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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 directly modulates KRIT1 expression. miR-21 expression was analyzed by Northern Blot-NB- (A, B) or qRT-PCR (C, D) analysis in 293T or HeLa or MC-1 or MDAMB231 cells transfected 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 at the indicated time points (h). U6 was used for normalization in NB and qRT-PCR analyses. KRIT1 protein (WB: E, F) or mRNA (qRT-PCR: G) expression in cells manipulated as in (A and C). Protein modulations in (E, F) were calculated relative to controls, normalized on controls (aspecific band [22] or actin), and expressed as percentages (%) of reduction. mRNA modulations in (G) were calculated as fold changes (mean ± SEM) relative to controls, normalized on GAPDH. (A–G) three independent analyses were performed in triplicate and representative results are shown.
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f0010: miR-21 directly modulates KRIT1 expression. miR-21 expression was analyzed by Northern Blot-NB- (A, B) or qRT-PCR (C, D) analysis in 293T or HeLa or MC-1 or MDAMB231 cells transfected 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 at the indicated time points (h). U6 was used for normalization in NB and qRT-PCR analyses. KRIT1 protein (WB: E, F) or mRNA (qRT-PCR: G) expression in cells manipulated as in (A and C). Protein modulations in (E, F) were calculated relative to controls, normalized on controls (aspecific band [22] or actin), and expressed as percentages (%) of reduction. mRNA modulations in (G) were calculated as fold changes (mean ± SEM) relative to controls, normalized on GAPDH. (A–G) three independent analyses were performed in triplicate and representative results are shown.

Mentions: Consequently to the anticorrelation found in human tumors for KRIT1 and miR-21, we investigated a possible functional direct link between these two genes, following miR-21 modulation in various tumor cell lines, HeLa, MDAMB231, MC-1 and 293T. The endogenous levels of miR-21 and KRIT1 were evaluated by Western Blot (WB) and qRT-PCR analyses and shown in Fig. 1A and B. 293T cells stably silenced (sh-KRIT1) or not (sh-CTRL) for KRIT1 were used in WB analysis to show anti-KRIT1 Ab specificity (80KDa band). miR-21 expression was increased or decreased by transfecting the cells with pre- or anti-miRs or their controls (pre-miR-21, anti-miR-21, pre- or anti-control) or with pWPT-empty or miR-21 overexpression lentivirus vectors (pWPT-ctrl, pWPT-miR-21). miR-21 expression was verified at different time points, 6–72 h post-transfection, by Northern Blot–NB (Fig. 2A and B) or qRT-PCR (Fig. 2C and D) analyses. When KRIT1, protein or mRNA expression, was evaluated, important inverse modulations were found, as shown and quantitated by WB or qRT-PCR (Fig. 2E–G).


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 expression. miR-21 expression was analyzed by Northern Blot-NB- (A, B) or qRT-PCR (C, D) analysis in 293T or HeLa or MC-1 or MDAMB231 cells transfected 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 at the indicated time points (h). U6 was used for normalization in NB and qRT-PCR analyses. KRIT1 protein (WB: E, F) or mRNA (qRT-PCR: G) expression in cells manipulated as in (A and C). Protein modulations in (E, F) were calculated relative to controls, normalized on controls (aspecific band [22] or actin), and expressed as percentages (%) of reduction. mRNA modulations in (G) were calculated as fold changes (mean ± SEM) relative to controls, normalized on GAPDH. (A–G) three independent analyses were performed in triplicate and representative results are shown.
© Copyright Policy
Related In: Results  -  Collection

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

f0010: miR-21 directly modulates KRIT1 expression. miR-21 expression was analyzed by Northern Blot-NB- (A, B) or qRT-PCR (C, D) analysis in 293T or HeLa or MC-1 or MDAMB231 cells transfected 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 at the indicated time points (h). U6 was used for normalization in NB and qRT-PCR analyses. KRIT1 protein (WB: E, F) or mRNA (qRT-PCR: G) expression in cells manipulated as in (A and C). Protein modulations in (E, F) were calculated relative to controls, normalized on controls (aspecific band [22] or actin), and expressed as percentages (%) of reduction. mRNA modulations in (G) were calculated as fold changes (mean ± SEM) relative to controls, normalized on GAPDH. (A–G) three independent analyses were performed in triplicate and representative results are shown.
Mentions: Consequently to the anticorrelation found in human tumors for KRIT1 and miR-21, we investigated a possible functional direct link between these two genes, following miR-21 modulation in various tumor cell lines, HeLa, MDAMB231, MC-1 and 293T. The endogenous levels of miR-21 and KRIT1 were evaluated by Western Blot (WB) and qRT-PCR analyses and shown in Fig. 1A and B. 293T cells stably silenced (sh-KRIT1) or not (sh-CTRL) for KRIT1 were used in WB analysis to show anti-KRIT1 Ab specificity (80KDa band). miR-21 expression was increased or decreased by transfecting the cells with pre- or anti-miRs or their controls (pre-miR-21, anti-miR-21, pre- or anti-control) or with pWPT-empty or miR-21 overexpression lentivirus vectors (pWPT-ctrl, pWPT-miR-21). miR-21 expression was verified at different time points, 6–72 h post-transfection, by Northern Blot–NB (Fig. 2A and B) or qRT-PCR (Fig. 2C and D) analyses. When KRIT1, protein or mRNA expression, was evaluated, important inverse modulations were found, as shown and quantitated by WB or qRT-PCR (Fig. 2E–G).

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