title: Combinatorial gene targeting hTERT and BI-1 in CNE-2 nasopharyngeal carcinoma cell line.
abstract: Nasopharyngeal carcinoma (NPC) is a common malignant tumor. In recent studies, we demonstrated that overexpression of the Bax inhibitor-1 (BI-1) induces cell transformation in NIH3T3 cells and that knockdown of BI-1 and human telomerase reverse transcriptase (hTERT) gene expression suppresses NPC cell proliferation and induces apoptosis. To evaluate the combination anti-tumor effects of siRNAs against hTERT and BI-1 in the CNE-2 NPC cell line, combined and separate short-hairpin (sh)RNA plasmids targeting hTERT and BI-1, respectively, were constructed. hTERT and BI-1 mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and western blot analysis. Cell proliferation, colony formation and migration ability were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), soft agar and wound healing assay. Cell apoptosis was observed by flow cytometry, Hoechst 33258 staining and caspase-3 activity. hTERT, BI-1 and combined shRNA plasmids were injected into xenograft NPC tumor tissues, and expression of hTERT and BI-1 was detected by real-time PCR and immunohistochemistry. Tumor growth was measured by tumor volume and apoptosisin vivowas confirmed by TdT-mediated dUTP nick end-labeling (TUNEL). Our results showed that combined shRNA specific for hTERT and BI-1 markedly suppressed hTERT and BI-1 gene expressionin vitroandin vivo. In addition, CNE-2 cell proliferation was inhibitedin vitroas well asin vivo. Following the knockdown of the two gene expressions, CNE-2 exhibited a decrease in colony formation and migration ability and an increase in the apoptotic rate compared to the control groups. Ourin vitroandin vivostudy showed that the combinative silencing of the two genes enhanced the therapeutic effect compared to the silencing of each individual shRNA. These data suggested that combinatorial gene therapy targeting hTERT and BI-1 may be beneficial as a tumor therapy strategy against human NPCs.
authors.affiliation: Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Guangdong Medical College, Dongguan, Guangdong 523808, P.R. China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liang
authors.affiliation: Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Guangdong Medical College, Dongguan, Guangdong 523808, P.R. China.
authors.forename: Xiangyong
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Guangdong Medical College, Dongguan, Guangdong 523808, P.R. China.
authors.forename: Rongwen
authors.initials: R
authors.lastname: Lin
authors.affiliation: Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Guangdong Medical College, Dongguan, Guangdong 523808, P.R. China.
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Guangdong Medical College, Dongguan, Guangdong 523808, P.R. China.
authors.forename: Huimin
authors.initials: H
authors.lastname: Wang
authors.affiliation: Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Guangdong Medical College, Dongguan, Guangdong 523808, P.R. China.
authors.forename: Ning
authors.initials: N
authors.lastname: Tan
authors.affiliation: Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Guangdong Medical College, Dongguan, Guangdong 523808, P.R. China.
authors.forename: Keshen
authors.initials: K
authors.lastname: Li
authors.affiliation: Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Guangdong Medical College, Dongguan, Guangdong 523808, P.R. China.
authors.forename: Xudong
authors.initials: X
authors.lastname: Tang
authors.affiliation: Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Guangdong Medical College, Dongguan, Guangdong 523808, P.R. China.
authors.forename: Keyuan
authors.initials: K
authors.lastname: Zhou
authors.affiliation: Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Guangdong Medical College, Dongguan, Guangdong 523808, P.R. China.
authors.forename: Tao
authors.initials: T
authors.lastname: Li
keywords: Bax inhibitor-1; gene therapy; human telomerase reverse transcriptase; nasopharyngeal carcinoma; short-hairpin RNA