1. Volume 59, Issue 2, Pages 243-257 (July 2015)
MicroRNA-101 Suppresses Tumor Cell Proliferation by Acting as an Endogenous Proteasome Inhibitor via Targeting the Proteasome Assembly Factor POMP 
Xin Zhang, Ramona Schulz, Shelley Edmunds, Elke Krüger, Elke Markert, Jochen Gaedcke, Estelle Cormet-Boyaka, Michael Ghadimi, Tim Beissbarth, Arnold J. Levine, Ute M. Moll, Matthias Dobbelstein 
Molecular Cell 
Volume 59, Issue 2, Pages (July 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 59, 243-257DOI: (10.1016/j.molcel.2015.05.036)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1 miR-101 Acts as an Endogenous Proteasome Inhibitor
(A) Immunoblot analysis of p53 in U2OS, HCT116, and MCF7 cells transfected with scrambled miRNA (scr) or miR-101. β-actin serves as loading control. The intensities of the bands were quantitated by ImageJ, and the relevant results are displayed beneath the bands.
(B) miR-101-mediated accumulation of modified p53 as a function of Mdm2. U2OS cells were transfected with control siRNA (si ctrl) or Mdm2 siRNA (si Mdm2). At 24 hr post-transfection, cells were further transfected with scr or miR-101 and incubated for another 48 hr.
(C) Detection of HA-ubiquitinated p53 upon miR-101 transfection. p53 was immunoprecipitated using anti-HA antibody in scr- or miR-101-transfected U2OS cells that stably express HA-tagged ubiquitin and was analyzed by immunoblot.
(D) Immunoblot analysis of p21, Mdm2, and p27 protein in scr- or miR-101-transfected cells (U2OS, HCT116, and MCF7).
(E) qRT-PCR of p53, p21, Mdm2, and p27 mRNA expression in scr- or miR-101-transfected U2OS cells. Data are presented as mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. = not significant.
(F) Cycloheximide (CHX) chase assays to determine the half-lives of p53, p21, and Mdm2 proteins following transfection of scr or miR-101. At 48 hr post-transfection, cells were treated with CHX (50 μg/ml) for the indicated time periods and then harvested for immunoblot analysis.
(G) Immunoblot analysis of endogenous poly-ubiquitinated proteins in scr- or miR-101-transfected cells (U2OS and HCT116) using an anti-ubiquitin antibody.
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4. Figure 2 miR-101 Targets the Proteasome Maturation Protein POMP
(A) Differential expression profile upon transfection with miR-101. Microarray data displayed by scatter diagram showing the relative gene expression in scr- versus miR-101-transfected U2OS cells. The raw signal level for each gene is displayed in relative fluorescence units (Log2 fl). Each value represents the average of three biological replicates.
(B) qRT-PCR of POMP mRNA expression in scr- or miR-101-transfected U2OS cells. Columns represent the mean ± SD of three independent experiments.
(C) POMP levels decrease in miR-101-transfected U2OS, MCF7, and HCT116 cells compared to scr-transfected controls, similar to targeted knockdown of POMP.
(D) POMP levels increase in miR-101 inhibitor-transfected HepG2 cells, compared to control-transfected cells. At 24 hr post-transfection, the cells were treated with 100 nM TPA for another 48 hr and then harvested for immunoblot analysis. The right panel quantitates the band intensity corresponding to POMP, normalized to the loading control.
(E) Dual-luciferase reporter assays (Promega) showing repression of the wild-type POMP 3′ UTR by miR-101. Mutant 3′ UTRs are depicted in Figure S3.
(F) Inverse correlation between miR-101 and POMP mRNA expression in ER+ breast tumor tissues, as derived from GEO: GSE29173 (Farazi et al., 2011).
Data are presented as mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. = not significant.
See also Figures S1–S4 and Tables S1, S2, and S3.
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5. Figure 3 miR-101 Interferes with Proteasome Assembly and Activity
(A) In vitro proteasome activity assay by native PAGE substrate overlay showing decreased activity of both 20S and 26S proteasome activity by miR-101 or si POMP.
(B) Quantitation of the in vitro proteasome activity, normalized to scr or si ctrl (n = 3). The values of scr and si ctrl were set as 100.
(C) Immunoblot analysis of proteasome assembly by native PAGE and immunoblotting using an α6 antibody that stains proteasome complexes and their assembly intermediates. GAPDH serves as a loading control.
(D) Protein levels of overexpressed POMP in U2OS cells that stably express POMP either with wild-type (wt) or mutant 3′ UTR (mut) compared to parental cells.
(E) mRNA levels of the same cells as above, as determined by qRT-PCR.
(F) Levels of POMP, EZH2, and poly-ubiquitinated proteins in scr- or miR-101-transfected U2OS cells that stably express POMP either with wild-type (wt) or mutant 3′ UTR (mut), as detailed in Figure S3.
(G) miR-101-resistant POMP restores proper turnover of proteasome substrates in the face of forced miR-101 expression. p53, p21, Mdm2, and p27 proteins in Scr- or miR-101-transfected U2OS cells stably expressing POMP either with wild-type (wt) or mutant 3′ UTR (mut) were analyzed by immunoblot.
Data are presented as mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See also Figure S3.
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6. Figure 4
miR-101 Suppresses Tumor Cell Growth and Acts as a Chemosensitizer
(A and B) Cell proliferation assay following scr, miR-101, si ctrl, or si POMP transfection. The curves represent cell numbers (A) and cell confluence (B).
(C) miR101-resistant POMP overexpression restores cell growth in the presence of miR-101. Cell proliferation following scr or miR-101 transfection in U2OS cells stably expressing POMP either with wild-type (wt) or mutant 3′ UTR (mut) was monitored by a Celigo Cell Cytometer.
(D) miR-101 arrests cells preferentially in the G2/M phase of the cell cycle. Flow cytometry analysis of U2OS cells transfected with Scr, miR-101, si ctrl, or si POMP.
(E) miR-101 induces increased apoptosis. Caspase activity assay showing increased apoptosis by miR-101 or si POMP in HCT116 cells, normalized to scr-transfected cells.
(F) Antagonizing miR-101 restores cell growth suppression by TPA. HepG2 cells were transfected with negative control inhibitor or miR-101 inhibitor. At 24 hr post-transfection, the cells were treated with DMSO or 100 nM TPA for another 24 hr. Then, the cell numbers were determined. The cell number for the anti-NC- and DMSO-treated sample was set as 100.
(G) POMP knockdown reverses the effects of miR-101 inhibitor on cell growth. HepG2 cells were transfected with negative control inhibitor, miR-101 inhibitor, si ctrl, or si POMP, alone or combined. At 24 hr post-transfection, the cells were treated with 100 nM TPA for another 24 hr, followed by cell counting as in (F).
(H and I) Chemosensitization by miR-101. Immunoblot analysis of cleaved caspase-3 and cleaved PARP was performed after transfecting U2OS (H) or HCT116 (I) cells with scr or miR-101. At 24 hr post-transfection, cells were treated with DMSO or CPT (200 nM, 500 nM) for another 24 hr.
Data are presented as mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. = not significant.
See also Figure S5.
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7. Figure 5
Correlation between miR-101 or POMP Expression and Survival of Breast Cancer Patients
(A) Kaplan-Meier analysis showing a positive correlation between miR-101 expression and survival of ER-positive, but not ER-negative, breast cancer patients.
(B) Kaplan-Meier analysis showing an inverse correlation between POMP expression and survival of ER-positive, but not ER-negative, breast cancer patients. ER status is derived from gene expression data.
(C and D) qRT-PCR analyzing the expression of representative estrogen-dependent genes (CXCL12, GREB1, and PGR) and POMP in the absence or presence of E2 in scr- or miR-101-transfected MCF7 cells (C) or si ctrl- or si POMP-transfected MCF7 cells (D).
(E and F) Colony formation assays showing estrogen-dependent and -independent cell growth suppression by miR-101 expression (E) and POMP knockdown (F) in MCF7 cells. The right panels quantitate the colony numbers, normalized to untreated scr- or si ctrl-transfected cells.
Data are presented as mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
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8. Figure 6
POMP Inhibition Suppresses Breast Cancer Growth and Triggers Tumor Regression
(A) Schematic diagram showing the TRIPZ-inducible shRNA expression system used for expressing POMP shRNA in vivo.
(B) Schematic diagram of subcutaneous cell injections into the flanks of female nude mice. The left flank received ctrl shRNA cells (shctrl), and the right flank received POMP shRNA cells (shPOMP).
(C and D) Xenograft experiments showing suppressed tumor growth by POMP inhibition. Female nude mice were injected subcutaneously into the flanks as indicated in (B). At 3 days prior to injection, mice were administered 17β-estradiol (0,67 μg/ml) via drinking water containing 5% sucrose with or without Dox (1 mg/ml). Graph represents the mean of tumor volumes (mm3) ± SD.
(E) Xenograft experiments showing tumor regression by POMP inhibition. Female nude mice were estradiol-treated and injected as in (B). At 5 weeks post-injection, we began to treat the mice with Dox in the drinking water (1 mg/ml).
(F) qRT-PCR showing POMP expression in harvested tumors at endpoint (9 weeks, 4 tumors each for shctrl and shPOMP) from (E).
(G) Accumulation of p53 and p21 in shPOMP-expressing tumors. Representative immunofluorescence image; scale bar, 50 μm.
(H) Reduced Ki67-positive cells in shPOMP-expressing tumors. Upper, representative immunohistochemical image; scale bar, 50 μm. Lower, the percentage of Ki67-positive cells was counted per high-power field (400×), three fields per tumor (n = 6).
(I) Accumulated cleaved caspase-3 in shPOMP-expressing tumors. Representative immunohistochemical image; scale bar, 50 μm.
Data are presented as mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
POMP Suppression Overcomes Tumor Cell Resistance to Bortezomib
(A) Proliferation of parental and bortezomib-resistant U2OS cells in the presence of 12.5 nM bortezomib. The curves represent cell numbers.
(B) Resistant cells undergo less apoptosis upon bortezomib. Immunoblot analysis of PARP cleavage for parental and resistant U2OS cells after treatment with bortezomib at the indicated concentrations for 48 hr.
(C) Resistant cells accumulate less poly-ubiquitinated proteins in response to bortezomib. Immunoblot analysis of poly-ubiquitinated proteins from parental and resistant U2OS cells after treatment with 20 nM bortezomib for the indicated times.
(D) Levels of 20S proteasome α/β subunits in parental and bortezomib-resistant U2OS cells.
(E) miR-101 or POMP knockdown by siRNA accumulates poly-ubiquitinated proteins in bortezomib-resistant cells. Immunoblot analysis of poly-ubiquitinated proteins from resistant U2OS cells transfected with miR-101 or si POMP twice for 3 days.
(F) Resistant cells no longer proliferate upon miR-101 expression or POMP knockdown. Proliferation of resistant U2OS cells transfected with si ctrl or si POMP. The curves represent cell numbers.
(G) miR-101 expression or POMP knockdown arrests cells in the G2/M phase in resistant cells. Flow cytometry of resistant U2OS cells, transfected with miR-101 or si POMP twice for 3 days.
(H) Combined treatment of miR-101 expression or POMP knockdown and bortezomib induces apoptosis in resistant cells. PARP cleavage after transfection with miR-101 or si POMP for 2 days, treated with or without 12.5 nM bortezomib.
Data are presented as mean ± SD of three independent experiments. See also Figure S7.
Molecular Cell  , DOI: ( /j.molcel )
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