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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.

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miR-21 regulates anchorage-independent growth opposite to KRIT1. (A) KRIT1 expression modulations were analyzed in MC-1 or MDAMB231 cells stably transduced with lentiviral vectors containing either specific shKRIT1 (pLKO.1-sh-KRIT1 #1 to #4) or a KRIT1 cDNA lacking its 3′UTR (pCLL-KRIT1 or pLVX-KRIT1) or empty controls (pLKO.1-sh-ctrl or pCLL-ctrl or pLVX-ctrl) by Western Blot (WB) analysis and actin was used as loading control. 3 independent analyses were performed in triplicate and representative results are shown. (B–E) Anchorage-independent growth of MDAMB231 and MC-1 cells transfected with miR-21 precursors or inhibitors or their negative controls (pre- and anti-miR-21 or control) or stably transduced with lentiviral vectors containing either specific shKRIT1 (pLKO.1-sh-KRIT1-3, -4) or a KRIT1 cDNA lacking its 3′UTR (pCLL-KRIT1 or pLVX-KRIT1) or empty controls (pLKO.1-sh-control or pCLL-ctrl or pLVX-ctrl). All results are shown as mean ± SEM of the area covered by colonies (pixels). Three independent experiments were performed in triplicate and representative results are shown. (F) Cartoon showing the proposed model for the role of KRIT1 in miR-21-mediated tumorigenesis.
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f0020: miR-21 regulates anchorage-independent growth opposite to KRIT1. (A) KRIT1 expression modulations were analyzed in MC-1 or MDAMB231 cells stably transduced with lentiviral vectors containing either specific shKRIT1 (pLKO.1-sh-KRIT1 #1 to #4) or a KRIT1 cDNA lacking its 3′UTR (pCLL-KRIT1 or pLVX-KRIT1) or empty controls (pLKO.1-sh-ctrl or pCLL-ctrl or pLVX-ctrl) by Western Blot (WB) analysis and actin was used as loading control. 3 independent analyses were performed in triplicate and representative results are shown. (B–E) Anchorage-independent growth of MDAMB231 and MC-1 cells transfected with miR-21 precursors or inhibitors or their negative controls (pre- and anti-miR-21 or control) or stably transduced with lentiviral vectors containing either specific shKRIT1 (pLKO.1-sh-KRIT1-3, -4) or a KRIT1 cDNA lacking its 3′UTR (pCLL-KRIT1 or pLVX-KRIT1) or empty controls (pLKO.1-sh-control or pCLL-ctrl or pLVX-ctrl). All results are shown as mean ± SEM of the area covered by colonies (pixels). Three independent experiments were performed in triplicate and representative results are shown. (F) Cartoon showing the proposed model for the role of KRIT1 in miR-21-mediated tumorigenesis.

Mentions: To evaluate the functions of miR-21 and KRIT1 in tumorigenesis, we performed anchorage independent growth experiments with MDAMB231 and MC-1 cells following miR-21 (Fig. 2C and D) or KRIT1 (Fig. 4A) modulations. miR-21 expression up- or down-regulations were obtained as presented above (Fig. 2C and D). Instead, to obtain KRIT1 modulations of expression, MC-1 or MDAMB231 cells were stably transduced with lentiviral vectors containing either specific shKRIT1 (pLKO.1-shKRIT1 #1–4) or a KRIT1 cDNA lacking its 3′UTR (pCLL-KRIT1 or pLVX-KRIT1) or empty controls (pLKO.1-shctrl or pCLL-ctrl or pLVX-ctrl) and expression was evaluated by Western Blot (WB) analysis; actin was used as loading control (Fig. 4A). miR-21 overexpression (pre-miR-21) resulted in increased (up to 3-folds) colony-formation compared to controls (pre-control), as shown in Fig. 4B, whereas inhibition of miR-21 led to decreased (2–3-folds) colony formation as in Fig. 4C. In parallel, when MC-1 cells were transduced with either specific shKRIT1 or control lentiviral vectors (pLKO-shKRIT1 #3 or #4 or pLKO-shctrl), the results obtained phenocopied miR-21 overexpression. Indeed, 1.5–2-fold increase in the area occupied by colonies (Fig. 4D) was observed. Instead, the opposite results were obtained when KRIT1 was overexpressed in MDAMB231 and MC-1 cells following transduction of KRIT1 cDNA or control (pCLL-ctrl and pCLL-KRIT1 or pLVX-ctrl and pLVX-KRIT1) expressing lentivirus vectors (Fig. 4E). In conclusion, we propose that KRIT1 is a functional player able to control tumor growth, downstream of miR-21 (Fig. 4F).


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 regulates anchorage-independent growth opposite to KRIT1. (A) KRIT1 expression modulations were analyzed in MC-1 or MDAMB231 cells stably transduced with lentiviral vectors containing either specific shKRIT1 (pLKO.1-sh-KRIT1 #1 to #4) or a KRIT1 cDNA lacking its 3′UTR (pCLL-KRIT1 or pLVX-KRIT1) or empty controls (pLKO.1-sh-ctrl or pCLL-ctrl or pLVX-ctrl) by Western Blot (WB) analysis and actin was used as loading control. 3 independent analyses were performed in triplicate and representative results are shown. (B–E) Anchorage-independent growth of MDAMB231 and MC-1 cells transfected with miR-21 precursors or inhibitors or their negative controls (pre- and anti-miR-21 or control) or stably transduced with lentiviral vectors containing either specific shKRIT1 (pLKO.1-sh-KRIT1-3, -4) or a KRIT1 cDNA lacking its 3′UTR (pCLL-KRIT1 or pLVX-KRIT1) or empty controls (pLKO.1-sh-control or pCLL-ctrl or pLVX-ctrl). All results are shown as mean ± SEM of the area covered by colonies (pixels). Three independent experiments were performed in triplicate and representative results are shown. (F) Cartoon showing the proposed model for the role of KRIT1 in miR-21-mediated tumorigenesis.
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f0020: miR-21 regulates anchorage-independent growth opposite to KRIT1. (A) KRIT1 expression modulations were analyzed in MC-1 or MDAMB231 cells stably transduced with lentiviral vectors containing either specific shKRIT1 (pLKO.1-sh-KRIT1 #1 to #4) or a KRIT1 cDNA lacking its 3′UTR (pCLL-KRIT1 or pLVX-KRIT1) or empty controls (pLKO.1-sh-ctrl or pCLL-ctrl or pLVX-ctrl) by Western Blot (WB) analysis and actin was used as loading control. 3 independent analyses were performed in triplicate and representative results are shown. (B–E) Anchorage-independent growth of MDAMB231 and MC-1 cells transfected with miR-21 precursors or inhibitors or their negative controls (pre- and anti-miR-21 or control) or stably transduced with lentiviral vectors containing either specific shKRIT1 (pLKO.1-sh-KRIT1-3, -4) or a KRIT1 cDNA lacking its 3′UTR (pCLL-KRIT1 or pLVX-KRIT1) or empty controls (pLKO.1-sh-control or pCLL-ctrl or pLVX-ctrl). All results are shown as mean ± SEM of the area covered by colonies (pixels). Three independent experiments were performed in triplicate and representative results are shown. (F) Cartoon showing the proposed model for the role of KRIT1 in miR-21-mediated tumorigenesis.
Mentions: To evaluate the functions of miR-21 and KRIT1 in tumorigenesis, we performed anchorage independent growth experiments with MDAMB231 and MC-1 cells following miR-21 (Fig. 2C and D) or KRIT1 (Fig. 4A) modulations. miR-21 expression up- or down-regulations were obtained as presented above (Fig. 2C and D). Instead, to obtain KRIT1 modulations of expression, MC-1 or MDAMB231 cells were stably transduced with lentiviral vectors containing either specific shKRIT1 (pLKO.1-shKRIT1 #1–4) or a KRIT1 cDNA lacking its 3′UTR (pCLL-KRIT1 or pLVX-KRIT1) or empty controls (pLKO.1-shctrl or pCLL-ctrl or pLVX-ctrl) and expression was evaluated by Western Blot (WB) analysis; actin was used as loading control (Fig. 4A). miR-21 overexpression (pre-miR-21) resulted in increased (up to 3-folds) colony-formation compared to controls (pre-control), as shown in Fig. 4B, whereas inhibition of miR-21 led to decreased (2–3-folds) colony formation as in Fig. 4C. In parallel, when MC-1 cells were transduced with either specific shKRIT1 or control lentiviral vectors (pLKO-shKRIT1 #3 or #4 or pLKO-shctrl), the results obtained phenocopied miR-21 overexpression. Indeed, 1.5–2-fold increase in the area occupied by colonies (Fig. 4D) was observed. Instead, the opposite results were obtained when KRIT1 was overexpressed in MDAMB231 and MC-1 cells following transduction of KRIT1 cDNA or control (pCLL-ctrl and pCLL-KRIT1 or pLVX-ctrl and pLVX-KRIT1) expressing lentivirus vectors (Fig. 4E). In conclusion, we propose that KRIT1 is a functional player able to control tumor growth, downstream of miR-21 (Fig. 4F).

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