Akademik Veri Yönetim Sistemi
Journal on Oncology, cilt.1, sa.2, ss.1-13, 2020 (Hakemli Dergi)
Aim: Our aim is to clone the GHRH cDNA and demonstrate the biological function on cell growth, proliferation via generating GHRH overexpressing MDA-MB-231 cells.
Methods: GHRH cDNA were amplified from LNCaP prostate cancer cells, that have highest transcript ional and translational GHRH expression. EcoRI and HindIII restriction enzymes were used to insert GHRH cDNA in pcDNA 3.1 vector and generated plasmid transformed to E. coli HB101 strain, positive clones were selected by colony PCR. Forced GHRH expressing MDA-MB-231 breast cancer cells were generated by lyposomal transfection of GHRH cDNA-PC3.1 plasmid and neomycin selection. Highest GHRH expressing MDA-MB-231 breast cancer cells were selected by qRT-PCR, and immunoblotting.
Results: Increased GHRH expression leads cell growth, proliferation, colony formation in time dependent-manner in MDA-MB-231 breast cancer cells. Moreover, significant difference on EMT pathway key molecules expression profile has been observed when compared to parental MDA-MB-231 breast cancer cells. Autocrine GHRH expression induced invasion-metastasis through upregulation of E-cadherin, Vimentin, c-jun, MMP-2 and MMP-9 expressions in MDA-MB-231 cells within 48 h.
Conclusion: we cloned GHRH variant 1 cDNA in pcDNA3.1 vector and stable cell line generated by GHRH cDNA inserted plasmid triggered invasion-metastatic profile in MDA-MB-231 breast cancer cells via EMT pathway.