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CBX 7 and miR‐9 are part of an autoregulatory loop controlling p16INK4a

O'Loghlen, Ana, Brookes, Sharon, Martin, Nadine, Rapisarda, Valentina, Peters, Gordon, Gil, Jesús·2015·Aging Cell
DOI 10.1111/acel.12404PMC PMC4693451
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Abstract

Summary: Polycomb repressive complexes (PRC 1 and PRC 2) are epigenetic regulators that act in coordination to influence multiple cellular processes including pluripotency, differentiation, cancer and senescence. The role of PRC s in senescence can be mostly explained by their ability to repress the INK 4/ARF locus. CBX 7 is one of five mammalian orthologues of Drosophila Polycomb that forms part of PRC 1. Despite the relevance of CBX 7 for regulating senescence and pluripotency, we have a limited understanding of how the expression of CBX 7 is regulated. Here we report that the miR‐9 family of micro RNA s (mi RNAS) downregulates the expression of CBX 7. In turn, CBX 7 represses miR‐9‐1 and miR‐9‐2 as part of a regulatory negative feedback loop. The miR‐9/CBX 7 feedback loop is a regulatory module contributing to induction of the cyclin‐dependent kinase inhibitor (CDKI) p16INK4a during senescence. The ability of the miR‐9 family to regulate senescence could have implications for understanding the role of miR‐9 in cancer and aging.

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Figure 1 miR‐9 targets human CBX7. (A) Schematic representation of CBX7 mRNA. Bioinformatics analysis for miRNAs targeting the 3′UTR of human CBX7 was performed using manual analysis and the Targetscan and microRNA.org resources. Potential target sites for miR‐125, miR‐9 and miR‐181 in the 3′UTR of human CBX7 are indicated. Nucleotide positions refer to the ENST00000216133 transcript. (B) A luciferase reporter assay shows that miR‐9 also regulates the 3′UTR of human CBX7. (C) Mutation of two sites in the 3′UTR makes CBX7 3′UTR reporter resilient to miR‐9 regulation. A luciferase reporter assay similar to (B) was carried out using either a CBX7 3′UTR wt reporter (black bars) or a CBX7 3′UTR 9.1/2 reporter (with two miR‐9 sites mutated, orange bars). Statistical significance was calculated using two‐tailed Student's t‐tests, **P < 0.01; *P < 0.05; n.s. nonsignificant. (D) Transfection of a miR‐9 mimic downregulates CBX7 expression, as shown by qRT–PCR. miR‐181 is used as a positive control for downregulating CBX7.
Figure 1 miR‐9 targets human CBX7. (A) Schematic representation of CBX7 mRNA. Bioinformatics analysis for miRNAs targeting the 3′UTR of human CBX7 was performed using manual analysis and the Targetscan and microRNA.org resources. Potential target sites for miR‐125, miR‐9 and miR‐181 in the 3′UTR of human CBX7 are indicated. Nucleotide positions refer to the ENST00000216133 transcript. (B) A luciferase reporter assay shows that miR‐9 also regulates the 3′UTR of human CBX7. (C) Mutation of two sites in the 3′UTR makes CBX7 3′UTR reporter resilient to miR‐9 regulation. A luciferase reporter assay similar to (B) was carried out using either a CBX7 3′UTR wt reporter (black bars) or a CBX7 3′UTR 9.1/2 reporter (with two miR‐9 sites mutated, orange bars). Statistical significance was calculated using two‐tailed Student's t‐tests, **P < 0.01; *P < 0.05; n.s. nonsignificant. (D) Transfection of a miR‐9 mimic downregulates CBX7 expression, as shown by qRT–PCR. miR‐181 is used as a positive control for downregulating CBX7.CC BY · PubMed Central
Figure 2 CBX7 represses the expression of miR‐9‐1 and miR‐9‐2. (A, B) CBX6, CBX7 and CBX8 associate with miR‐9‐1 (A) and miR‐9‐2 (B). ChIP‐seq for CBX6, CBX7 and CBX8 was performed in Hs68 human fibroblasts (left panel). Their binding profiles on miR‐9‐1 (A) and miR‐9‐2 (B) are shown. The location of primer sets used (primer sets PS1.1, PS3.1 and PS4.1 for miR‐9‐1 and PS1.2, PS2.2, PS3.2 and PS4.2 for miR‐9‐2) is also depicted (left). ChIP in human fibroblast cells confirming that CBX7 binds to miR‐9‐1 (A) and miR‐9‐2 (B) is shown (right). (C) CBX7 overexpression reduces miR‐9 levels in IMR90 cells as shown by qRT–PCR. (D) CBX7 knockdown results in an increase in miR‐9 as assessed by qRT–PCR. (E) Knockdown of CBX7 results in upregulated levels of pri‐miR‐9‐1 and pri‐miR‐9‐2 as assessed by qRT–PCR. Statistical significance was calculated using two‐tailed Student's t‐tests, **P < 0.01; *P < 0.05; n.s. nonsignificant.
Figure 2 CBX7 represses the expression of miR‐9‐1 and miR‐9‐2. (A, B) CBX6, CBX7 and CBX8 associate with miR‐9‐1 (A) and miR‐9‐2 (B). ChIP‐seq for CBX6, CBX7 and CBX8 was performed in Hs68 human fibroblasts (left panel). Their binding profiles on miR‐9‐1 (A) and miR‐9‐2 (B) are shown. The location of primer sets used (primer sets PS1.1, PS3.1 and PS4.1 for miR‐9‐1 and PS1.2, PS2.2, PS3.2 and PS4.2 for miR‐9‐2) is also depicted (left). ChIP in human fibroblast cells confirming that CBX7 binds to miR‐9‐1 (A) and miR‐9‐2 (B) is shown (right). (C) CBX7 overexpression reduces miR‐9 levels in IMR90 cells as shown by qRT–PCR. (D) CBX7 knockdown results in an increase in miR‐9 as assessed by qRT–PCR. (E) Knockdown of CBX7 results in upregulated levels of pri‐miR‐9‐1 and pri‐miR‐9‐2 as assessed by qRT–PCR. Statistical significance was calculated using two‐tailed Student's t‐tests, **P < 0.01; *P < 0.05; n.s. nonsignificant.CC BY · PubMed Central
Figure 3 miR‐9 expression is upregulated during replicative senescence of human fibroblast. The levels of miR‐9 (left), INK4a mRNA (coding for p16INK 4a, centre) and CBX7 mRNAs (right) were analysed by qRT–PCR comparing either IMR90 (A) or WI38 (B) young and senescent cells.
Figure 3 miR‐9 expression is upregulated during replicative senescence of human fibroblast. The levels of miR‐9 (left), INK4a mRNA (coding for p16INK 4a, centre) and CBX7 mRNAs (right) were analysed by qRT–PCR comparing either IMR90 (A) or WI38 (B) young and senescent cells.CC BY · PubMed Central
Figure 4 miR‐9 expression triggers cellular senescence. (A, B) Overexpression of miR‐9‐1 results in decreased proliferation of IMR90 cells. IMR90 cells infected with miR‐9‐1 (or miR‐181a/b, or control vectors) were selected, and cells seeded at low density. Plates were stained with crystal violet 2 weeks later. (A) Representative plates and (B) quantification of crystal violet staining (average of five independent experiments) are shown. Statistical significance was calculated using two‐tailed Student's t‐tests, ***P < 0.001. (C) miR‐9 triggers arrest of IMR90 cells. BrdU incorporation was assessed by IF in IMR90 cells transfected with miR‐9 or miR‐181 mimics or with anti‐miR‐9. (D) IMR90 cells infected with the indicated vectors were subjected to SA‐β‐Gal (left) and DAPI staining (right) to visualize SAHFs. Representative images (top) and quantification of the percentages positive for SA‐β‐Gal or containing SAHFs are shown (bottom). (E) Expression of miR‐9 induces p16INK 4a. The levels of p16INK 4a were assessed by quantitative IF in IMR90 cells infected with a retroviral vector expressing miR‐9.
Figure 4 miR‐9 expression triggers cellular senescence. (A, B) Overexpression of miR‐9‐1 results in decreased proliferation of IMR90 cells. IMR90 cells infected with miR‐9‐1 (or miR‐181a/b, or control vectors) were selected, and cells seeded at low density. Plates were stained with crystal violet 2 weeks later. (A) Representative plates and (B) quantification of crystal violet staining (average of five independent experiments) are shown. Statistical significance was calculated using two‐tailed Student's t‐tests, ***P < 0.001. (C) miR‐9 triggers arrest of IMR90 cells. BrdU incorporation was assessed by IF in IMR90 cells transfected with miR‐9 or miR‐181 mimics or with anti‐miR‐9. (D) IMR90 cells infected with the indicated vectors were subjected to SA‐β‐Gal (left) and DAPI staining (right) to visualize SAHFs. Representative images (top) and quantification of the percentages positive for SA‐β‐Gal or containing SAHFs are shown (bottom). (E) Expression of miR‐9 induces p16INK 4a. The levels of p16INK 4a were assessed by quantitative IF in IMR90 cells infected with a retroviral vector expressing miR‐9.CC BY · PubMed Central
Figure 5 miR‐9‐induced senescence is rescued by CBX7 overexpression. (A) Control and IMR90 cells overexpressing murine CBX7 (Cbx7) were infected with pMSCV‐miR‐9‐1, miR‐181a/b or control vectors, cells selected and seeded at low density. Crystal violet stained plates are shown. (B–D) Cells derived from the same experiments were used to measure (B) BrdU incorporation, (C) SA‐β‐Gal positive cells and (D) the percentage of cells showing SAHFs. (E) The levels of the INK4a transcript (encoding for p16INK 4a) were analysed by qRT–PCR in the indicated cells.
Figure 5 miR‐9‐induced senescence is rescued by CBX7 overexpression. (A) Control and IMR90 cells overexpressing murine CBX7 (Cbx7) were infected with pMSCV‐miR‐9‐1, miR‐181a/b or control vectors, cells selected and seeded at low density. Crystal violet stained plates are shown. (B–D) Cells derived from the same experiments were used to measure (B) BrdU incorporation, (C) SA‐β‐Gal positive cells and (D) the percentage of cells showing SAHFs. (E) The levels of the INK4a transcript (encoding for p16INK 4a) were analysed by qRT–PCR in the indicated cells.CC BY · PubMed Central
Figure 6 miR‐9‐induced senescence is p16INK 4a‐dependent. (A) IMR90 cells were infected with a vector knocking down p16INK 4a expression (shp16) or the corresponding control (vector). The derived cells were subsequently infected with viruses expressing miR‐9 or controls. The expression of p16INK 4a was assessed by IF. Representative pictures (left) and quantification (right) is shown. (B, C) Knocking down p16INK 4a expression suppressed miR‐9‐induced senescence. The percentage of (B) cells incorporating BrdU and (C) cells positive for SA‐β‐Gal staining is shown. (D) Scheme depicting the regulatory network formed by CBX7 and miR‐9 to control p16INK 4a expression and regulate senescence.
Figure 6 miR‐9‐induced senescence is p16INK 4a‐dependent. (A) IMR90 cells were infected with a vector knocking down p16INK 4a expression (shp16) or the corresponding control (vector). The derived cells were subsequently infected with viruses expressing miR‐9 or controls. The expression of p16INK 4a was assessed by IF. Representative pictures (left) and quantification (right) is shown. (B, C) Knocking down p16INK 4a expression suppressed miR‐9‐induced senescence. The percentage of (B) cells incorporating BrdU and (C) cells positive for SA‐β‐Gal staining is shown. (D) Scheme depicting the regulatory network formed by CBX7 and miR‐9 to control p16INK 4a expression and regulate senescence.CC BY · PubMed Central

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Metadata

Authors
O'Loghlen, A., Brookes, S., Martin, N., Rapisarda, V., Peters, G., & Gil, J.
Affiliations
  • Cell Proliferation Group MRC Clinical Sciences Centre Imperial College London Hammersmith Campus London W12 0NN UK
  • Epigenetics &amp; Cellular Senescence Group Blizard Institute Barts and The London School of Medicine and Dentistry Queen Mary University of London 4 Newark Street London E1 2AT UK
  • Molecular Oncology Laboratory CRUK London Research Institute 44 Lincoln's Inn Fields London WC2A 3LY UK
Venue
Aging Cell
Publisher
Wiley
Published
2015-09-29
Volume
14
Issue
6
Pages
1113-1121
ISSN
  • 1474-9718
  • 1474-9726
DOI
10.1111/acel.12404
PMC
PMC4693451
Cited by
23
References
6
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
  • Medical Research Council (A652 5PZ00)
  • European Molecular Biology Organization
  • Marie Curie fellowships
  • London Research Institute by Cancer Research UK

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CoChat. (2026). Citation check: CBX 7 and miR‐9 are part of an autoregulatory loop controlling p16INK4a. https://app.cochat.ai/papers/10.1111/acel.12404
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O'Loghlen, A., Brookes, S., Martin, N., Rapisarda, V., Peters, G., & Gil, J. (2015). CBX 7 and miR‐9 are part of an autoregulatory loop controlling p16INK4a. Aging Cell. https://doi.org/10.1111/acel.12404