c-erbB-2反义寡核苷酸转染对子宫内膜癌HEC-1A细胞系的疗效

Efficacy of c-erbB-2 antisense oligonucleotide transfection on uterine endometrial cancer HEC-1A cell lines.

作者信息

Zhao F J, Zhang S L, Ma L, Gao H, Zhong Z H

机构信息

Obstetrics and Gynecology Department, Shengjing Hospital, China Medical University, Shenyang, China.

出版信息

Eur J Gynaecol Oncol. 2007;28(4):263-9.

PMID:17713089

Abstract

OBJECTIVE

Antigene therapy targeting only one oncogene has made much progress although it still has some limitations. To explore the potential for antigene therapy in uterine endometrial cancer, we examined the in vitro inhibitory effects of liposmal anti-sense phosphorothioate oligonucleotides targeting c-erbB-2 in the human uterine endometrial cancer HEC-1A cell line.

METHODS

To detect c-erbB-2 protein expression on HEC-1A cell membranes by immunohisto- chemistry. 2) To assay cellular growth inhibition by MTT after transfecting 0.1-0.6 microM ASODN. 3) To observe cellular and ultra-structural changes under transmission electron microscope and to assay the cellular apoptotic rate by flow cytometry and c-erbB-2 mRNA, and protein expression by RT-PCR and Western blot after transfecting 0.3 microM ASODN.

RESULTS

c-erbB-2 protein expression was positive on HEC-1A cell membranes. 2) With the increase of the transfecting ASODN concentration from 0.1-0.6 microM, HEC-1A cellular growth inhibition was also enhanced. The results of MTT showed that when the transfecting concentration of ASODN was 0.3 microM, the HEC-1A cellular growth inhibition rate was 50% while when the transfecting concentration of ASODN was 0.6 microM, the HEC-1A cell growth inhibition rate was 75%. 3) When the concentration of transfecting ASODNs was 0.3 microM, there were obvious vacuolar degenerations in the plasma of HEC-1A cells, disappearance of organelle and nuclear structure and obvious shrinkage of nuclei under transmission electron microscope. The cellular apoptotic rate was 62.80%, while c-erbB-2 mRNA and protein expression were 47.18% and 33.60%, respectively, compared with those of the normal control cells.

CONCLUSION

Transfecting c-erbB-2 ASODNs can obviously suppress the mRNA and protein expression in HEC-1A cells, cause cellular apoptosis and inhibit cell growth. It may be a more useful gene therapy for endometrial cancer.

摘要

目的

尽管仅靶向一种癌基因的反基因疗法仍存在一些局限性，但已取得了很大进展。为了探索反基因疗法在子宫内膜癌中的应用潜力，我们检测了脂质体反义硫代磷酸寡核苷酸对人子宫内膜癌HEC-1A细胞系中c-erbB-2的体外抑制作用。

方法

1）通过免疫组织化学检测HEC-1A细胞膜上c-erbB-2蛋白的表达。2）转染0.1 - 0.6微摩尔反义寡脱氧核苷酸（ASODN）后，用MTT法检测细胞生长抑制情况。3）转染0.3微摩尔ASODN后，在透射电子显微镜下观察细胞及超微结构变化，并用流式细胞术检测细胞凋亡率，用逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测c-erbB-2 mRNA和蛋白表达。

结果

1）HEC-1A细胞膜上c-erbB-2蛋白表达呈阳性。2）随着转染ASODN浓度从0.1 - 0.6微摩尔增加，HEC-1A细胞生长抑制作用增强。MTT结果显示，当ASODN转染浓度为0.3微摩尔时，HEC-1A细胞生长抑制率为50%；当ASODN转染浓度为0.6微摩尔时，HEC-1A细胞生长抑制率为75%。3）当转染ASODN浓度为0.3微摩尔时，透射电子显微镜下可见HEC-1A细胞胞质中有明显的空泡变性，细胞器和核结构消失，细胞核明显皱缩。细胞凋亡率为62.80%，与正常对照细胞相比，c-erbB-2 mRNA和蛋白表达分别为47.18%和33.60%。