| ObjectiveTo investigate the biological function and molecular mechanism of WWOX gene in nasopharyngeal carcinoma cells.Methods 1.The lentiviral vectors carrying WWOX gene fragment and empty fragment were designed and packaged.A lentiviral vector carrying WWOX gene and empty fragment were transfected into NPC cells CNE1 in vitro to construct experimental group CNE1/WWOX and blank group CNE1/CON,and positive cell clones were selected and amplified,respectively.Transfected empty lentiviral vector cell(empty group)and untransfected cel(blank contrast group)were served as control groups.After 72 h transfection,it was observed,photographed and calculated transfection efficiency under the inverted fluorescence microscope.The WWOX mRNA and WWOX protein expression in three groups were determined by real-time fluorescencequantitative reverse transcription polymerase chain reaction and Western Blot.2.The effects of WWOX overexpression on the migration and invasion ability of the three groups were studied by using wound healing assay and/or transwell migration assay and Transwell invasion,respectively.The mRNA expression of E-cadherin was detected by RT-PCR.3.The growth and proliferation ability of three groups of cells were detected by the cell colony formation assay and MTT assay.The cell cycle and cell apoptosis assay with Annexin V-APC/ 7-AAD bifluorescence marker was used to detect cell cycle ability and apoptosis in three groups of cells.The protein expression of AKT,p-akt,caspase-3 and Cleaved caspase-3 was detected by Western Blot,respectively.Results 1.The transfection efficiency of CNE1/CON and CNE1/WWOX was 96.7 % and 95.8 %,respectively.RT-PCR results showed that the mRNA relative expression levels of WWOX in CNE1,CNE1/CON and CNE1/WWOX were 1.000±0.001、1.010±0.041 and 104.691±0.418,respectively.The relative expression of the WWOX mRNA in the experimental group was significantly higher than that in the empty group and blank control group(P<0.05).Western blot results revealed that the relative expression of WWOX protein in CNE1,CNE1/CON and CNE1/WWOX were 0.151 ± 0.042,0.139 ± 0.10 and 1.263 ±0.195,respectively.The relative protein expression of WWOX in the experimental group was significantly higher than that in the empty group and blank control group(P <0.05).2.The wound healing assay results showed that rate of relative wound healing CNE1,CNE1/CON and CNE1/WWOX was(55.67 ± 2.08)%,(58.67 ± 1.15)% and(11.67 ± 1.15)%,respectively.Compared with the empty group and the blank control group,the wound healing rate of the experimental group was significantly reduced.And the difference was statistically significant(P<0.05).The results of Transwell migration showed that the number of cell migration in CNE1,CNE1/CON and CNE1/WWOX was206.8 ± 3.7,201.4 ± 3.1 and 58.8 ± 3.0.The number of cells in the experimental group was significantly lower than that in the empty group and the blank control group.And the difference was statistically significant(P<0.05).The results of Transwell invasion showed that the number of cell invasion in CNE1,CNE1/CON and CNE1/WWOX was 114.2 ± 8.7,109.0± 5.9,and 31.0 ± 2.2.The number of cells in the experimental group significantly decreased,compared with that in the empty group and blank control group.And the difference was statistically significant(P<0.05).The mRNA expression levels of E-cadherin was detected by RT-PCR,and the results showed that the mRNA expression levels of E-cadherin in CNE1,CNE1/CON and CNE1/WWOX was1.000±0.000、1.009±0.015 and 1.393±0.080,respectively.The relative expression of E-cadherin mRNA in the experimental group was significantly increased compared with that in the empty group and the blank control group.And the difference was statistically significant(P<0.05).3.Cell clone formation experiments showed that the rate of cell clone formation in CNE1,CNE1/CON and CNE1/WWOX were(71.9±3.4)%、(70.1%±2.3)% and(27.9%±1.7)%,respectively.The growth of the cells in the experimental group was slow,and theformation of clones was small,and the rate of cell clone formation was significantly less than that of the empty group and the blank control group.And the difference was statistically significant(P<0.05).MTT results showed that OD value in three groups on the same day was no statistically significant difference(P > 0.05).Starting from 24 h,the OD value of experimental group was significantly lower than the each time point of the empty group and the blank control group.And the difference was statistically significant(P < 0.05).The wound healing assay results showed that rate of relative wound healing CNE1,CNE1/CON and CNE1/WWOX was(55.67 ± 2.08)%,(58.67 ± 1.15)%and(11.67 ± 1.15)%,respectively.Compared with the empty group and the blank control group,the wound healing rate of the experimental group was significantly reduced.And the difference was statistically significant(P<0.05).Cell cycle was measured by flow cytometry.The results showed that the number of G0/G1 phase in CNE1,CNE1/CON and CNE1/WWOX was(62.31 ±1.26)%,(59.60 ± 1.87)% and(40.95 ± 2.29)%;the number of CNE1,CNE1/CON and CNE1/WWOX cells in G2/M phase was(8.38 ± 0.23)%,(8.88± 0.87)% and(22.06 ± 1.34)%,respectively;the number of CNE1,CNE1/CON and CNE1/WWOX cells in S phase was(29.30 ± 1.48)%,(31.52 ± 1.28)% and(36.99 ± 1.78)%,respectively.The number of cells in the G0/G1 phase of the experimental group was significantly lower than that in the empty group and the blank control group.And the difference was statistically significant(P<0.05).The number of cells in the G2/M phase of experimental group was significantlyhigher compared with the empty group and blank control group.And the difference was statistically significant(P<0.05).However,there was no significant difference among the three groups in S stage(P >0.05).The cell apoptosis rate was detected by flow cytometry with Annexin V-APC/7-AAD dyework cells.The results showed that the apoptosis rate of CNE1,CNE1/CON,CNE1/WWOX cells was(4.13 ± 0.10)%,(4.15 ± 0.04)% and(16.54 ± 0.07)%,respectively.The apoptosis rate of the experimental group was significantly increased compared with that in the empty group and the blank control group.And the difference was statistically significant(P<0.05).Western blot was used to detect the expression levels of AKT,p-AKT protein and Caspase3 and cleaved Caspase3 protein.The results showed that the relative expression levels of p-AKT protein in CNE1,CNE1/CON and CNE1/WWOX was 0.842 ± 0.009,0.843 ± 0.010 and 0.604 ± 0.006,respectively.The relative expression of p-AKT protein in the experimental group was significantly lower than that in the empty group and the blank control group.And the difference was statistically significant(p <0.05).However,the protein relative expression of AKT in the three groups showed no significant changed.And the difference was not statistically significant(P>0.05).In CNE1,CNE1/CON and CNE1/WWOX,the relative expression of Caspase3 protein was 0.548 ± 0.10,0.546 ± 0.008 and0.147 ± 0.06,respectively.The protein relative expression of Caspase3 in the experimental group was significantly lower than that in the empty group and the blank control group.And the difference was statistically significant(P<0.05).However,the relative expression of cleaved Caspase3 protein in the three groups was 0.285 ± 0.008,0.287 ± 0.007 and 0.533 ± 0.004,respectively.The relative expression of cleaved-Caspase3 protein in the experimental group was significantly increased compared with that in the empty group and the blank control group.And the difference was statistically significant(P<0.05).Conclusions 1.The CNE1/WWOX cell line of WWOX overexpression and the CNE1/CON cell line of the empty group were successfully constructed.2.The WWOX overexpression can make the expression of E-cadherin up-regulated and inhibit the migration and invasion ability of nasopharyngeal carcinoma cells.3.WWOX overexpression can significantly inhibit the growth and proliferation of nasopharyngeal carcinoma cells.WWOX overexpression may suppress the proliferation of nasopharyngeal carcinoma cells by inducing cell cycle arrest in the G2/M phase.WWOX overexpression may make AKT activation down-regulated p-AKT(Ser473)and/or by activating the protein expression level of caspase 3,make the cleaved-Caspase3 protein expression levels up-regulated,thus promotes the apoptosis of nasopharyngeal carcinoma CNE1 cells. |
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