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. 2021 Jan 14;13:379–392. doi: 10.2147/CMAR.S287676

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© 2021 Cheng et al.

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UBA6-AS1 acts as a molecular sponge for miR-760 in GBM cells. (A) The localization of UBA6-AS1 was predicted by lncLocator. (B) Nucleus-cytoplasm fractionation assay was used to test the subcellular distribution of UBA6-AS1 in U251 and T98 cells. (C) The potential miRNAs targeting UBA6-AS1 were predicted by the ENCORI and miRDB databases. (D) The expression of miRNAs (miR-378a-3p, miR-378b/c/d/e/f/h/I, miR-422a and miR-760) was detected in UBA6-AS1-depleted U251 and T98 cells via RT-qPCR analysis. (E) RT-qPCR was implemented to measure UBA6-AS1 expression in miR-760 mimic-transfected or miR-NC-transfected U251 and T98 cells. (F) miR-760 expression in 49 GBM tissues and 13 normal brain tissue samples was analyzed by RT-qPCR. (G) Pearson’s correlation analysis was used to determine the correlation between the expression of miR-760 and that of UBA6-AS1 in 49 GBM tissues. (H) Wild-type and mutant binding sites between miR-760 and UBA6-AS1. (I) Luciferase reporter gene assay was conducted to confirm the targeting association between miR-760 and UBA6-AS1. Luciferase activity was measured in U251 and T98 cells transfected with miR-760 mimic or miR-NC in combination with UBA6-AS1-wt or UBA6-AS1-mut. (J) Radioimmunoprecipitation assays revealed Ago2 antibody enrichment of miR-760 and UBA6-AS1 in U251 and T98 cells. (K) U251 and T98 cells were transfected with bio-miR-760 and bio-miR-NC. After transfection, RT-qPCR was performed to assess the relative enrichment of UBA6-AS1 in the formed bio-miRNA-lncRNA complexes. **P < 0.01.

Abbreviations: GBM, glioblastoma; RT-qPCR, reverse transcription-quantitative PCR; miR-NC, negative control miRNA mimic; miR, microRNA; UBA6-AS1, UBA6 antisense RNA 1; si-NC, negative control small interfering RNA; si-UBA6-AS1, small interfering RNA targeting UBA6-AS1; wt, wild-type; mut, mutant; Ago2, Argonaute 2.