miR-21 coordinates tumor growth and modulates KRIT1 levels.
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.
Affiliation: Molecular Biotechnology Center, University of Torino, Torino, Italy.Show MeSH
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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).
Affiliation: Molecular Biotechnology Center, University of Torino, Torino, Italy.