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DEK基因RNA干扰慢病毒表达载体的构建及其对肝癌细胞生物学行为的影响

Construction of RNA interference (RNAi) lentiviral expression vector of DEK gene and its effect on the biological behavior of liver cancer cells

摘要:

目的:构建DEK基因的RNA干扰(RNAi)慢病毒表达载体,并探讨其对肝癌细胞生物学行为的影响。方法:采用RNAi技术,根据DEK基因的干扰序列,合成Oligo DNA,退火形成双链DNA,将其克隆到酶切后的小干扰RNA表达载体pLKO.1上,构建重组慢病毒载体pLKO.1-sh hDEK,经293T细胞包装,收集病毒上清液,感染细胞。采用实时逆转录PCR(RT-PCR)和免疫蛋白印迹法(Western blot)检测人肝癌细胞Bel-7402、Huh-7、SMMC-7721和HepG2中DEK的表达及感染慢病毒的各组细胞中DEK的敲低效率。分别通过细胞计数试剂盒-8(CCK8)增殖实验、流式细胞术、划痕实验检测细胞增殖能力、克隆形成能力、凋亡及迁移能力。两组间均数比较采用 t检验,多组间比较采用单因素方差分析(one-way ANOVA)。 结果:酶切鉴定和DNA测序结果证实,重组慢病毒载体pLKO.1-sh hDEK1及pLKO.1-sh hDEK3构建成功。RT-PCR和Western blot结果显示,肝癌细胞Bel-7402和Huh7中DEK的表达较高,pLKO.1-sh hDEK3能更有效地抑制DEK基因的表达( P < 0.05),因此选择pLKO.1-sh hDEK3重组慢病毒感染肝癌细胞Bel-7402和Huh7进行后续的功能实验。CCK8细胞增殖实验结果显示肝癌细胞Bel-7402和Huh7感染重组慢病毒后,细胞增殖能力与空白对照及阴性对照相比减弱( P < 0.05);细胞凋亡结果显示敲低组细胞凋亡率高于空白对照及阴性对照组( P < 0.05);细胞划痕实验结果显示敲低组划痕愈合率低于空白对照及阴性对照组( P < 0.05),差异均有统计学意义;而空白对照及阴性对照组比较,差异无统计学意义。 结论:靶向沉默肝癌细胞中DEK的表达,能够抑制细胞增殖及迁移能力,并诱导凋亡,为进一步研究DEK基因在肝癌中的作用奠定基础。

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abstracts:

Objective:To construct RNA interference (RNAi) lentiviral expression vector of DEK gene, and to explore its effect on the biological behavior of liver cancer cells.Methods:Double-stranded oligo DNAs were annealed and synthesized according to the interference sequence of DEK gene by RNAi technology. Small interfering RNA expression vector pLKO.1 was cloned after enzymatic digestion. The recombinant lentiviral pLKO.1-sh hDEK was constructed, and then the virus supernatant was collected, packed and infected by 293T cells. Real-time reverse transcription PCR (RT-PCR) and Western blot were used to detect DEK expression in human liver cancer cells Bel-7402, Hu-7, SmMC-7721 and HepG2, and DEK knockdown efficiency in each group of lentivirus-infected cells. Cell proliferation ability, cloning ability, apoptosis and migration ability were detected by cell counting kit-8 (CCK8), flow cytometry and scratch test, respectively. The t-test was used to compare the mean between the two groups, and one-way ANOVA was used to compare the multiple groups.Results:Enzymatic digestion and DNA sequencing results confirmed that the recombinant lentiviral vectors pLKO.1-sh hDEK1 and pLKO.1-sh hDEK3 were successfully constructed. RT-PCR and Western blot results showed that the expression of DEK in human liver cancer cells BEL-7402 and Huh7 cells was higher, and pLKO.1-sh hDEK3 was more effective in inhibiting the DEK gene expression ( P < 0.05). Therefore, pLKO.1-sh hDEK3 was selected to infect BEL-7402 and Huh7 cells for subsequent functional experiments. CCK8 cell proliferation test result showed that the cell proliferation ability of BEL-7402 and Huh7 cells infected with recombinant lentivirus was weakened when compared with blank control and negative control group ( P < 0.05). Apoptosis results showed that the apoptosis rate of knockdown group was higher than that of blank and the negative control group ( P < 0.05). Cell scratch test result showed that the wound healing rate of knockdown group was lower than that of blank control and negative control group ( P < 0.05), and the difference was statistically significant; however, there was no statistically significant difference between blank control and negative control group. Conclusion:Targeting DEK expression in silent liver cancer cells can inhibit the cell proliferation, migration ability, and induce apoptosis, which lays the foundation for further study of the role of DEK gene in liver cancer.

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作者: 李术华 [1] 侯宇 [2] 陈哲 [2] 吴韦铷 [2] 吴传新 [3] 孙航 [1]
作者单位: 重庆医科大学病毒性肝炎研究所 重庆医科大学附属第二医院感染科 400010 [1] 陆军军医大学第一附属医院,重庆 400010 [2] 重庆医科大学附属第二医院肝胆外科 400010 [3]
期刊: 《中华肝脏病杂志》2020年28卷10期 868-875页 MEDLINEISTICPKUCSCDCA
栏目名称: 肝癌
DOI: 10.3760/cma.j.issn.1007-3418.2020.03.000
发布时间: 2020-11-16
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