Fig 1: Identification of the direct relationship between miR‐375 and circ_0000654 or E2F3. (a) Venn diagram showing the putative miRNAs identified by both CircInteractome and ENCORI prediction programs. (b) RNA pull‐down assays on cell extracts using bio‐miR‐375, bio‐miR‐346 or bio‐NC. (c) RIP experiments on cell extracts using anti‐Ago2 or anti‐IgG antibody. (d) qRT‐PCR of miR‐375 in cells after transfection by miR‐375 mimic or miR‐NC mimic. (e) The putative complementary sequence for miR‐375 in circ_0000654, the mutation in the target sequence, and the sequence of mature miR‐375. (f) Luciferase reporter assay showing the effect of miR‐375 on the expression of WT‐circ_0000654 and MUT‐circ_0000654. (g) The predicted binding sequence for miR‐375 in E2F3 3′UTR, the mutated sites in seed region, and the sequence of miR‐375. (h) Luciferase reporter assay showing the effect of miR‐375 on the expression of WT‐E2F3 3′UTR and MUT‐E2F3 3′UTR. (i) qRT‐PCR of miR‐375 in cells after transfection by in‐miR‐375 or in‐miR‐NC. (j) Western blot showing E2F3 protein level in cells transfected with miR‐NC mimic, miR‐375 mimic, in‐miR‐NC or in‐miR‐375. (k) Western blot of E2F3 protein in cells after transfection by sh‐circ_0000654, sh‐circ_NC, sh‐circ_0000654 + pcDNA or sh‐circ_0000654 + E2F3. (l) Relative miR‐375 expression by qRT‐PCR analysis in matched tumor and normal tissues from the same patients with ESCC (n = 44). (m and n) expression correlation analysis between miR‐375 and circ_0000654 or E2F3 mRNA using Pearson's correlation coefficients. (o) qRT‐PCR analysis of miR‐375 in HET‐1A, KYSE‐450, TE‐1, and ECA‐109 cells. **p < 0.01, ***p < 0.001
Fig 2: LOC102553417 silencing suppresses MTDH expression via competitively binding to miR-30e. Alignment of the binding site between (A) LOC102553417, miR-30e and the designed MUT sequence, and (B) miR-30e, MTDH and the designed MUT sequence. Binding of (C) miR-30e to LOC102553417 and (D) miR-30e to MTDH were assessed via dual-luciferase reporter assay. (E) Binding of miR-30e to LOC102553417 was assessed via RNA binding protein immunoprecipitation. mRNA expression levels of (F) miR-30e and (G) MTDH were assessed via RT-qPCR. (H) Representative western blotting images of MTDH, Akt, p-Akt, p53, caspase-3 and cleaved caspase-3 protein bands. (I) Statistical analysis of MTDH, Akt, p-Akt, p53, caspase-3 and cleaved caspase-3 protein expression levels assessed via western blotting. Results are expressed as the mean ± SD (n=3). **P<0.01; ***P<0.001; ns, no significant difference; WT, wild-type; MUT, mutant; NC, negative control; Ago2, Argonaute RNA-induced silencing complex catalytic component 2; p, phosphorylated; miR, microRNA; MTDH, metadherin; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; KD, knockdown.
Fig 3: Circ_0006174 acted as a sponge of miR-138-5p. (A and B) Relative enrichments of circ_0006174, U6 and 18S rRNA in cytoplasm and nucleus were detected by RT-qPCR. (C and D) Ago2 RIP assay was employed to assess the combination of Ago2 and circ_0006174 in CRC cells. (E) The potential target miRNAs of circ_0006174 were predicted by Circbank and Starbase 2.0. (F and G) RNA pull down assay was performed to evaluate the target relationship between miRNAs and circ_0006174. (H) The online tool StarBase 2.0 was used to predict the binding sites between circ_0006174 and miR-138-5p. (I) RT-qPCR assay was performed to analyze miR-138-5p expression in CRC cells transfected with miR-138-5p or miR-NC. (J and K) Dual-luciferase reporter assay was performed to verify the interaction between miR-138-5p and circ_0006174. (L) Relative expression of miR-138-5p in CRC cells transfected with sh-circ #1 or sh-NC was detected by RT-qPCR. Experiments were performed in triplicate, and data were presented as means ± SD. *P<0.05.
Fig 4: Circ_0097010 sponges miR-769-5p. (A) Sequence of miR-769-5p, the potential miR-769-5p complementary sites at circ_0097010, and the mutated target sequence in circ_0097010. (B) qRT-PCR showing the transfection efficiency of miR-769-5p mimic. (C) Luciferase activity of the wild-type (WT) or mutant (MUT) reporter constructs after transfection with miR-769-5p mimic or miR-NC mimic. (D) RIP experiments on extracts of hPDLCs using an antibody against Ago2 or IgG. (E) RNA pull-down assays on extracts of hPDLCs using bio-miR-769-5p or bio-miR-NC. (F) Relative miR-769-5p expression by qRT-PCR in gingival tissue samples of patients with periodontitis (n = 22) and healthy volunteers (n = 19). (G) Expression correlation analysis of miR-769-5p and circ_0079010 in periodontitis samples (n = 22) using Pearson's correlation coefficient. (H) qRT-PCR of miR-769-5p in hPDLCs after exposure to 0 or 2 μg/mL of LPS for 24 h. ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.
Fig 5: KLF6 is a direct miR-769-5p target and circ_0097010 controls KLF6 expression by miR-769-5p. (A) Alignment between miR-769-5p and the 3′UTR of human KLF6 and the mutations in the target sequence. (B) Luciferase activity of the wild-type (WT) or mutant (MUT) KLF6 3′UTR reporter constructs transfected into hPDLCs in the presence of miR-796-5p mimic or mimic control. (C) RIP experiments on extracts of hPDLCs using an antibody against Ago2 or IgG. (D) RNA pull-down assays on extracts of hPDLCs using bio-miR-769-5p or bio-miR-NC. (E) Quantification of KLF6 mRNA expression by qRT-PCR in gingival tissue samples of patients with periodontitis (n = 22) and healthy volunteers (n = 19). (F) Correlation analysis between KLF6 mRNA and miR-769-5p expression in periodontitis samples (n = 22) using Pearson's correlation coefficient. (G) Western blot of KLF6 protein in gingival tissue samples of patients with periodontitis (n = 3) and healthy volunteers (n = 3). (H) KLF6 protein expression by Western blot of lysates from hPDLCs after exposure to 0 or 2 μg/mL of LPS for 24 h. (I) Representative Western blot showing KLF6 protein level in LPS-treated hPDLCs after transfection by anti-miR-769-5p + si-circ_0097010, anti-miR-NC + si-circ_0097010, si-circ_0097010 or si-NC. ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.
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