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. 2006 Nov;26(21):8191-201.
doi: 10.1128/MCB.00242-06. Epub 2006 Aug 28.

Mir-17-5p regulates breast cancer cell proliferation by inhibiting translation of AIB1 mRNA

Anwar Hossain  1 , Macus T Kuo, Grady F Saunders
Affiliations

Mir-17-5p regulates breast cancer cell proliferation by inhibiting translation of AIB1 mRNA

Anwar Hossain et al. Mol Cell Biol. 2006 Nov.

Abstract

MicroRNAs are an extensive family of approximately 22-nucleotide-long noncoding RNAs expressed in a wide range of eukaryotes, including humans, and they are important in development and disease. We found that microRNA Mir-17-5p has extensive complementarity to the mRNA of AIB1 (named for "amplified in breast cancer 1"). Cell culture experiments showed that AIB1 expression was downregulated by Mir-17-5p, primarily through translational inhibition. Expression of Mir-17-5p was low in breast cancer cell lines. We also found that downregulation of AIB1 by Mir-17-5p resulted in decreased estrogen receptor-mediated, as well as estrogen receptor-independent, gene expression and decreased proliferation of breast cancer cells. Mir-17-5p also completely abrogated the insulin-like growth factor 1-mediated, anchorage-independent growth of breast cancer cells. Our results reveal that Mir-17-5p has a role as a tumor suppressor in breast cancer cells.

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Figures

FIG. 1.
FIG. 1.
AIB1 is a target of Mir-17-5p. (A) Sequence alignment and diagram of Mir-17-5p and its complementary sites in the AIB1 cDNA. The hatched area indicates the coding region; the black square indicates the region cloned at the end of the luciferase reporter gene. (B) Activity of luciferase reporters containing the Mir-17-5p complementary site from human AIB1 (LucMir-17-5p), a mutant Mir-17-5p complementary site from human AIB1 (mutLucMir-17-5p), or the perfect antisense sequence of Mir-17-5p (LucMir-17-5pAS). The reporters were cotransfected with the indicated amounts of 2′-O-methyl Mir-17-5p or Mir-95. Firefly luciferase activity was normalized to that of β-galactosidase. The data shown are the mean of three independent experiments, and error bars indicate the standard deviations. (C) Activity of luciferase reporters containing the perfect antisense sequence of Mir-95 (LucMir-95AS). The reporters were cotransfected with the indicated amounts of 2′-O-methyl Mir-17-5p or Mir-95. Firefly luciferase activity was normalized to that of β-galactosidase. The data shown are means of three independent experiments, and error bars indicate standard deviations.
FIG. 2.
FIG. 2.
Mir-17-5p regulates translation of endogenous AIB1. (A) Northern blot of RNA from untreated MCF-7 cells and cells treated with Mir-17-5p, Mir-95, or anti-AIB1 siRNA. The RNA transcript levels were equal in all experimental samples, as shown by GAPDH and ethidium bromide staining (bottom two panels). (B) Western blot of AIB1 protein in untreated MCF-7 cells and cells treated with Mir-17-5p, Mir-95, anti-AIB1 siRNA, or scrambled siRNA. Anti-β-actin antibody was used as a loading control (bottom panel). (C and D) MCF-7 cells were transfected with Mir-17-5p, Mir-95, or vehicle only. Cells were starved in methionine-free medium and then pulse-labeled with [35S]methionine. The cells were then harvested and immunoprecipitated with anti-AIB1 antibody. Precipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by using a phosphorimager. (E and F) Level of Mir-17-5p mature transcript (E) and AIB1 protein (F) in HeLa cells treated with siRNA targeted to the loop region of pre-Mir-17p transcript or a scrambled siRNA (control). U6 snRNA (snU6) and β-actin were used as internal controls.
FIG. 3.
FIG. 3.
Mir-17-5p suppresses ER-mediated signaling. (A) Luciferase activity in CHO-K1 cells transiently transfected with an ER expression vector, a luciferase reporter plasmid, and 2′-O-methyl Mir17p or Mir-95, in the absence or presence of 10 nM E2. The data shown are means of triplicate experiments, and error bars indicate the standard deviations. (B) Luciferase activity in cells cotransfected with AIB1-expressing reporter plasmids containing wild-type (AIB3′UTR) or mutant (AIBm3′UTR) AIB1 3′UTRs, ER, ERE-Luc, and 2′-O-methyl Mir-17-5p or 2′-O-methyl Mir-95. The data shown are means of triplicate experiments, and error bars indicate the standard deviations.
FIG. 4.
FIG. 4.
Expression of Mir-17-5p is reduced or silent in some cancer cell lines. (A) Northern blot sequentially assayed with radiolabeled probes specific for Mir-17-5p, with U6 snRNA used as a control. Lanes 1 to 17 show RNA from the cell lines ZR-75-1, MCF-7, BT-20, MDA-MB-361, BT-474, T-47D, MDA-MB-468, MDA-MB-453, BG-1, Hs 748.T, BT-549, SW527, EB-3, HeLa, OV90, HEL60, and K562. Lanes 1 to 12 show breast cancer cell lines, and lanes 13 to 17 show other types of cancer cells. (B) Western blot of AIB1 protein in different cell lines. Cell lysates (50 μg) of the different cell lines were separated by SDS-PAGE, transferred onto nitrocellulose membrane, and probed with anti-AIB1 antibody. The same blot was stripped and reprobed with anti-β-actin antibody and was used as a loading control (bottom panel).
FIG. 5.
FIG. 5.
Mir-17-5p regulates translation of endogenous E2F1. Cell lysates (50 μg) from MCF-7 (A) and ZR-75-1 (D) were separated by SDS-PAGE, transferred onto nitrocellulose membrane, and probed with anti-AIB1 and anti-E2F1 antibody. The same blot was stripped and reprobed with anti-β-actin antibody and used as a loading control (bottom panel). (B and C) MCF-7 cells were transfected with Mir-17-5p, Mir-95, or vehicle only. Cells were starved in methionine-free medium and then pulse-labeled with [35S]methionine. The cells were then harvested and immunoprecipitated with anti-E2F1 antibody. Precipitated proteins were separated by SDS-PAGE and visualized by using a phosphorimager.
FIG. 6.
FIG. 6.
Mir-17-5p represses the expression of some ER and E2F1 target genes. Cells were treated with the indicated 2′-O-methyl RNA, and cell lysates (50 μg) were prepared and separated by SDS-PAGE, transferred onto nitrocellulose membrane, and probed with the indicated antibodies. Cells were treated with (B) or without (A) E2 to check target gene expression. The control reflects cells treated with phosphate-buffered saline only. The same blot was stripped and reprobed with anti-β-actin antibody and used as a loading control (bottom panel).
FIG. 7.
FIG. 7.
Mir-17-5p suppresses both estrogen-stimulated and estrogen/ER-independent breast cancer cell proliferation. (A) Reduction of AIB1 protein in stably transfected MCF-7 cells expressing Mir-17-5p but not Mir-95 compared to pSilencer vector only (control). Anti-β-actin antibody was used as a loading control. (B) Estrogen (E2)-stimulated cell proliferation assay. MCF-7 cells were stably transformed with pSilencer empty vector (control), pSilencerMir-17-5p (Mir-17-5p), or pSilencerMir-95 (Mir-95) and treated with 10 nM E2 or vehicle only. pcAIBm3′UTR was reintroduced by transient transfection in the stably transformed MCF-7 cells described above, which were treated with 10 nM E2. Cell proliferation was measured by the MTT assay. The data shown are means of triplicate experiments, and error bars indicate the standard deviations. (C) 2′-O-methyl Mir-17-5p or 2′-O-methyl Mir-95 was transiently transfected into BT-474 cells and treated with 10 nM E2 or vehicle only. Cell growth was measured by the MTT assay after 96 h. The data shown are means of triplicate experiments, and error bars indicate the standard deviations. (D) ZR75-1cells were treated with siRNA targeted to the loop region of pre-Mir-17p transcript or a scrambled siRNA (control) and treated with 10 nM E2 or vehicle only. Cell growth was measured by MTT assay after 96 h. The data shown are means of triplicate experiments, and error bars indicate the standard deviations.
FIG. 8.
FIG. 8.
Mir-17-5p suppresses estrogen/ER-independent breast cancer cell proliferation. 2′-O-Methyl Mir-17-5p or 2′-O-methyl Mir-95 with or without pcAIBm3′UTR was transiently transfected into BT-20 cells, and cells were maintained in hormone-deprived medium. (A) Western blot of AIB1 protein. Cell lysates (50 μg) of transfected cells were separated by SDS-PAGE, transferred onto nitrocellulose membrane, and probed with anti-AIB1 antibody. β-Actin was used as a loading control. (B) Cell proliferation assay. Cell growth was measured by the MTT assay after 96 h. The data shown are means of triplicate experiments, and error bars indicate the standard deviations.
FIG. 9.
FIG. 9.
Reduction of endogenous AIB1 by Mir-17-5p inhibits the anchorage-independent growth of MCF-7 Cells. (A) Mir-17-5p and Mir-95 overexpressing stably transfected MCF-7 cells were grown in improved minimal essential medium and 1% charcoal-stripped calf serum in 0.35% soft agar dishes in the absence or presence of 100 ng of IGF-1/ml. (B) Mean numbers of colonies with a diameter >80 μm. The data shown are means of triplicate experiments, and error bars indicate the standard deviations.
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