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 apoptosis in vivo was 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 expression in vitro and in vivo. In addition, CNE-2 cell proliferation was inhibited in vitro as well as in 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. Our in vitro and in vivo study 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.

Keywords: Bax inhibitor-1; gene therapy; human telomerase reverse transcriptase; nasopharyngeal carcinoma; short-hairpin RNA.

Figure 1

Downregulation of human telomerase reverse transcriptase (hTERT) and Bax inhibitor-1 (BI-1) gene expression after shRNA transfection is shown. hTERT and BI-1 gene expression in CNE-2 cells was suppressed following transfection with sequence-specific short-hairpin (sh)RNA against hTERT, BI-1 and a combination of the two gene plasmids. Untreated, transfection reagent Lipofectamine™ 2000, empty vector pcDNA3.1(+) and vector carrying scrambled shRNA sequence was used as the negative control. After transfection CNE-2 cells were collected and used for RNA and protein isolation, respectively. (A) hTERT and (B) BI-1 mRNA and protein levels were analyzed using real-time PCR and western blot analysis. Results of the analysis are shown in the bar graph. The labels for the bars are: control, untreated, lip transfection reagent Lipofectamine™ 2000, pcDNA3.1: empty vector pcDNA3.1(+), hTERT or BI-1 siRNA: hTERT- or BI-1-specific shRNA-contained plasmids, hTERT-BI-1 siRNA: hTERT-BI-1 combination shRNA-contained plasmids, hTERT-s, BI-1-s or hTERT-BI-1-s siRNA: hTERT, BI-1 or hTERT-BI-1 scramble shRNA-containing plasmid. The western blot analysis was stripped and re-probed with β-actin antibody to check for equal loading of total protein. Data are shown as the mean ± SD of three independent experiments. ^*P<0.05 vs. the parental group.

Figure 2

Simultaneous knockdown of human telomerase reverse transcriptase (hTERT) and Bax inhibitor-1 (BI-1) inhibited CEN-2 cell growth and migration (A) Cell viability of CNE-2 cells following transfection with shRNA against hTERT, BI-1 or hTERT-BI-1, using MTT assay. The values are normalized to the control (100%). (B) Colony formation ability of CNE-2 cells following transfection with various plasmids was assayed in soft agar. At day 21, cell colonies were counted in 10 randomly chosen microscope fields and the colony formation rate was calculated. (C) Effect of hTERT, BI-1 or hTERT-BI-1 combinative shRNA on CNE-2 cell migration is shown. CNE-2 cells were seeded in 6-well plates and wounded the following day. Images were captured at 0, 24 and 48 h, respectively, after the wound was made. Data were normalized to NS control and are the means ± SD of triplicates. Asterisks are representative of significant differences compared to the control. (^*P<0.05, ^**P<0.01).

Figure 3

Induction of apoptosis following short hairpin (sh)RNA treatments is shown. (A) Cellular apoptosis was observed using Hoechst 33258 fluorescence staining to detect chromosomal condensation and nuclear fragmentation. (B) Early CNE-2 cells apoptosis rate was analyzed by flow cytometry. (C) Caspase-3 activity was measured and described as a percentage of change in the mean values derived from three separate experiments compared to the control group. Data were normalized to NS control and represent the means ± SD of triplicates. Asterisks are representative of statistically significant differences compared to the control. (^*P<0.05, ^**P<0.01).

Figure 4

Therapeutic effect of shRNA on the tumor obtained from inoculated CNE-2 cells in nude mice is shown. (A) The appearance of the xenograft subcutaneous nasopharyngeal carcinoma (NPC) in the control and various treatment groups is shown. The xenograft tumors treated with specific targeting short-hairpin (sh)RNA were markedly smaller compared to the control group of mice. (B) Growth curves of subcutaneous NPC in nude mice with various treatments demonstrates that tumor growth was markedly retarded compared to the control group. Compared to the single targeting group, the combinative shRNA-treated group showed enhanced therapeutic effects. (C) Inhibition rate of the control and various treatment groups is shown. As compared to the control group, the inhibitory rate of the three shRNA treatment groups was markedly increased and the combinative shRNA treatment group showed a higher inhibitory effect (**P<0.01). (D) Hematoxylin and eosin (HE) staining results of the tumors formed in nude mice, which are typical nasopharyngeal squamous cell carcinomas.

Figure 5

Specific short-hairpin (sh)RNA downregulated hTERT and BI-1 gene expression and induced apoptosis in tumor tissue are shown. (A) Expression of hTERT and BI-1 in subcutaneous nasopharyngeal carcinomas (NPCs) treated with shRNA targeting hTERT or BI-1 was suppressed, as shown by results of real-time RT-PCR. (B) Similar results were also obtained by immunohistochemistry (magnification, ×200) (*P<0.05). Compared to the single knockdown group, combinative shRNA simultaneously suppressed hTERT and BI-1 gene expression. (C) Apoptotic cells were increased in subcutaneous NPCs that were treated with shRNA targeting hTERT, BI-1 or combined hTERT/BI-1 detected by the TUNEL (TdT-mediated dUTP nick end-labeling) method (magnification, ×200) Combinative shRNA-treated group demonstrated a higher apoptotic rate (*P<0.05).