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. 2022 Mar-Mar;21(6):618-629.
doi: 10.1080/15384101.2022.2030168. Epub 2022 Jan 25.

miR-26a-5p suppresses nasopharyngeal carcinoma progression by inhibiting PTGS2 expression

Binlin Cai  1 , Xiu Qu  2 , Dan Kan  1 , Yi Luo  1
Affiliations

miR-26a-5p suppresses nasopharyngeal carcinoma progression by inhibiting PTGS2 expression

Binlin Cai et al. Cell Cycle. 2022 Mar-Mar.

Abstract

Nasopharyngeal carcinoma (NPC) has a low five-year survival rate, and its pathogenesis remains unclear. There is an urgent need to improve our understanding of the genetic regulation of NPC tumorigenesis and development. The role of miR-26a-5p in NPC growth regulation and the expression of its target, PTGS2, was analyzed. Quantitative Real-time PCR assay was used to detect miR-26a-5p and PTGS2 expression in human NPC tissues and cell lines. The RNA pull-down dual-luciferase reporter assay was used to determine the association between miR-26a-5p and PTGS2. The effects of miR-26a-5p and PTGS2 on NPC cell viability, proliferation, migration, and invasion were measured by CCK-8, BrdU, and Transwell assays. miR-26a-5p expression in NPC tissues and cell lines was significantly decreased. The overexpression of miR-26a-5p inhibited the viability, proliferation, migration, and invasion of NPC cells. miR-26a-5p bound to the 3-'untranslated region of PTGS2, thus reducing PTGS2 protein levels. miR-26a-5p inhibited NPC development by reducing the expression of its target PTGS2.

Keywords: Nasopharyngeal carcinoma; PTGS2; invasion; miR-26a-5p; migration; proliferation.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
The identification of PTGS2 and miR-26a-5p in NPC. (a) STRING showed the PPI network analysis of the top 10 most upregulated genes from GSE12452 data analysis (Criteria: adjusted P < 0.01, logFC≥1.5). (b) The intersection between the predicted target miRNAs of PTGS2 by Targetscan algorithm and the significantly downregulated miRNAs from GSE118613 data analysis (adjusted P < 0.05, logFC≤-1.5). (c) qRT-PCR analysis was employed to measure miR-26a-5p expression in NPC tissues and normal tissues. **P < 0.001. (d) qRT-PCR analysis was employed to measure miR-223-3p expression in NPC tissues and normal tissues. **P < 0.05.
Figure 2.
Figure 2.
Overexpression of miR-26a-5p inhibits viability and proliferation of NPC cells. (a) MiR-26a-5p expression in NPC cell lines (TW03, C666-1, 5–8 F) and normal nasopharyngeal epithelial cell line (NP69) was detected by qRT-PCR. The error bars indicate SD. n = 3. **P < 0.001 suggested a statistically significant difference in comparison with NP69. (b) qRT-PCR showed miR-26a-5p expression was significantly increased in C666-1 and 5–8 F cells after upregulation of miR-26a-5p. (c) CCK-8 assay showed the proliferation in C666-1 and 5–8 F cells were inhibited after upregulation of miR-26a-5p. (d) BrdU assay showed that the viability of C666-1 and 5–8 F cells were restrained after miR-26a-5p was upregulated. (b-d) The error bars indicate SD. n = 3. *P < 0.05, **P < 0.001 suggested a statistically significant difference in comparison with blank group.
Figure 3.
Figure 3.
Overexpression of miR-26a-5p inhibits migration and invasion of NPC cells. (a) Transwell migration assay revealed that cell migration ability were decreased in C666-1 and 5–8 F cells after miR-26a-5p was upregulated. (b) Transwell invasion assay revealed that cell invasion in C666-1 and 5–8 F cells were decreased after upregulation of miR-26a-5p. The error bars indicate SD. n = 3. **P < 0.001 suggested a statistically significant difference in comparison with blank group.
Figure 4.
Figure 4.
PTGS2 was targeted by miR-26a-5p in NPC Cells. (a) The binding site between miR-26a-5p and PTGS2 was presented from targetscan. (b) Luciferase reporter assay demonstrated that miR-26a-5p mimic significantly inhibited the luciferase activity of vectors that carried the wild-type of 3’-UTR of PTGS2 in C666-1 and 5–8 F cells. The error bars indicate SD. n = 3. **P < 0.001 suggested a statistically significant difference in comparison with miR-NC co-transfected with PTGS2-WT. (c) RNA pull-down assay showed that PTGS2 enrichment level increased in C666-1 and 5–8 F cells treated with biotin labeled miR-26a-5p. The error bars indicate SD. n = 3. **P < 0.001 suggested a statistically significant difference in comparison with Bio-NC group. (d) qRT-PCR analysis was employed to measure PTGS2 mRNA expression in NPC tissues and normal tissues. **P < 0.001. (e) qRT-PCR analysis was employed to test PTGS2 mRNA expression in NPC cell lines and NP69 cell line. The error bars indicate SD. n = 3. **P < 0.001 suggested a statistically significant difference in comparison with NP69. (f) The correlation between miR-26a-5p and PTGS2 was conducted by Pearson correlation analysis. The error bars indicate SD.
Figure 5.
Figure 5.
Overexpression of PTGS2 canceled out the inhibitory effect of upregulation of miR-26a-5p on the viability and proliferation of NPC cells in vitro. (a) Western blot revealed that overexpression of miR-26a-5p and PTGS2 plasmid reversed the down-regulation of PTGS2 protein induced by overexpression of miR-26a-5p in C666-1 and 5–8 F cells. (b) CCK-8 assay showed that overexpression of miR-26a-5p and PTGS2 plasmid reversed the inhibition of C666-1 and 5–8 F cell viability induced by upregulation of miR-26a-5p. (c) BrdU assay showed that overexpression of miR-26a-5p and PTGS2 plasmid reversed the inhibition of C666-1 and 5–8 F cell proliferation induced by upregulation of miR-26a-5p. The error bars indicate SD. n = 3. *P < 0.05, **P < 0.001 suggested a statistically significant difference in comparison with blank group. #P < 0.05, ##P < 0.001 suggested a statistically significant difference in comparison with OE + mimic group.
Figure 6.
Figure 6.
Overexpression of PTGS2 canceled out the inhibitory effect of upregulation of miR-26a-5p on the migration and invasion of NPC cells in vitro. (a) Transwell migration assay showed that the inhibition of migration ability of C666-1 and 5–8 F cells caused by miR-26a-5p overexpression was reversed by up-regulation of miR-26a-5p and PTGS2. (b) Transwell invasion assay showed that the inhibition of invasion ability of C666-1 and 5–8 F cells caused by miR-26a-5p overexpression was reversed by up-regulation of miR-26a-5p and PTGS2. The error bars indicate SD. n = 3. **P < 0.001 suggested a statistically significant difference in comparison with blank group. ##P < 0.001 suggested a statistically significant difference in comparison with OE + mimic group.
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