Limits...
miR-21 coordinates tumor growth and modulates KRIT1 levels.

Orso F, Balzac F, Marino M, Lembo A, Retta SF, Taverna D - Biochem. Biophys. Res. Commun. (2013)

Bottom Line: Here, we show that expression of miR-21 in primary tumors anticorrelates with KRIT1/CCM1, an interacting partner of the Ras-like GTPase Rap1, involved in Cerebral Cavernous Malformations (CCM).We present evidences that miR-21 silences KRIT1 by targeting its mRNA 3'UTR and that this interaction is involved in tumor growth control.In fact, miR-21 over-expression or KRIT1 knock-down promote anchorage independent tumor cell growth compared to controls, whereas the opposite is observed when anti-miR-21 or KRIT1 overexpression are employed.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biotechnology Center, University of Torino, Torino, Italy.

Show MeSH

Related in: MedlinePlus

miR-21 directly modulates KRIT1 3′UTR. (A) Scheme showing the two putative miR-21 binding sites in human KRIT1 3′UTR at positions 494 (highly conserved) and 1270 (poorly conserved) paired with miR-21 seed. (B) Luciferase assays in 293T or MC-1 or HeLa cells cotransfected with pMIR-luciferase constructs containing either a 973 nt-long portion of KRIT1 3′UTR (position 340–1313) or a synthetic sequence, which includes three perfect miR-21 binding sites (miR-21 sensor), together with miR-21 precursors or inhibitors or their negative controls (pre- and anti-miR-21 or control) or with pWPT-ctrl or miR-21 overexpression (pWPT-miR-21) vectors. Results are shown as mean ± SEM of Firefly Luciferase activity relative to controls, normalized on Renilla Luciferase activity. Three independent analyses were performed in triplicate and representative results are shown.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750217&req=5

f0015: miR-21 directly modulates KRIT1 3′UTR. (A) Scheme showing the two putative miR-21 binding sites in human KRIT1 3′UTR at positions 494 (highly conserved) and 1270 (poorly conserved) paired with miR-21 seed. (B) Luciferase assays in 293T or MC-1 or HeLa cells cotransfected with pMIR-luciferase constructs containing either a 973 nt-long portion of KRIT1 3′UTR (position 340–1313) or a synthetic sequence, which includes three perfect miR-21 binding sites (miR-21 sensor), together with miR-21 precursors or inhibitors or their negative controls (pre- and anti-miR-21 or control) or with pWPT-ctrl or miR-21 overexpression (pWPT-miR-21) vectors. Results are shown as mean ± SEM of Firefly Luciferase activity relative to controls, normalized on Renilla Luciferase activity. Three independent analyses were performed in triplicate and representative results are shown.

Mentions: Bioinformatics analysis revealed that KRIT1 3′UTR contained two putative miR-21 binding sites for miR-21 (Fig. 3A). To examine whether miR-21 binds at its putative binding site, 293T or MC-1 or HeLa cells were transiently transfected with a Luciferase reporter vector containing a 973 bps portion (including both sites) of the 3′UTR for KRIT1 in miR-21 overexpression (pre-miR-21 or pWPT-miR-21) or silencing (anti-miR-21) conditions and luciferase activity measured and compared to the negative controls (pre- or anti-controls; pWPT-ctrl). As shown in Fig. 3B, luciferase activity was significantly reduced in presence of miR-21 overexpression in 293T and MC-1 cells. Conversely, it was increased following miR-21 silencing in HeLa cells. As a positive control, a miR-21-sensor construct, containing 3 perfect bindings for miR-21, was used for each experiment. Results were normalized on Renilla activity.


miR-21 coordinates tumor growth and modulates KRIT1 levels.

Orso F, Balzac F, Marino M, Lembo A, Retta SF, Taverna D - Biochem. Biophys. Res. Commun. (2013)

miR-21 directly modulates KRIT1 3′UTR. (A) Scheme showing the two putative miR-21 binding sites in human KRIT1 3′UTR at positions 494 (highly conserved) and 1270 (poorly conserved) paired with miR-21 seed. (B) Luciferase assays in 293T or MC-1 or HeLa cells cotransfected with pMIR-luciferase constructs containing either a 973 nt-long portion of KRIT1 3′UTR (position 340–1313) or a synthetic sequence, which includes three perfect miR-21 binding sites (miR-21 sensor), together with miR-21 precursors or inhibitors or their negative controls (pre- and anti-miR-21 or control) or with pWPT-ctrl or miR-21 overexpression (pWPT-miR-21) vectors. Results are shown as mean ± SEM of Firefly Luciferase activity relative to controls, normalized on Renilla Luciferase activity. Three independent analyses were performed in triplicate and representative results are shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750217&req=5

f0015: miR-21 directly modulates KRIT1 3′UTR. (A) Scheme showing the two putative miR-21 binding sites in human KRIT1 3′UTR at positions 494 (highly conserved) and 1270 (poorly conserved) paired with miR-21 seed. (B) Luciferase assays in 293T or MC-1 or HeLa cells cotransfected with pMIR-luciferase constructs containing either a 973 nt-long portion of KRIT1 3′UTR (position 340–1313) or a synthetic sequence, which includes three perfect miR-21 binding sites (miR-21 sensor), together with miR-21 precursors or inhibitors or their negative controls (pre- and anti-miR-21 or control) or with pWPT-ctrl or miR-21 overexpression (pWPT-miR-21) vectors. Results are shown as mean ± SEM of Firefly Luciferase activity relative to controls, normalized on Renilla Luciferase activity. Three independent analyses were performed in triplicate and representative results are shown.
Mentions: Bioinformatics analysis revealed that KRIT1 3′UTR contained two putative miR-21 binding sites for miR-21 (Fig. 3A). To examine whether miR-21 binds at its putative binding site, 293T or MC-1 or HeLa cells were transiently transfected with a Luciferase reporter vector containing a 973 bps portion (including both sites) of the 3′UTR for KRIT1 in miR-21 overexpression (pre-miR-21 or pWPT-miR-21) or silencing (anti-miR-21) conditions and luciferase activity measured and compared to the negative controls (pre- or anti-controls; pWPT-ctrl). As shown in Fig. 3B, luciferase activity was significantly reduced in presence of miR-21 overexpression in 293T and MC-1 cells. Conversely, it was increased following miR-21 silencing in HeLa cells. As a positive control, a miR-21-sensor construct, containing 3 perfect bindings for miR-21, was used for each experiment. Results were normalized on Renilla activity.

Bottom Line: Here, we show that expression of miR-21 in primary tumors anticorrelates with KRIT1/CCM1, an interacting partner of the Ras-like GTPase Rap1, involved in Cerebral Cavernous Malformations (CCM).We present evidences that miR-21 silences KRIT1 by targeting its mRNA 3'UTR and that this interaction is involved in tumor growth control.In fact, miR-21 over-expression or KRIT1 knock-down promote anchorage independent tumor cell growth compared to controls, whereas the opposite is observed when anti-miR-21 or KRIT1 overexpression are employed.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biotechnology Center, University of Torino, Torino, Italy.

Show MeSH
Related in: MedlinePlus