultured for 14d,the cell clone formation rate was measured by plate clone formation assay in 3 groups.The cell scratch healing rate of MCF-7 cells was observed by scratch test.Transwell Migration Assay was used to detect the number of cells permeating Transwell membrane in 3 groups in 24h.PI staining method was used to detect the distribution of cell cycle in 3 groups 48h after transfection.The cells apoptosis rate were invesgated by FITC Annexin V-PI double staining method in 3 groups 48h or 72h after transfection.Results24h after transfection,the expression level of METTL13m`NA was 1,0.903±0.173,0.175±0.076,0.196±0.034,0.330±0.091 respectively in group NC,group MOCK,group FEATsiRNAl,group FEATsiRNA2 and group FEATsiRNA3.The expression level of METTL13mRNA in group FEATsiRNAl,group FEATsiRNA2 and group FEATsiRNA3 was lower than group NC and group MOCK significantly(P=0.000).48h after transfection,the expression level of FEAT protein was 1,1.1127±0.1128,0.2513±0.0432,0.7167±0.0759,0.628±0.0548 respectively in group NC,group MOCK,group FEATsiRNAl,group FEATsiRNA2 and group FEATsiRNA3.The expression level of FEAT protein in group FEATsiRNAl,group FEATsiRNA2 and group FEATsiRNA3 was lower than group NC and group MOCK significantly(P=0.000).MTT assay showed that OD value was lower in group siRNAFEAT than group NC and group MOCK 24h and 48h after transfection,but the difference is not significant(P=0.098);72h and 96h after transfection,OD value in group siRNAFEAT was lower than group NC and group MOCK significantly(P=0.000).Plate clone formation assay showed that cell clone formation rate were 26.22%±1.89%;26.67%±2.19%,7.45%±0.69%respectively in group NC,group MOCK and group siRNAFEAT,cell clone formation rate in group siRNA FEAT was lower than group NC and group MOCK significantly(P<0.05).Scratch test showed that cell scratch healing rate were 53.63±6.66%,56.74±3.71%,37.50±6.15%respectively in group NC,group and MOCKgroup siRNAFEAT 24h after transfection,and 100.00±0.00%,100.00±0.00%,62.06±5.11%respectively 48h after transfection;cell scratch healing rate in group siRNAFEAT was lower than group NC and group MOCK significantly(P<0.05).Transwell Migration Assay showed that the number of cells permeating Transwell membrane were 267.67±27.46,255.33±56.20,46.33±21.27 respectively in group NC,group MOCK and group siRNAFEAT 24h after inoculating in Transwell chamber,the number of cell permeating Transwell membrane in group siRNAFEAT was lower than group NC and group MOCK significantly(P<0.05).48h after transfection,PI staining showed the cell with 66.43±3.85%G1 phase,26.40±3.62%S phase and 7.17±0.55%G2/M phase in group NC;the cell with 65.49±1.25%G1 phase,27,51±1.11%S phase and 6.70±0.23%G2/M phase in group MOCK;the cell with 82.63±1.62%G1 phase,11.37±1.70%S phase and 6.01±0.49%G2/M phase in group siRNAFEAT;Cells proportion with G1 phase in group siRNAFEAT was higher than group NC and group MOCK significantly(P<0.05),but cells proportion with S phase was lower significantly(P<0.05).Annexin V-FITC/PI double staining showed the apoptosis rate was 5.80±1.23%,5.37±0.86%and 6.63±1.07%respectively in group NC,group MOCK and group siRNAFEAT 48h after transfection;the apoptosis rate was 6.07±0.60%,6.03±0.90%and 5.90±1.14%respectively in 3 group 72h after transfection,the apoptosis rate is not different significantly in 3 groups(P>0.05).ConclusionFEAT siRNA can downregulate the expression of METTL13 gene in breast cancer cell line MCF-7,then the level of FEAT protein decreased in MCF-7 cells.After downregulating the FEAT protein,the proliferation of MCF-7 cells is inhibited with the decrease of cell migration ability,also cell cycle is arrested at G1 phase.However,the apoptosis was not affected by the downregulation of FEAT protein.PART ?Impacts of FEAT on drug sensitivity and protein expression in cell line MCF-7ObjectiveWhen the expression of FEAT protein is downregulated in the MCF-7 cells,we observed the change of drug sensitivity for paclitaxel,epirubicin and cyclophosphamide.Also combined actions of downregulation of FEAT protein and these chemotherapeutic drugs on cell apoptosis and necrosis were observed.Then the effect of downregulation of FEAT protein on the expression of proteins related to apoptosis and cell cycle was invesgated.MethodsMCF-7 cells were divided into group NC and group siRNAFEAT.MCF-7 cell were treated with different concentration of cyclophosphamide(0mg/L,100mg/L,200mg/L,400mg/L,800mg/L,1600mg/L),epirubicin(O?M,0.2?M,1?M,5?M,25vM,125?M)or paclitaxel(0,0.08?M,0.4?M,2?M,10?M,50?M)for 48 hours,then the absorbance value was detected by MTT assay and the IC50 value was calculated for 2 groups of cells.Cells in group NC and group siRNAFEAT were treated with 800mg/L cyclophosphamide,1.5?M epirubicin or 4.5pM paclitaxel respectivly for 36h,the apoptosis and necrosis were detected by FITC Annexin V-PI double staining.The experiments were divided into 3 groups:group NC,group MOCK,group siRNAFEAT.The total protein of MCF-7 cells was extracted 48h after transfection,then protein concentration was measured.The different expressions of Caspase-3,p21,AKT,p-AKT and CDK2 were detected by Western blot in 3 groups of cells.ResultsThe IC50 value of cyclophosphamide is 1446.43±9.82mg/L and 999.56±139.43mg/L in group NC and group siRNAFEAT.The IC50 value of epirubicin is 2.25±0.17?M and 1.04±0.28?M in group NC and group siRNAFEAT.The IC50 value of paclitaxel is 40.47±14.23?M and 10.36±5.02?M in group NC and group siRNAFEAT.IC50 values of three drugs in the group siRNAFEAT were lower than the NC group significantly(P<0.05).Cell necrosis rate were 6.07%±1.86 and 45.23%±5.45 respetivly in group NC and group siRNAFEAT after cells were treated with 800mg/L cyclophosphamide for 36h.Cell necrosis rate were 6.77%±2.72 and 53.60%±3.87 respetivly in group NC and group siRNAFEAT after cells were treated with 1.5?M epirubicin for 36h.Cell necrosis rate were 7.13%±1.32 and 49.67%±2.98 respetivly in group NC and group siRNAFEAT after cells were treated with 4.5?M paclitaxel for 36h.Cell necrosis rate for three drugs in the group siRNAFEAT were higher than the NC group significantly(P<0.05).There was no significant difference in Caspase-3,AKT,p-AKT and dk2 expression among the 3 groups,but the expression of p21 protein in the groups siRNAFE/AT was significantly higher than group NC and group MOCK.ConclusionsDownregulation of FEAT protein can increase the sensitivity of MCF-7 cells to cyclophosphamide,epirubicin and paclitaxel,also promote the necrosis of MCF-7 cells induced by cyclophosphamide,epirubicin and paclitaxel.The reduced expression of FEAT protein can affected the process of cell cycle by upregulating the expression of p21 protein in MCF-7 cells. |
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