engleski

Nm23-H1 gene silencing in CAL 27 cells

The metastatic process is a major characteristic of cancer invasion. There is a huge amount of evidence showing that metastatic potential is greatly influenced by expression of nm23-H1 gene in some cancer types. The product of nm23-H1 gene is a subunit of nucleosid-diphosphate kinase (NDPK), which also exhibits protein kinase and nuclease activity. It has been reported that upregulation of nm23-H1 reduces cell migration and/or invasion in several tumor cell lines tested. Likewise, the application of cells overexpressing nm23-H1 into mice or rat, reduces formation of distinct metastasis compared to cells with normal or reduced nm23-H1 expression. In the light of these arguments we decided to create a model of posttranscriptionally silenced nm23-H1 expression in CAL 27 (squamous cell carcinoma of the tongue) to proceed with our project on the role of Nm23-H1 in oral squamous cell carcinoma. To silence nm23-H1 a plasmid based system (Openbiosystems) of retroviral origin, pSM2c, will be used. The plasmid is carrying a inserted sequence for shRNA which should silence the gene of our interest. The nm23-H1 silencing vector (V2HS_76045) and the control vector (pSM2c) were multiplied in pir+ E.coli. Due to tendency of viral vectors to recombine, the level of background recombination was checked using PCR and restriction enzyme digest. In addition the shRNA gene insert was verified by sequencing. Our further experimental procedure will include transfection of CAL 27 cells in order to produce transient and stable transfectants with downregulated Nm23-H1. The positive clones will be selected on the basis of puromycine resistance. The level of efficient silencing in corresponding transfectants will be verified by Western blotting, real-time PCR, and possibly immunocytochemistry.

nm23 ; Cal27 ; gene silencing ; shRNA

nije evidentirano