慢病毒介导高表达OTUD7B对乳腺癌MCF-7细胞生物学行为影响

Construction of high expression OTUD7B and its effect on the biological behavior of breast cancer MCF-7 cells

目的去泛素化酶OTUD7B与肿瘤的发生、发展密切相关。为了明确OTUD7B在乳腺癌中所发挥的作用,实验利用慢病毒构建高表达OTUD7B载体感染MCF-7乳腺癌细胞后对其生物学行为的影响。方法构建带有绿色荧光蛋白标签的人OTUD7B表达质粒的慢病毒(pEGFP-hOTUD7B)及对照(pEGFP-CI)感染MCF-7乳腺癌细胞;于荧光倒置显微镜下观察病毒转染效果及应用蛋白质印迹法及免疫组化法检测OTUD7B的表达水平;MTS法检测实验组(pEGFP-hOTUD7B)、阴性对照组(pEGFP-CI)和正常对照组对MCF-7乳腺癌细胞增殖能力的影响;应用细胞划痕实验检测细胞迁移能力;流式细胞仪检测细胞凋亡。结果感染病毒后于荧光倒置显微镜下观察病毒感染效率,可见病毒感染成功。应用蛋白质印迹法检测病毒感染率并找出最适病毒感染复数(multiplicity of infection,MOI),当MOI=30时,实验组、阴性对照组和正常对照组灰度值分别为3.81±0.08、2.12±0.078和2.05±0.15,差异有统计学意义,F=402.03,P<0.001。应用免疫组化法可见感染OTUD7B表达水平。MTS法结果显示,实验组、阴性对照组和正常对照组细胞24hA值分别为0.36±0.08、0.56±0.25和0.69±0.17,F=11.819,P<0.001;48hA值分别为0.65±0.17、1.45±0.48和1.82±0.63,F=23.752,P<0.001;在72hA值分别为0.73±0.21、1.58±0.63和1.99±0.27,F=35.563,P<0.001。细胞划痕试验显示,24h后实验组组迁移率为(7.7±0.91)%,阴性对照组和正常对照组迁移率分别为(13.4±1.52)%和(12.1±1.32)%,F=49.36,P<0.001,48h后实验组迁移率为(12.4±1.29)%,阴性对照组及正常对照组迁移率分别为(32.9±1.71)%和(31.8±1.59)%,F=504.50,P<0.001。流式细胞仪检测细胞凋亡结果提示,实验组与阴性对照组及正常对照组相比明显使细胞阻滞在G_0/G_1期,促进其凋亡,F_(G_0/G_1)期=425.102,F_S期=135.063,均P<0.001。结论成功构建了能够高表达OTUD7B的慢病毒载体,明显抑制了MCF-7乳腺癌细胞增殖、迁移,并促进了其凋亡。

OBJECTIVE OTUD7 Bis closely related to the occurrence and development of the tumor.In order to clarify the role of OTUD7 Bin breast cancer,we constructed a high expression OTUD7 Bvector in MCF-7and explored its effect on the biologic behavior of human Breast cancer MCF-7cells.METHODS Targeting expression plasmid with green fluorescent protein tagged human OTUD7B（pEGFP-hOTUD7B）and control（pEGFP-CI）were constructed,then human breast cancer MCF-7cells were infected with these vectors.Under fluorescence microscope the expression was observed.Afterwards,we used Western blot and immunohistochemistry to detect the expression of OTUD7 B,MTS colorimetric assay to measure the cell proliferation,and cell scratch test for cell migration and flow cytometiy analysis for apoptosis in cells treated with TNF-α.RESULTS We observed the efficiency under fluorescence microscope after infecting,which were successful,then we used Western blot to find the optimal multiplicity of infection virus（multiplicity of infection,MOI）,MOI=30,the gray level of the experimental group,negative control group and normal control group were 3.81±0.08,0.15±0.078 and 2.05±2.12,respectively,the difference was statistically significant,F=402.03,P〈0.001.Immunohistochemistryl method showed the expression level of OTUD7 Binfection.MTS results indicted that compared with the negative control group and the normal control group,the experimental group could significantly inhibit the proliferation of MCF-7cells,F（24h）=11.819,P〈0.001,F（48h）=23.752,P〈0.001,F（72h）=35.563,P〈0.001.In cell scratch test after 24 h,the migration rate in OTUD7 Bexpression group was（7.7±0.91）%,negative control group and normal group migration rates were（13.4±1.52）% and（12.1±1.32）%,F=49.36,P〈0.001,after 48 h,OTUD7Bexpression group mobility（12.4±1.29）%,negative control group and normal group migration rates were（32.9±1.71）% and（31.8±1.59）%,F=504.50,P〈0.001.The apoptosis was detected by flow cytometry indicated that with the comparison of negative control group and the normal control group,G0/G1 phase cell of experimental group was obviously increased.It indicated OTUD7 Bexpression could promote cell apoptosis,F（G0/G1）=425.102,FS=135.063,P〈0.001.CONCLUSION We have successfully constructed a recombinant lentiviral vector which can efficiently express OTUD7 Band it can inhibit the proliferation,migration of tumor cells,and promote cell apoptosis under the condition of TNF-a treatment.