Oncogenic role of microRNA-20a in human multiple myeloma

Figures & data

Figure 1 miR-20a is upregulated in MM patient bone marrow and MM cell lines.

Notes: QPCR detection of miR-20a in bone marrow samples of HDs and MM patients (A) and in nPCs and MM cell lines (B). Data are mean ± SD of three independent experiments. *p<0.05.
Abbreviations: MM, multiple myeloma; HDs, healthy donors; SD, standard deviation; QPCR, quantitative polymerase chain reaction; nPCs, normal plasma cells; miR, microRNA.

Figure 2 Role of miR-20a in MM cells proliferation and cell cycle progression.

Notes: (A and B) Relative expression of miR-20a in NCI-H929 cells after transfection with miR-20a mimics (miR-20a, A) or in U266 cells after transfection with miR-20a inhibitor (anti-miR-20a, B) determined by QPCR. (C–H) MM cells proliferation (C and D), apoptosis rate (E and F), cell cycle distribution (G and H) change after transfection with miR-20a mimics (C, E, and G, performed in NCI-H929 cells) or miR-20a inhibitor (D, F, and H, performed in U266 cells) determined by MTT assay or flow cytometry. Data are mean ± SD of three independent experiments.
Abbreviations: con, control; MM, multiple myeloma; SD, standard deviation; QPCR, quantitative polymerase chain reaction; OD, optical density; miR, microRNA; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide.

Figure 3 miR-20a targets EGR2 3′-UTR.

Notes: (A) Sequence of mature miR-20a and its predicted target sequence of EGR2 3′-UTR and positions of mutation (EGR2 3′-UTR-mut). (B) EGR2 expression of U266 cells transfected with miR-20a mimics or inhibitor determined by Western blot. β-actin was used as the loading control. (C) Dual-luciferase assay of U266 cells co-transfected with the WT (or mut) EGR2 3′-UTR-psiCHECK2 reporter vector and miR-20a mimics or miR-20a-in. *p<0.05.
Abbreviations: miR-20a, microRNA-20a; WT, wild-type; EGR2, early growth response protein 2; 3′-UTR, 3′ untranslated region; mut, mutated; NC, negative control; miR, microRNA; in, inhibitor.

Figure 4 Effect of EGR2 on U266 cells proliferation and apoptosis.

Notes: (A) Efficiency of small RNA interference to EGR2 determined by QPCR. Cell proliferation (B), apoptosis (C), and cell cycle distribution (D) of U266 cells with EGR2 knockdown measured by MTT assay or flow cytometry. Data are mean ± SD of three independent experiments. *p<0.05.
Abbreviations: si-NC, scrambled negative control of si-EGR2; si-EGR2, small RNA interference to EGR2; SD, standard deviation; OD, optical density; QPCR, quantitative polymerase chain reaction; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; EGR2, early growth response protein 2.

Figure 5 miR-20a alters p21 and cyclin D1 expression in U266 cells.

Notes: QPCR (A) and Western blot (B) detection of cyclin D1 and p21 expression in U266 cells as forced miR-20a overexpression or inhibition. β-Actin served as the loading control in Western blot. *p<0.05.
Abbreviations: QPCR, quantitative polymerase chain reaction; miR, microRNA; NC, negative control; in, inhibitor.

Figure 6 Oncogenic activity of miR-20a in MM xenograft SCID/NOD mice.

Notes: miR-20a mimics and inhibitor significantly promoted and suppressed xenograft tumor growth, respectively. Representative xenograft mice treated with miR-20 mimics or inhibitor or control oligos (A) and their isolated tumor nodule (B; the samples were separated in 3 groups, each group contained 2 mice). (C) Tumor volumes of xenograft mice treated with miR-20a mimics or miR-20a inhibitor.
Abbreviations: MM, multiple myeloma; miR, microRNA; NC, negative control; in, inhibitor.

Figure S1 U266 cells transfected with miR-20a have decreased sensitivity to bortezomib activity.

Abbreviations: bort, bortezomib; cont, control; miR, micro RNA; OD, optical density; scr, scrambled.