Functional effects of SKIL overexpression in prostate cancer cell lines
Tuominen, Joonas (2015)
2015
Biokemia - Biochemistry
BioMediTech - BioMediTech
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Hyväksymispäivämäärä
2015-06-03
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:uta-201507152101
https://urn.fi/URN:NBN:fi:uta-201507152101
Tiivistelmä
Background and aims: Prostate cancer is among the most common malignancies in the world affecting millions of men worldwide. Molecular mechanisms behind the prostate cancer are still poorly understood. Genomic copy-number alterations and gene fusions are common in prostate cancer and for example TMPRSS2-ERG gene fusion is one of the common known subtypes of prostate cancer. Previously we found TMPRSS2-SKIL gene fusion from clinical sample collected from prostate cancer patient. This patient was also negative for ETS fusions so this predicts that this cancer may be caused by TMPRSS2-SKIL gene fusion. The aim of this study was to generate prostate cancer cell lines with stable SKIL overexpression and to investigate whether SKIL overexpression would have any effect on the phenotype of the cells.
Methods: Plasmid with SKIL and control plasmid without it were ordered from Addgene. DU-145 and RWPE-1 were transfected either with SKIL plasmid or with control plasmid. Then cell were let to grow with geneticin® selective antibiotic (Gibco) to select only the cells with plasmid. With DU-145 cells after selection single cell clones were created by using very low cells/ml dilution. Both mRNA and proteins were collected from both cell lines and RT-qPCR were performed for mRNAs and western blotting for proteins. Proliferation assays were performed for both cell lines using microscopy and digital image analysis. Invasion assays were performed for both cell lines using BD BioCoat Matrigel Invasion Chambers (BD Biosciences).
Results: In this study we created cell lines with stable transfections of SKIL and control plasmid. We showed that SKIL is overexpressed in mRNA level when compared to control cells. Expression levels were differentiating quite a lot between DU-145 single cell clones. However we failed to show that this expression is also carried to protein level (in both DU-145 and RWPE-1 cells). We showed that SKIL overexpression does not induce any proliferation difference but it induces invasion in RWPE-1 cells and with some DU-145 clones.
Conclusion: Our results showed that it is possible for SKIL overexpression to induce different phenotype for cell lines used. In this case there was clear difference with RWPE-1 cells and with some DU-145 cells. This indicates that it is be possible that SKIL overexpression alone could induce prostate cancer growth. However there were no clear SKIL overexpression in protein level and this leaves questions about mechanisms which cause the phenotype. Further investigations needs to be carried out to find out precise functions of SKIL and it’s overexpression in prostate cancer.
Methods: Plasmid with SKIL and control plasmid without it were ordered from Addgene. DU-145 and RWPE-1 were transfected either with SKIL plasmid or with control plasmid. Then cell were let to grow with geneticin® selective antibiotic (Gibco) to select only the cells with plasmid. With DU-145 cells after selection single cell clones were created by using very low cells/ml dilution. Both mRNA and proteins were collected from both cell lines and RT-qPCR were performed for mRNAs and western blotting for proteins. Proliferation assays were performed for both cell lines using microscopy and digital image analysis. Invasion assays were performed for both cell lines using BD BioCoat Matrigel Invasion Chambers (BD Biosciences).
Results: In this study we created cell lines with stable transfections of SKIL and control plasmid. We showed that SKIL is overexpressed in mRNA level when compared to control cells. Expression levels were differentiating quite a lot between DU-145 single cell clones. However we failed to show that this expression is also carried to protein level (in both DU-145 and RWPE-1 cells). We showed that SKIL overexpression does not induce any proliferation difference but it induces invasion in RWPE-1 cells and with some DU-145 clones.
Conclusion: Our results showed that it is possible for SKIL overexpression to induce different phenotype for cell lines used. In this case there was clear difference with RWPE-1 cells and with some DU-145 cells. This indicates that it is be possible that SKIL overexpression alone could induce prostate cancer growth. However there were no clear SKIL overexpression in protein level and this leaves questions about mechanisms which cause the phenotype. Further investigations needs to be carried out to find out precise functions of SKIL and it’s overexpression in prostate cancer.