MicroRNA miR-21 regulates the metastatic behavior of B16 melanoma cells - PubMed (original) (raw)

MicroRNA miR-21 regulates the metastatic behavior of B16 melanoma cells

Chuan He Yang et al. J Biol Chem. 2011.

Abstract

MicroRNA-21 (miR-21) is overexpressed in many human tumors and has been linked to various cellular processes altered in cancer. miR-21 is also up-regulated by a number of inflammatory agents, including IFN, which is of particular interest considering the close relationship between inflammation and cancer. Because miR-21 appears to be overexpressed in human melanoma, we examined the role of miR-21 in cancer development and metastasis in B16 mouse melanoma cells. We found that miR-21 is a member of an IFN-induced miRNA subset that requires STAT3 activation. To characterize the role of miR-21 in melanoma behavior, we transduced B16 cells with lentivirus encoding a miR-21 antagomir and isolated miR-21 knockdown B16 cells. miR-21 knockdown or IFN treatment alone inhibited B16 cell proliferation and migration in vitro, and in combination they had an enhanced effect. Moreover, miR-21 knockdown sensitized B16 cells to IFN-induced apoptosis. In B16 cells miR-21 targeted tumor suppressor (PTEN and PDCD4) and antiproliferative (BTG2) proteins. To characterize the role of miR-21 in vivo, empty vector- and antagomiR-21-transduced B16 melanoma cells were injected via tail vein into syngeneic C57BL/6 mice. Although empty vector-transduced B16 cells produced large lung metastases, miR-21 knockdown cells only formed small lung lesions. Importantly, miR-21 knockdown tumor-bearing mice exhibited prolonged survival compared with empty vector tumor-bearing mice. Thus, miR-21 regulates the metastatic behavior of B16 melanoma cells by promoting cell proliferation, survival, and migration/invasion as well as by suppressing IFN action, providing important new insights into the role of miR-21 in melanoma.

PubMed Disclaimer

Figures

FIGURE 1.

FIGURE 1.

Characterization of IFN-induced miR-21 expression in B16 melanoma cells in vitro. B16 cells were treated with IFN at 1,000 units/ml for the indicated times (A) or at the indicated doses for 6 h (B), and miR-21 was quantified by qPCR. IFN-induced ISG15 induction is shown for comparison (n = 3). Error bars, S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 2.

FIGURE 2.

Role of STAT3 in IFN-induced miRNA expression in B16 melanoma cells in vitro. B16 cells transduced with EV or F705-STAT3 were treated with IFN (1,000 units/ml) and cell lysates were prepared and immunoblotted as indicated (A), or after 6 h of IFN treatment the indicated miRNAs were quantified by qPCR (n = 3) (B). Error bars, S.D. **, p < 0.01. NS, not significant.

FIGURE 3.

FIGURE 3.

Effects of miR-21KD on miRNA expression and IFN-induced gene expression in B16 cells in vitro. B16 cells transduced with anti-miR-21 lentivirus (miR-21KD) or with empty vector (EV) were treated with IFN (1,000 units/ml), and total RNA assayed for miRNA expression (A) or IFN-induced gene expression by qPCR (n = 3) (B). Error bars, S.D. **, p < 0.01; ***, p < 0.001.

FIGURE 4.

FIGURE 4.

Effects of miR-21KD on B16 melanoma cell proliferation, apoptosis, migration, and protein expression in vitro. A–C, EV and miR-21KD B16 cells were treated with IFN (1,000 units/ml), and at daily intervals cell numbers were determined in a Coulter Counter (n = 3) (A), or after 48 h apoptosis was determined by cell death ELISA or by Annexin V-staining (n = 3) (B), or after 24 h cell migration was determined by transwell migration assays (n = 3) (C). D, EV and miR-21KD B16 cells were lysed, and the expression of key target proteins was determined by immunoblotting with anti-PTEN, PDCD4, BTG2, and SPRY2. Error bars, S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 5.

FIGURE 5.

Decreased metastatic potential of miR-21KD B16 melanoma cells in vivo. Mice were injected into the tail vein with 106 EV or miR-21KD B16 cells. A, bioluminescent imaging of mice injected with luciferin performed at day 16 (n = 8). B, Kaplan-Meier analysis of survival data (n = 8). C, mean body weight of mice at day 18 (n = 8). Error bars, S.D. *, p < 0.05; **, p < 0.01.

Similar articles

Cited by

References

    1. Bartel D. P. (2004) Cell 116, 281–297 - PubMed
    1. Calin G. A., Croce C. M. (2006) Nat. Rev. Cancer 6, 857–866 - PubMed
    1. Si M. L., Zhu S., Wu H., Lu Z., Wu F., Mo Y. Y. (2007) Oncogene 26, 2799–2803 - PubMed
    1. Volinia S., Calin G. A., Liu C. G., Ambs S., Cimmino A., Petrocca F., Visone R., Iorio M., Roldo C., Ferracin M., Prueitt R. L., Yanaihara N., Lanza G., Scarpa A., Vecchione A., Negrini M., Harris C. C., Croce C. M. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 2257–2261 - PMC - PubMed
    1. Folini M., Gandellini P., Longoni N., Profumo V., Callari M., Pennati M., Colecchia M., Supino R., Veneroni S., Salvioni R., Valdagni R., Daidone M. G., Zaffaroni N. (2010) Mol. Cancer 9, 12. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources