Research Paper Volume 14, Issue 10 pp 4376—4389

Figure 3. has_circ_0069313 directly targeted miR-325-3p. (A) The relative RNA level of miR-325-3p of indicated cells. (Student’s two-tailed paired test, ^***p < 0.001). (B) RNA probe was used in RNA pull-down assay and the circRNA level and miRNA level were detected (Student’s two-tailed paired test, ^***p < 0.001). (C) RIP assay was applied using Ago2 antibody. The relative has_circ_0069313 and miR-325-3p were detected and normalized (Student’s two-tailed paired test, ^***p < 0.001). (D) FISH using the junction specific probe and miR-325-3p probes, scale 20 μm. (E) The graphic illustration of has_circ_0069313 WT and MUT establishing strategy. (F) The detailed Mut allele establishment strategy. (G) SCC-9 sh-1 cells were transfected with the has_circ_0069313 Mut allele and named as Mut rescue. Cal-27 was transfected with the has_circ_0069313 Mut allele and was named as MUT. Cells were subjected to qRT-PCR and has_circ_0069313 was detected and normalized (Student’s two-tailed paired test, ^***p < 0.001). (H) miR-325-3p was detected and normalized in cells with indicated modifications (Student’s two-tailed paired test, ^***p < 0.001). (I) RNA pull-down assay was applied using junction-specific primers and the complex was subjected to qRT-PCR and miR-325-3p was detected (Student’s two-tailed paired test, ^***p < 0.001). (J) The relative miR-325-3p level in our in-house database (Student’s two-tailed paired test, ^***p < 0.001). (K) The regression analysis between has_circ_0069313 and miR-325-3p in our in-house database (R = 0.49, p < 0.001).