1.05 MicroRNA-21 Regulates Melanoma Invasion via Inhibition of Tissue Inhibitor of Metalloproteinases-3

N. Latchana3, S. Martin Del Campo3, V. Grignol3, K. Levine3, E. Fairchild6, A. Ganju3, C. Jaime-Ramirez3, T. Dao5, V. Karpa5, M. Carson3, A. Chan5, W. Carson3  3Ohio State University,Columbus, OH, USA 5Wright State University,Dayton, OH, USA 6Nationwide Children’s Hospital,Columbus, OH, USA

Introduction: Melanoma accounts for the highest number of skin cancer associated deaths annually yet the progression towards a metastatic phenotype is not completely understood.  Increased cellular invasion through the extracellular matrix is a prerequisite for metastasis and is enhanced by matrix metalloproteinases (MMPs), MMPs are negatively regulated by the tissue inhibitor of metalloproteinases (TIMP) protein family such as TIMP3.  MicroRNAs (miRs) are small, noncoding RNAs that inhibit gene expression and regulate many cellular processes.  It was previously shown by our group that miR-21, a potential regulator of TIMP3, is over-expressed in cutaneous melanoma.  It was therefore hypothesized that increased levels of miR-21 expression would lead to decreased expression of TIMP3 and thereby promote the invasiveness of melanoma cells.

Methods: WM1552c, WM793b, A375 and MEL39 melanoma cell lines were transfected with control miR, pre-miR-21, or anti-TIMP3.  Oligonucleotide uptake was assessed by Real-Time PCR and transfection efficiency was determined using fluorescent microscopy with a FAM-miR construct during transfection.  Transfected cells were used for invasion, proliferation, and migration assays.  Immunoblot analysis was performed on transfected cells for targets of miR-21 including programmed cell death protein (PDCD4), tropomyosin-1 (TM1), phosphatase and tensin homolog (PTEN).  A375 cells were transfected with oligonucleotides against miR-21 or a control miR before injection into the flanks of 01B74 Athymic NCr-nu/nu mice.  Tumor growth was analyzed over three weeks.  Another group of mice were injected with untransfected A375 cells in the flank and monitored until tumor growth reached an average of 100mm3 before injections with PBS alone (control) or anti-miR 21 every three days for four cycles.  Tumor growth was monitored and tumor specimens were analyzed for TIMP3 expression by immunohistochemistry using a goat anti-TIMP3.

Results: Fluoroscopic evaluation revealed >90% transfection efficiency of miRs into melanoma cells with oligonucleotide uptake also confirmed by PCR.  Immunoblot analysis of miR-21 overexpressing cells revealed reduced expression of TIMP3.  This in turn led to an increase in the invasiveness of the 4 melanoma cell lines as wells as an additional melanoma cell line, 1174 MEL.  miR-21 transfection did not change the proliferation or migration capacity of melanoma cells.  Reduced expression of TIMP3 was achieved by siRNA knockdown and significantly enhanced invasion of melanoma cells, mimicking the effects of miR-21 over-expression.  Treatment of tumor cells in vivo with an antagomir to miR-21 inhibited tumor growth with a corresponding increased expression of TIMP3 on immunohistochemistry.  Intra-tumoral injections of anti-miR-21 in vivo produced similar effects.

Conclusion: Increased expression of miR-21 enhanced the invasive potential of melanoma cell lines through TIMP3 inhibition. Therefore, inhibition of miR-21 in melanoma may reduce melanoma invasiveness.