沉默CDK7对子宫内膜癌细胞顺铂敏感性的影响

Construction of CDK7 siRNA expression vector and its effect on cisplatin sensitivity of endometrial carcinoma cells

目的:研究沉默CDK7对子宫内膜癌细胞HEC-1-A顺铂化疗敏感性的影响。方法:根据CDK7的基因序列设计合成不同的CDK7 siRNA片段并转染子宫内膜癌细胞HEC-1-A,通过实时定量PCR和Western blot验证RNA干扰的效果后,选择效果最优的CDK7 siRNA片段特异性沉默子宫内膜癌细胞中CDK7的表达,采用MTT细胞毒性实验、流式细胞仪及Hochest/PI双染色荧光显微镜技术检测转染前后该细胞系对化疗药物顺铂敏感性的变化。结果:共选择了4个位点设计CDK7 siRNA并成功转染子宫内膜癌细胞系HEC-1-A,实时定量PCR及Western blot验证了各组的干扰作用,证实CDK7-423干扰效果最强,达70%以上。选择CDK7-423转染HEC-1-A,MTT细胞毒性实验结果发现在CDK7表达被抑制后,DDP的IC50由45.122 g/mL降为3.200 g/mL,细胞毒性显著增高(P<0.05)。流式细胞仪检测结果:CDK7低表达组细胞平均凋亡率细胞为37.57%,与高表达组细胞(11.66%)相比,凋亡率明显增加(P<0.05)。Hochest/PI双染色荧光显微镜下可见CDK7低表达组HEC-1-A细胞与亲代细胞比较,凋亡小体明显增多。结论:通过CDK7 siRNA转染下调子宫内膜癌细胞中CDK7的表达以后,可以明显增强子宫内膜癌细胞对DDP的化疗敏感性,使子宫内膜癌细胞凋亡增加。可作为子宫内膜癌治疗的新靶点进行更加深入的研究。

Objective： This study was aimed to investigate the influence of CDK7 siRRA on the sensitivity of endometrial carcinoma cell line HEC-1-A to cisplatin （DDP）-based chemotherapy. Methods： Different CDK7 siRRA fragments were synthesized based on the designs of the CDK gene sequence and were transfected into HEC-1-A. Real time reverse transcription polymerase chain reaction （RT-PCR） and Western blot analysis were employed to demonstrate the effects of transfection. The best CDK7 siRRA was chosen to specifically silence CDK7 expression in HEC-1-A.The sensitivity of the cells to DDP therapy before and after transfection was determined by methyl thiazol tetrazolium （MTT） cytotoxicity assay, flow cytometry, and Hoechst/PI double-staining fluorescence microscopy. Results： A total of four different CDK7 siRRA segments were designed and successfully transfected into HEC-1-A cells. The interference effect in each group was confirmed by real time RT-PCR and Western blot assays. CDK7-423 was determined as the best performing CDK7 siRRA （over 70%） to transfect into HEC-1-A cells. MTT cytotoxicity test showed that IC50 of DDP decreased to a range from 45.122 μg/mL and 3.200 μg/mL after inhibition of CDK7 expression. DDP toxicity to the endometrial carcinoma cells significantly increased （P〈0.05）. Flow cytometry revealed that the average cell apoptosis rate significantly increased after the inhibition of CDK7 expression （11.66 % to 37.57%, P〈0.05）. Similar results were observed using Hoechst/PI double-staining fluorescence microscopy, and the number of apoptotic corpuscle demonstrated an apparent increase in the low CDK7-expressing group compared with the parental cells. Conclusion： After the downregulation of CDK7 expression by CDK7 siRRA transfection, DDP chemotherapy sensitivity and apoptosis of endometrial carcinoma cells significantly increased. Further research is anticipated on the use of CDK7 as a new treatment target for endometrial carcinoma.