MicroRNA-384 inhibits nasopharyngeal carcinoma growth and metastasis via binding to Smad5 and suppressing the Wnt/β-catenin axis

Abstract

Nasopharyngeal carcinoma (NPC) is a major otorhinolaryngological disease with limited effective therapeutic options. This work focused on the function of microRNA-384 (miR-384) on the NPC pathogenesis and the molecules involved. miR-384 expression in cancer tissues and cells was detected. Gain- and loss-of-functions of miR-384 were performed to identify its role in NPC progression. The target mRNA of miR-384 was predicted on an online system and validated through a luciferase reporter assay. The activity of Wnt/β-catenin signaling was detected. Consequently, miR-384 was found to be poorly expressed in NPC tissues and cell lines and was linked to unfavorable survival rates in patients. Overexpression of miR-384 in 6-10B cells suppressed growth, migration, invasion and resistance to apoptosis of cells, but inverse trends were presented in C6661 cells where miR-384 was downregulated. miR-384 targeted Smad5 mRNA. Upregulation of Smad5 counteracted the roles of miR-384 mimic in cells. The NPC-inhibiting effects of miR-384 mimic were also blocked by Wnt/β-catenin activation. To conclude, miR-384 targets Smad5 and inactivates the Wnt/β-catenin pathway, which exerts a suppressing role in NPC cell behaviors as well as tumor growth in vivo. The findings may offer novel thoughts into NPC therapy.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-021-00458-3.

Keywords: Apoptosis; MicroRNA-384; Nasopharyngeal carcinoma; Proliferation; Smad5; Wnt/β-catenin signaling pathway.

Fig. 1

Poor expression of miR-384 indicates unfavorable prognosis in NPC patients. a miR-384 expression in 43 pairs of NPC and paracancerous tissues determined by RT-qPCR; b the correlation between miR-384 expression and survival rate of NPC patients evaluated by Kapla-Meier analysis; c miR-384 expression in NP69 and in NPC cell lines (C6661, SUNE1, SUNE2 and 6-10B) determined by RT-qPCR; d, 6-10B with lowest miR-384 expression among the four NPC cell lines was transfected with miR-384 mimic or mimic control, while C6661 cells with highest miR-384 expression was given miR-384 inhibitor or inhibitor control, after which miR-384 expression in cells was measured by RT-qPCR. Data are exhibited as mean ± SD from three independent experiments; Data in panel a were analyzed using paired t test, while data in panels c, d were analyzed using one-way ANOVA and Tukey’s multiple comparison test; **p < 0.01 vs. paracancerous tissues or the NP69 group; #p < 0.05 vs. the InC group

Fig. 2

Overexpression of miR-384 inhibits viability of NPC cells. a proliferation activity of 6-10B and C6661 cells determined by EdU labeling assay; b proportion of live and dead cells measured by AO/EB staining; c number of apoptotic cells determined by Hoechst 33,258 staining; d ratio of apoptotic cells detected by flow cytometry. Data are exhibited as mean ± SD from three independent experiments; Data were analyzed using one-way ANOVA and Tukey’s multiple comparison test; *p < 0.05; **p < 0.01 vs. the Mock group; #p < 0.05, ## p < 0.01 vs. the InC group

Fig. 3

miR-384 mimic inhibits EMT, migration and invasion of NPC cells. a levels of mesenchyme marker protein Vimentin and epithelium-marker protein E-cadherin in cells measured by western blot analysis; b, c number of migrated (b) and invaded (c) cells determined by Transwell assays. Data are exhibited as mean ± SD from three independent experiments; in panel (a), data were analyzed using two-way ANOVA, while data in panels (b) and (c) were analyzed by one-way ANOVA, and Tukey’s multiple comparison test was used for the post-hoc test after ANOVA; *p < 0.05; **p < 0.01 vs. the Mock group; #p < 0.05 vs. the InC group

Fig. 4

miR-384 inhibits Smad5 expression to inactivate the Wnt/β-catenin signaling pathway. a, b, binding relationship between miR-384 and Smad5 predicted on StarBase (http://starbase.sysu.edu.cn/) and validated through a dual luciferase reporter gene assay; c, d, mRNA (c) and protein (d) expression of Smad5 assessed by RT-qPCR and western blot analysis, respectively; (e), expression of Wnt1 and β-catenin in cells determined by western blot analysis. Data are exhibited as mean ± SD from three independent experiments; in panels (b) and (e), data were analyzed using two-way ANOVA, while data in panels (c) and (d) were analyzed by one-way ANOVA, and Tukey’s multiple comparison test was used for the post-hoc test after ANOVA; *p < 0.05; **p < 0.01 vs. the Mock group; #p < 0.05 vs. the InC group

Fig. 5

Overexpression of Smad5 or activation of β-catenin partially blocks the effects of miR-384 on NPC cells. LV overexpressing Smad5 was transfected into 6-10B cells pre-transfected with miR-384 mimic, while a Wnt/β-catenin-specific antagonist, IWR-1, was introduced into C6661 cells with silenced miR-384. a Smad5 expression in Smad5 cells and Wnt/β-catenin activation in C6661 cells determined by western blot analysis; b proliferation ability of cells determined by EdU labeling assay; c ratio of apoptotic cells determined by flow cytometer; d, e, number of migrated (d) and invaded (e) cells measured by Transwell assays. Data are exhibited as mean ± SD from three independent experiments; in panel (a), data were analyzed using unpaired t test or two-way ANOVA, while data in panels b, c, d and e were analyzed by one-way ANOVA, and Tukey’s multiple comparison test was used for the post-hoc test after ANOVA; *p < 0.05; **p < 0.01 vs. the miR-384 + Lv-NC group; #p < 0.05, ##p < 0.01 vs. the Inhibitor + DMSO group

Fig. 6

Overexpression of miR-384 inhibits growth of xenograft tumor in vivo. 6-10B cells with stable miR-384 mimic or mimic control transfection were implanted in nude mice. a change of tumor volume after cell implantation in mice; b tumor weight in mice on the 35th day after animal euthanasia; c Ki-67 expression in mouse tumors detected by IHC staining; Repetition = 3. Data are exhibited as mean ± SD; n = 5 in each group; in panel a, data were analyzed using two-way ANOVA and Tukey’s multiple comparison test, while data in panels b and c were analyzed by the unpaired t test; *p < 0.05; **p < 0.01 vs. Mock group