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

KRIT1 and miR-21 expression in tumor cell lines. KRIT1 (A) and miR-21 (B) expression was evaluated in HeLa, MDAMB231, MC-1 and 293T (wild type or silenced-sh- for KRIT1 and its sh-control-ctrl) human tumor cells by Western Blot (WB) and qRT-PCR analyses. An aspecific band was used as loading control in WBs as in [22]. For the qRT-PCRs, results are shown as fold changes (mean ± SEM) relative to expression in 293T cells, normalized on GAPDH for KRIT1 or U6 for miR-21 levels. Two independent analyses were performed in triplicate and a representative one is shown.
© Copyright Policy
Related In: Results  -  Collection

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

f0005: KRIT1 and miR-21 expression in tumor cell lines. KRIT1 (A) and miR-21 (B) expression was evaluated in HeLa, MDAMB231, MC-1 and 293T (wild type or silenced-sh- for KRIT1 and its sh-control-ctrl) human tumor cells by Western Blot (WB) and qRT-PCR analyses. An aspecific band was used as loading control in WBs as in [22]. For the qRT-PCRs, results are shown as fold changes (mean ± SEM) relative to expression in 293T cells, normalized on GAPDH for KRIT1 or U6 for miR-21 levels. Two independent analyses were performed in triplicate and a representative one is 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)

KRIT1 and miR-21 expression in tumor cell lines. KRIT1 (A) and miR-21 (B) expression was evaluated in HeLa, MDAMB231, MC-1 and 293T (wild type or silenced-sh- for KRIT1 and its sh-control-ctrl) human tumor cells by Western Blot (WB) and qRT-PCR analyses. An aspecific band was used as loading control in WBs as in [22]. For the qRT-PCRs, results are shown as fold changes (mean ± SEM) relative to expression in 293T cells, normalized on GAPDH for KRIT1 or U6 for miR-21 levels. Two independent analyses were performed in triplicate and a representative one is shown.
© Copyright Policy
Related In: Results  -  Collection

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

f0005: KRIT1 and miR-21 expression in tumor cell lines. KRIT1 (A) and miR-21 (B) expression was evaluated in HeLa, MDAMB231, MC-1 and 293T (wild type or silenced-sh- for KRIT1 and its sh-control-ctrl) human tumor cells by Western Blot (WB) and qRT-PCR analyses. An aspecific band was used as loading control in WBs as in [22]. For the qRT-PCRs, results are shown as fold changes (mean ± SEM) relative to expression in 293T cells, normalized on GAPDH for KRIT1 or U6 for miR-21 levels. Two independent analyses were performed in triplicate and a representative one is 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