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. 2021 Mar 26;13:2821–2834. doi: 10.2147/CMAR.S293322

Figure 2.

Figure 2

NOP14-AS1 competitively sponges miR-665 in TSCC. (A) Cellular sublocalization of NOP14-AS1 in TSCC cells was examined by cell cytoplasmic/nuclear fractionation with qRT-PCR analysis. (B) Intersection of predicted miRNAs targeting NOP14-AS1 and weakly expressed miRNAs in TCGA-HNSC database. (C) qRT-PCR indicated the expression of these miRNAs in CAL-27 and SCC-15 cells with NOP14-AS1 knockdown. (D) qRT-PCR was conducted to detect miR-665 expression in 56 pairs of TSCC tissues and adjacent normal tissues. (E) Pearson’s correlation coefficient analysis of the correlation between NOP14-AS1 and miR-665 in 56 TSCC tissues. (F) Complementary WT and MUT binding sequences between NOP14-AS1 and miR-665. (G) Luciferase reporter assay was performed to verify the direct binding between NOP14-AS1 and miR-665. Luciferase activity was detected in CAL-27 and SCC-15 cells cotransfected with miR-665 mimic or NC mimic in parallel with WT-NOP14-AS1 or MUT-NOP14-AS1. (H) RIP assay was performed in CAL-27 and SCC-15 cells using anti-Ago2 or anti-IgG antibodies followed by quantification of NOP14-AS1 and miR-665 in immunoprecipitated RNA. **P < 0.01, compared with control.

Abbreviations: NOP14-AS1, NOP14 antisense RNA 1; TSCC, tongue squamous cell carcinoma; si-NC, negative control small interfering RNA; si-NOP14-AS1, small interfering RNA targeting NOP14-AS1; miRNA, microRNA; WT, wild-type; MUT, mutant; Ago2, Argonaute 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; U6, small nuclear RNA U6; NC mimic, negative control miRNA mimic;; IgG, immunoglobulin G.