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. 2019 Sep 19;9(23):6745–6763. doi: 10.7150/thno.34674

Figure 3.

Figure 3

P21 is the target gene of miR-20a, -93, and 106b, which were suppressed by CA. (A) CA-caused expression change of the miR-17 family members is presented as the heat map, in which red or green means a relatively high or low expression of the genes, respectively; hsa, Homo sapiens. (B) Expression change of the miR-17 family members by CA in Huh7 (left) or H446 (right) cells was verified by qRT-PCR. Huh7 and H446 cells were treated with CA (25 or 50 μM for 24 h) followed by various measurements; data shown are mean ± SEM of 3 independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, CA treated vs untreated). (C) Seed sequence of miR-17 family complemented to the p21 mRNA 3' UTR was identified by using existing bio-information websites; the seed sequence of the miR-17 members 5'-AAAGUGC-3' and the complementary sequence in the p21 mRNA 3' UTR are marked in red. (D) p21 mRNA expression in Huh7 (left) and H446 (right) cells was assessed by RT- qPCR following treatment of cells with inhibitors of the miR-17 members; data shown are mean ± SEM of 3 independent experiments (* p < 0.05, ** p < 0.01 and *** p < 0.001, miRs inhibitor vs negative control inhibitor, nc inhibitor). (E) P21 protein expression in the Huh7 (left) and H446 (right) cells treated with miR-17 inhibitor by Western blotting. (F) P21 was identified as a target gene of miR-17 members, using dual luciferase reporter assay; three pairs of vectors were constructed, and the study fragments were obtained using specific primers, forward: 5'-GAAATTTGTGATGCTATTGC-3' and reverse: 5'-GCAATAGCATCACAAATTT-3', followed by sequencing. P1 represents a successful insertion into the oligonucleotide sequence of p21 3' UTR at position 468-474 (GCACTTT), the binding site of the miR-17 members (solid red line box), and mP1 is an insertion into the mutant sequence of p21 3' UTR at position 468-474 (GCATCC, dashed red box). P2 represents an insertion into the oligonucleotide sequence of p21 3' UTR at position 1148-1154, the binding site of the miR-17 members, and mP2 is an insertion into the mutant sequences of p21 3' UTR at position 1148-1154 (GCATCC). P1P2 are insertions into the oligonucleotide sequences of p21 3' UTR at both positions 468-474 and 1148-1154, the binding sites of miR-17 members, and mP1mP2 represent insertions into the mutant sequences of p21 3' UTR at positions 468-474 and 1148-1154 (GCATCC). The data were normalized to EV as 100%. EV stands for empty vector.