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. 2018 Jan 1;8(4):1084–1105. doi: 10.7150/thno.21740

Figure 3.

Figure 3

Over-expression of PIWIL3, piR-30188and knockdown of OIP5-AS1 inhibited CEBPA expression by down-regulating miR-367-3p. (A) Quantitative real-time PCR (qRT-PCR) analysis for PIWIL3, piR-30188, and OIP5-AS1-regulated miR-367-3p expression in U87 and U251 cells (data are presented as the mean ± SD (n =3, each group); *#ΔP < 0.05 relative to normal control (NC)groups; &#ΨP< 0.05 relative to PIWIL3(+), pre-piR-30188 and sh-OIP5-AS1 groups). (B) OIP5-AS1 expression levels altered by over-expression or knockdown of miR-367-3p (data are presented as the mean ± SD (n =3, each group); **P < 0.01 relative to pre-NC group; ##P < 0.01 relative to anti-NC group). (C) The predicted miR-367-3p binding site in OIP5-AS1 (OIP5-AS1-Wt) and the designed mutant sequence (OIP5-AS1-Mut) are illustrated. (D) Luciferase reporter assay of HEK 293T cells co-transfected with OIP5-AS1-Wt or OIP5-AS1-Mut and pre-miR-367-3p or pre-NC (data are presented as the mean ± SD (n = 3, each group);**P< 0.01 relative to OIP5-AS1-Wt+pre-NC group). (E, F) RIP confirmed that OIP5-AS1 and miR-367-3p were in the RISC complex. (G, H) Effects of over-expression of PIWIL3, piR-30188 as well as knockdown of OIP5-AS1 on CEBPA expression. The integrated density values (IDVs) of CEBPA are shown using GAPDH as an endogenous control (data are presented as the mean ± SD (n =3, each group);*#P < 0.05 relative to NC groups; ΔΔP< 0.01 relative to NC groups; &%ΨP< 0.05 relative to PIWIL3(+), pre-piR-30188, and sh-OIP5-AS1 groups). (I-K) qRT-PCR and Western blot analysis for miR-367-3p-regulated CEBPA expression in U87 and U251 cells. The relative expression of CEBPA is shown using GAPDH as an endogenous control. The IDVs of CEBPA are shown using GAPDH as an endogenous control (data are presented as the mean ± SD (n =3, each group); **P < 0.01 relative to pre-NC group; ##P<0.01 relative to anti-NC group). (L) The predicted miR-367-3p binding site in CEBPA (CEBPA-Wt) and the designed mutant sequence (CEBPA-Mut) are illustrated. (M) Luciferase reporter assay of HEK 293T cells co-transfected with CEBPA-Wt or CEBPA-Mut and pre-miR-367-3p or pre-NC (data are presented as the mean ± SD (n = 3, each group);**P< 0.01 relative to CEBPA-Wt+pre-NC group).