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miR-185-3p regulates nasopharyngeal carcinoma radioresistance by targeting WNT2B in vitro.

Li G, Wang Y, Liu Y, Su Z, Liu C, Ren S, Deng T, Huang D, Tian Y, Qiu Y - Cancer Sci. (2014)

Bottom Line: Luciferase reporter assays confirmed that miR-185-3p directly targeted the coding region of WNT2B.Furthermore, we found radioresistance decreased in WNT2B-silenced NPC cells.We concluded that miR-185-3p contributed to the radioresistance of NPC via modulation of WNT2B expression in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology Head and Neck Surgery, Xiangya Hospital, Central South University, Changsha, China; Otolaryngology Major Disease Research Key Laboratory of Hunan Province, Changsha, Hunan, China.

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MiR-185-3p directly targets the coding region of WNT2B. (a) The potential second structure of WNT2B and miR-185-3p and the minimum free energy required for this hybridization. (b) A mutation was generated in the WNT2B coding region. In particular, the mutation was located in the complementary site for the seed region of miR-185-3p as indicated. The wild-type WNT2B coding region and mutant WNT2B coding region were subcloned into a luciferase reporter construct, as shown. Relative luciferase activity in 5-8F cells was determined after the WNT2B coding region or mutant plasmids were co-transfected with miR-185-3p mimics or a negative control (**P < 0.01).
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fig05: MiR-185-3p directly targets the coding region of WNT2B. (a) The potential second structure of WNT2B and miR-185-3p and the minimum free energy required for this hybridization. (b) A mutation was generated in the WNT2B coding region. In particular, the mutation was located in the complementary site for the seed region of miR-185-3p as indicated. The wild-type WNT2B coding region and mutant WNT2B coding region were subcloned into a luciferase reporter construct, as shown. Relative luciferase activity in 5-8F cells was determined after the WNT2B coding region or mutant plasmids were co-transfected with miR-185-3p mimics or a negative control (**P < 0.01).

Mentions: As we predicted, WNT2B is most likely the target gene of miR-185-3p. A series of experiments was performed to confirm our prediction. Hybridization of miR-185-3p and WNT2B mRNA can be predicted using RNAhybrid software and the minimum free energy required for this hybridization is −35.2 kcal/mol (Fig. 5a). Thus, specific targeting of WNT2B by miR-185-3p was examined using luciferase reporter assays. A mutant WNT2B reporter gene was constructed by deleting the seed sequence GGT GCG GAG GAA GCU GCG CAG CUC CC and mutating this sequence to GCT GGG CTC CTT TCT CGG GTC GGG GC (Fig. 5b). Our data revealed that disruption of the binding sites between miR-185-3p and the coding region of WNT2B mRNA abolished the miR-185-3p-mediated inhibition of WNT2B luciferase activity (P < 0.01; Fig. 5b). Taken together, these data indicate that miR-185-3p inhibits the expression of WNT2B protein via specific binding to the coding region of its mRNA.


miR-185-3p regulates nasopharyngeal carcinoma radioresistance by targeting WNT2B in vitro.

Li G, Wang Y, Liu Y, Su Z, Liu C, Ren S, Deng T, Huang D, Tian Y, Qiu Y - Cancer Sci. (2014)

MiR-185-3p directly targets the coding region of WNT2B. (a) The potential second structure of WNT2B and miR-185-3p and the minimum free energy required for this hybridization. (b) A mutation was generated in the WNT2B coding region. In particular, the mutation was located in the complementary site for the seed region of miR-185-3p as indicated. The wild-type WNT2B coding region and mutant WNT2B coding region were subcloned into a luciferase reporter construct, as shown. Relative luciferase activity in 5-8F cells was determined after the WNT2B coding region or mutant plasmids were co-transfected with miR-185-3p mimics or a negative control (**P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4317952&req=5

fig05: MiR-185-3p directly targets the coding region of WNT2B. (a) The potential second structure of WNT2B and miR-185-3p and the minimum free energy required for this hybridization. (b) A mutation was generated in the WNT2B coding region. In particular, the mutation was located in the complementary site for the seed region of miR-185-3p as indicated. The wild-type WNT2B coding region and mutant WNT2B coding region were subcloned into a luciferase reporter construct, as shown. Relative luciferase activity in 5-8F cells was determined after the WNT2B coding region or mutant plasmids were co-transfected with miR-185-3p mimics or a negative control (**P < 0.01).
Mentions: As we predicted, WNT2B is most likely the target gene of miR-185-3p. A series of experiments was performed to confirm our prediction. Hybridization of miR-185-3p and WNT2B mRNA can be predicted using RNAhybrid software and the minimum free energy required for this hybridization is −35.2 kcal/mol (Fig. 5a). Thus, specific targeting of WNT2B by miR-185-3p was examined using luciferase reporter assays. A mutant WNT2B reporter gene was constructed by deleting the seed sequence GGT GCG GAG GAA GCU GCG CAG CUC CC and mutating this sequence to GCT GGG CTC CTT TCT CGG GTC GGG GC (Fig. 5b). Our data revealed that disruption of the binding sites between miR-185-3p and the coding region of WNT2B mRNA abolished the miR-185-3p-mediated inhibition of WNT2B luciferase activity (P < 0.01; Fig. 5b). Taken together, these data indicate that miR-185-3p inhibits the expression of WNT2B protein via specific binding to the coding region of its mRNA.

Bottom Line: Luciferase reporter assays confirmed that miR-185-3p directly targeted the coding region of WNT2B.Furthermore, we found radioresistance decreased in WNT2B-silenced NPC cells.We concluded that miR-185-3p contributed to the radioresistance of NPC via modulation of WNT2B expression in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology Head and Neck Surgery, Xiangya Hospital, Central South University, Changsha, China; Otolaryngology Major Disease Research Key Laboratory of Hunan Province, Changsha, Hunan, China.

Show MeSH
Related in: MedlinePlus