MiR-1204 sensitizes nasopharyngeal carcinoma cells to paclitaxel both in vitro and in vivo

Abstract

Nasopharyngeal carcinoma (NPC) is an endemic tumor with a relatively high incidence in Southern China and Southeast Asia. Paclitaxel combination chemotherapy has been used for treatment of advanced NPC. However, treatment failure often occurs due to development of acquired paclitaxel resistance. In this study, we first established a paclitaxel-resistant CNE-1/Taxol, HNE-2/Taxol and 5-8F/Taxol cell sublines by treating the parental CNE-1, HNE-2 and 5-8F cells with increasing doses of paclitaxel for about 5 months, respectively. Then, microRNA arrays were used to screen differentially expressed miRNAs between the CNE-1/Taxol cells and the parental CNE-1 cells. We found 13 differentially expressed miRNAs, of which miR-1204 was significantly downregulated in the paclitaxel-resistant CNE-1/Taxol cells. We restored miR-1204 expression in the CNE-1/Taxol, HNE-2/Taxol and 5-8F/Taxol cells and found that restoration of miR-1204 re-sensitized the paclitaxel-resistant CNE-1/Taxol, HNE-2/Taxol and 5-8F/Taxol cells to paclitaxel both in vitro. Finally, we demonstrated that restoration of miR-1204 in significantly inhibits tumor growth in vivo. Thus, our study provides important information for the development of targeted gene therapy for reversing paclitaxel resistance in NPC.

Keywords: EBV, Epstein-Barr virus; Lv-miR-1204, Lentiviruses containing miR-1204; NPC, nasopharyngeal carcinoma; chemotherapy; miR-1204; miRNAs, MicroRNAs; microRNAs; nasopharyngeal carcinoma; paclitaxel.

Figure 1.

Establishment of paclitaxol-resistant CNE-1/Taxol, HNE-2/Taxol and 5–8F/Taxol cell sublines. Cell morphology of the parental CNE-1, HNE-2 and 5–8F cells and paclitaxol-resistant CNE-1/Taxol (A), HNE-2/Taxol (B) and 5–8F/Taxol (C) cell sublines (left panel). Paclitaxel sensitivity was estimated by MTT assay (right panel). The established CNE-1/Taxol, HNE-2/Taxol and 5–8F/Taxol cell sublines were all high resistant to paclitaxel. (*P value < 0.05).

Figure 2.

Expression of miR-1204 is significantly different in the paclitaxel-resistant nasopharyngeal carcinoma cell sublines. (A) Volcano plot showing differences in the miRNAs detected by microarray for the paclitaxel-resistant CNE-1/Taxel cells (n=3 , N2, N4, N6) and the parental CNE-1 cells (n=3 , Q1, Q3, Q5). The -Log₁₀ (p-value) is plotted against the fold change difference in expression, the vertical lines correspond to fold2- up and down, respectively, and the horizontal line represents a p-value of 0.05, so the red points in the plot represent the differentially expressed miRNAs with statistical significance. (B) Comparion of miR-1204 expression levels between CNE-1, HNE-2 and 5–8F cells and CNE-1/Taxol, HNE-2/Taxol and 5–8F/Taxol cells. (*P value < 0.05).

Figure 3.

Up-regulation of miR-1204 expression in CNE-1/Taxol, HNE-2/Taxol and 5–8F/Taxol cells was successful. The CNE-1/Taxol (A), HNE-2/Taxol (B) and 5–8F/Taxol (C) cells were infected with Lv-miR-1204 (Lv- miR-1204) or negative control (Lv-NC) labeled with GFP gene sequences, respectively. GFP expression was observed under a fluorescence microscope after being infected for 6 d (left panel). Real-time qRT-PCR was performed to determinate expression levels of miR-1204 in stably infected CNE-1/Taxol, HNE-2/Taxol and 5–8F/Taxol cells (right panel), Lv-NC groups were taken as the control. (*P value< 0.05).

Figure 4.

MiR-1204 sensitizes CNE-1/Taxol, HNE-2/Taxol and 5–8F/Taxol cells to paclitaxel in vitro. The impacts of miR-1204 on drug sensitivity of CNE-1/Taxol (A), HNE-2/Taxol (B) and 5–8F/Taxol (C) cells at different paclitaxel doses (0, 2, 4, 6, 8 and 10 ng/ml) were determined by MTT assay. The CNE-1/Taxol (A), HNE-2/Taxol (B) and 5–8F/Taxol (C) cells were treated with a final concentration of 10 ng/ml paclitaxel for 24 h, and the impact of miR-1204 on drug sensitivity was determined by colony formation assay. (*P value < 0.05).

Figure 5.

MiR-1204 sensitizes CNE-1/Taxol cells to paclitaxel in vivo. BALB/C nude mice were subcutaneously inoculated with CNE-1/Taxol cells with overexpressed miR-1204 (Lv-miR-1204, n=5 ) and negative controls (Lv-miR-NC, n=5 ), respectively. After 61 days, paclitaxel (10 mg kg^-1) was then intravenously injected into mice once a day for 5 d (A) Tumor volume (mm³) was calculated every 4 d (B) Two days after complete paclitaxel treatments, all mice were euthanized and the tumors were excised and imaged under a light microscope. (*P value < 0.05).