Isolation and characterization of novelRECKtumor suppressor gene splice variants

// Marina Trombetta-Lima 1, 2 , Sheila Maria Brochado Winnischofer 3 , Marcos Angelo Almeida Demasi 1, 2 , Renato Astorino Filho 1, 2 , Ana Claudia Oliveira Carreira 1, 2 , Beiyang Wei 5 , Thais de Assis Ribas 2 , Michelle Silberspitz Konig 2 , Christian Bowman-Colin 2, 4 , Sueli Mieko Oba-Shinjo 2, 6 , Suely Kazue Nagahashi Marie 2, 6 , William Stetler-Stevenson 5 , Mari Cleide Sogayar 1, 2 1 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo 05508-000 SP, Brazil 2 NUCEL-NETCEM-Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, 05360-120, Brazil 3 Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, 81531-990, Brazil 4 Dana Farber Cancer Institute, Harvard Medical School, Cambridge, MA, 02138, USA 5 Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892-4605, USA 6 Departmento de Neurologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, 01246-000, Brazil Correspondence to: Mari Cleide Sogayar, e-mail: mcsoga@iq.usp.br Keywords: RECK, GBM, splicing, isoforms, MMP Received: March 18, 2015 Accepted: September 15, 2015 Published: September 28, 2015 ABSTRACT Glioblastoma multiforme is the most common and lethal of the central nervous system glial-derived tumors. RECK suppresses tumor invasion by negatively regulating at least three members of the matrix metalloproteinase family: MMP-9, MMP-2, and MT1-MMP. A positive correlation has been observed between the abundance of RECK expression in tumor samples and a more favorable prognosis for patients with several types of tumors. In the present study, novel alternatively spliced variants of the RECK gene: RECK-B and RECK-I were isolated by RT-PCR and sequenced. The expression levels and profiles of these alternative RECK transcripts, as well as canonical RECK were determined in tissue samples of malignant astrocytomas of different grades and in a normal tissue RNA panel by qRT-PCR. Our results show that higher canonical RECK expression, accompanied by a higher canonical to alternative transcript expression ratio, positively correlates with higher overall survival rate after chemotherapeutic treatment of GBM patients. U87MG and T98G cells over-expressing the RECK-B alternative variant display higher anchorage-independent clonal growth and do not display modulation of, respectively, MMP-2 and MMP-9 expression. Our findings suggest that RECK transcript variants might have opposite roles in GBM biology and the ratio of their expression levels may be informative for the prognostic outcome of GBM patients.