Silencing of long non-coding RNA TUC338 inhibits the malignant phenotype of nasopharyngeal cancer cells via modulating the miR-1226-3p/FGF2 axis

Abstract

Long noncoding RNAs (lncRNAs) have been suggested as essential regulators in the cancer progression. LncRNA TUC338 was found to promote the malignancy of various cancers, however, the involvement of TUC338 in nasopharyngeal cancer (NPC) has not been well characterized. Here, our results found the significant overexpression of TUC338 in NPC tissues. Higher level of TUC338 was also observed in NPC cells. Interestingly, NPC patients harboring overexpressed TUC338 have worse prognosis. Functional study indicated that down-regulated TUC338 remarkably suppressed the NPC cell proliferation and cell migration. Notably, depletion of TUC338 significantly inhibited the in vivo tumor growth. Mechanistically, TUC338 acted as molecular sponge of miR-1226-3p and attenuated the negative regulation of miR-1226-3p on the expression of fibroblast growth factor 2 (FGF2). Down-regulation of TUC338 inhibited FGF2 expression in NPC cells and tumor tissues. Overexpression of FGF2 attenuated the suppressed NPC proliferation upon the depletion of TUC338. Our results demonstrated the novel function of TUC338/miR-1226-3p/FGF2 axis in NPC progression, suggesting the potential diagnosis and therapeutics significance of TUC338 in NPC.

Keywords: FGF2; NPC; TUC338; miR-1226-3p.

Fig. 1

TUC338 was overexpressed in NPC. A Total RNA was extracted from the NPC tissues and adjacent tissues. The TUC338 expression was determined by RT-qPCR. B The compassion for the levels of TUC338 in NPC cell line CNE-1, SUNE-1, 5-8F and HONE-1, and the human immortalized nasopharyngeal epithelial cell lines NP69. C The correlation between the expression of TUC338 and the 5-year OS of NPC patients

Fig. 2

Depleted TUC338 slowed down NPC cell growth. A The depletion of TUC338 in CNE-1 and 5-8F cells was validated by RT-qPCR. B, C CCK-8 assay was done to investigate the proliferation of CNE-1 and 5-8F cells following transfection of control-siRNA or siRNA-TUC338. D Down-regulation of TUC338 in CNE-1 and 5-8F cells induced cell cycle arrest in G₁ phase. E Depletion of TUC338 increased the apoptosis of both CNE-1 and 5-8F cells. F Knockdown of TUC338 inhibited NPC cell migration. Scale bar, 50 μm

Fig. 3

TUC338 knockdown inhibited tumor growth in vivo. A The knockdown efficiency of TUC338 in tumors was detected by RT-qPCR. B–D Knockdown of TUC338 significantly inhibited the tumor size and weight

Fig. 4

TUC338 was a ceRNA of miR-1226-3p in NPC. A The predicted binding sites of miR-1226-3p within the sequence of TUC338. B, C Both CNE-1 and 5-8F cells were transfected with miR-1226-3p and WT/Mutant TUC338. And the luciferase activity was detected. D Ago 2-IP was performed using Ago 2 specific antibody and the enrichment of TUC338 in Ago 2 IP components was detected by RT-qPCR normalized to control IgG. E The antisense oligonucleotide (ASO) pull-down assay was performed. TUC338 was specifically enriched using oligos against TUC338 (left panel). miR-1226-3p was significantly enriched in TUC338 pull-down components in both cell lines. F The level of miR-1226-3p was significantly decreased with the overexpression of TUC338 in NPC cells. G Down-regulation of TUC338 up-regulated the expression of miR-1226-3p in CNE-1 and 5-8F cells. H The abundance of miR-1226-3p in tissues was determined by RT-qPCR. I The correlation between the level of miR-1226-3p and TUC338 was investigated by the Spearman’s test

Fig. 5

MiR-1226-3p targeted FGF2 in NPC cells. A The overexpression of miR-1226-3p after transfection was examined by RT-qPCR. B, C Overexpression of miR-1226-3p inhibited CNE-1 and 5-8F cell proliferation. D The possible binding of miR-1226-3p in the 3′-UTR of FGF2 as predicted by the bioinformatics analysis. E, F Overexpression of miR-1226-3p decreased the luciferase activity of cells expressing WT instead of mutant 3′-UTR of FGF2. G, H Transfection of miR-1226-3p decreased FGF2 in NPC cells. I FGF2 expression in NPC was significantly increased compared with adjacent normal tissues. J, K The correlation between expressions of TUC338, miR-1226-3p with FGF2 was analyzed with Spearman correlation test

Fig. 6

Recovered FGF2 attenuated the inhibitory effects of TUC338 depletion on NPC cell proliferation. A, B Cells were transfected with control-siRNA or siRNA-TUC338. The mRNA and protein levels of FGF2 were detected. C, D The mRNA and protein levels of FGF2 in tumor tissues were examined. E, F FGF2 was recovered by transfecting pcDNA-FGF2 into NPC cells. G, H Overexpression of FGF2 attenuated the suppressed proliferation of NPC cells induced by TUC338 depletion. I Restoration of FGF2 significantly reversed TUC338 knockdown-induced apoptosis of both CNE-1 and 5-8F cells