OTUB1基因对宫颈癌Hela细胞顺铂敏感性的影响及机制

Effects and Mechanisms of OTUB1 Gene on Chemotherapy Sensitivity to Cisplatin of Cervical Cancer Hela Cell

目的研究去泛素化酶OTUB1基因对宫颈癌Hela细胞株顺铂敏感性的影响及可能的机制。方法用慢病毒包装的干扰质粒（SH组）和阴性对照质粒（NC。组）、过表达质粒（OE组）和相应的阴性对照质粒（NC1组）分别转染宫颈癌Hela细胞，建立稳转细胞系。用实时定量PCR（RT—PCR）法及Western blot法验证OTUB1基因的表达效率；用CCK8法检测细胞增殖；用半数抑制浓度（IC50）、克隆形成评价Hela细胞对顺铂的化疗敏感性；用流式细胞仪检测顺铂作用后的细胞调亡情况；用Western blot法检测凋亡相关蛋白P53、活性Caspase3和Caspase8的表达情况。结果RT—PCR和Westernblot结果提示稳转细胞系已建成。使用顺铂处理后，各组细胞抑制率随着药物浓度增加而升高。其中，OE组抑制率明显比NC2高，SH组抑制率低于NC1组（P〈0．05）。IC50和克隆形成结果提示OTUB1基因的表达可以增加Hela细胞对顺铂的敏感性（P〈0．05）。流式细胞仪结果提示OTUB1基因的表达可以促进细胞凋亡（P〈0．05）。Westernblot检测进一步发现过表达OTUB1基因细胞中P53、活性Caspase3和Caspase8蛋白表达升高（P〈0．05）。结论OTUB1基因表达可以增加Hela细胞对顺铂的化疗敏感性，这可能与去泛素化酶OTUB1蛋白促进细胞凋亡有关。

Objective To investigate effects and mechanisms of deubiquitinase OTUB1 gene on chemotherapy sensitivity to cisplatin of cervical cancer Hela cell. Methods SH - EGFP - OTUB1 （ SH group） and SH - EGFP（ NC1 group） , pZsGreen- OTUB1 （OE group）and pZsGreen（ NC2 group）assembled by lentivirus were transfected into Hela cell lines. RT - PCR and Western blot were used to detect the expression efficiency of OTUB1 gene. CCK8 assay was employed to analysis cell proliferation. Half maximal inhibitory concentration（IC50） and clone formation were used to assess chemotherapy sensitivity to cisplatin of cervical cancer Hela cell. Flow cytometry was performed to evaluate cel- lular apoptosis. Apoptosis - related protein P53, active Caspase3 and Caspase8 were measured by western blot. Results Both of RT - PCR and western blot results proved that the stable transfected cell lines were successfully established. Cell inhibition rates of all groups were ascended in a cisplatin dosage manner. Specifically, cell inhibition rates in OE group were higher than those in NC2 group, while inhibitory rates in SH group were lower than those in NC1 group （P 〈 O. 05）. The results of IC50 and clone formation indicated that the expression of OTUB1 gene could increase the sensitity to cisplatin（P 〈 0.05）. Flow cytometry results showed that OTUB1 gene could promote cell apoptosis. Western blot showed that expression of apoptosis - related protein P53, active Caspase 3 and Caspase 8 increased （ P 〈 0.05）. Conclusion Expression of OTUB1 gene can increase chemotherapy sensitivity to cisplatin of cervical cancer Hela cell,which may relate to cellular apoptosis regulated by OTUB1 gene.