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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 To ubiquitin enzyme OTUD7B and tumor occurrence and development are closely related. In order to clarify the role of OTUD7B in breast cancer, we used the lentivirus to construct a highly expressed OTUD7B vector to affect the biological behavior of MCF-7 breast cancer cells. Methods The lentivirus (pEGFP-hOTUD7B) and control (pEGFP-CI) with green fluorescent protein tagged human OTUD7B expression plasmid were used to infect MCF-7 breast cancer cells. The transfection efficiency was observed under a fluorescence inverted microscope and Western blot The effect of pEGFP-hOTUD7B, pEGFP-CI and normal control group on the proliferation of MCF-7 breast cancer cells was detected by MTS assay and immunohistochemistry. The cell migration assay was used to detect the cell migration and apoptosis was detected by flow cytometry. Results After infected with virus, the virus infection efficiency was observed under a fluorescence inverted microscope, showing that the virus infection was successful. Western blotting was used to detect the virus infection rate and find out the optimal multiplicity of infection (MOI). When the MOI was 30, the gray values of the experimental group, negative control group and normal control group were 3.81 ± 0.08, 2.12 ± 0.078 and 2.05 ± 0.15, the difference was statistically significant, F = 402.03, P <0.001. The expression of OTUD7B was detected by immunohistochemistry. The results of MTS assay showed that the 24hA values of experimental group, negative control group and normal control group were 0.36 ± 0.08,0.56 ± 0.25 and 0.69 ± 0.17 respectively, F = 11.819, P <0.001; 48hA values were 0.65 ± 0.17,1.45 ± 0.48 and 1.82 ± 0.63, F = 23.752, P <0.001; values at 72 hA were 0.73 ± 0.21, 1.58 ± 0.63 and 1.99 ± 0.27, respectively, F = 35.563, P <0.001. The cell scratch test showed that the migration rate of the experimental group was (7.7 ± 0.91)% after 24 hours, (13.4 ± 1.52)% and (12.1 ± 1.32)%, F = 49.36 respectively in the negative control group and the normal control group, The migration rate of the experimental group was (12.4 ± 1.29)% at P <0.001 and 48h, the positive rate was (32.9 ± 1.71)% and (31.8 ± 1.59)% respectively in the negative control group and the normal control group, F = 504.50, P <0.001 . Flow cytometry results of apoptosis showed that the experimental group compared with the negative control group and the normal control group obviously blocked the cells in the G_0 / G_1 phase, and promoted the apoptosis, F_ (G_0 / G_1) = 425.102, F_S Period = 135.063, all P <0.001. Conclusion The lentiviral vector that can overexpress OTUD7B was successfully constructed, which significantly inhibited the proliferation, migration and apoptosis of MCF-7 breast cancer cells.