Specific and reliable detection of Myosin 1C isoform A by RTqPCR in prostate cancer cells.

Autor:	Saidova AA; Biological Faculty, M.V. Lomonosov Moscow State University, Moscow, Russia., Potashnikova DM; Biological Faculty, M.V. Lomonosov Moscow State University, Moscow, Russia.; Laboratory of Atherothrombosis, Moscow State University of Medicine and Dentistry, Moscow, Russia., Tvorogova AV; A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Moscow, Russia., Maly IV; Department of Physiology and Biophysics, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, United States of America., Hofmann WA; Department of Physiology and Biophysics, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, United States of America., Vorobjev IA; Biological Faculty, M.V. Lomonosov Moscow State University, Moscow, Russia.; A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Moscow, Russia.; Department of Biology, School of Science and Technology, Nazarbayev University, Astana, Kazakhstan.
Jazyk:	angličtina
Zdroj:	PeerJ [PeerJ] 2018 Nov 20; Vol. 6, pp. e5970. Date of Electronic Publication: 2018 Nov 20 (Print Publication: 2018).
DOI:	10.7717/peerj.5970
Abstrakt:	Background: Prostate cancer (PC) diagnostics and treatment often present a challenging task due to cancer subtype heterogeneity and differential disease progression in patient subgroups. Hence, the critical issue is finding a reliable and sensitive diagnostic and prognostic PC marker, especially for cases of biopsies with low percentages of cancer cells. Isoform A of myosin 1C was shown to be expressed in PC cells and responsible for their invasive properties, however, its feasibility for diagnostic purposes remains to be elucidated. Methods: To verify the role of myosin 1C isoform A mRNA expression as a putative prostate cancer marker we performed RT qPCR normalized by three reference genes ( GAPDH, YWHAZ, HPRT1 ) on PC3, RWPE-1, LNCaP and 22Rv1 cell lines. Myosin 1C isoform A detection specificity was confirmed by immunofluorescence staining, cancer and non-cancer prostate cell lines were immunophenotyped by flow cytometry. Results: Median normalized mRNA expression level of myosin 1C isoform A in PC cells (PC3 and 22Rv1) is two orders of magnitude higher compared to RWPE-1 cells, which functionally correspond to benign prostate cells. Myosin 1C isoform A expression allows PC cell detection even at a dilution ratio of 1:1000 cancer to non-cancer cells. At the protein level, the mean fluorescence intensity of myosin 1C isoform A staining in PC3 nuclei was only twice as high as in RWPE-1, while the immunophenotypes of both cell lines were similar (CD44+/CD90-/CD133-/CD57-/CD24+-). Conclusions: We report a distinct difference in myosin 1C isoform A mRNA levels in malignant (PC3) and benign (RWPE-1) prostate cell lines and suggest a combination of three reference genes for accurate data normalization. For the first time we provide an immunophenotype comparison of RWPE-1 and PC3 cells and demonstrate that RT qPCR analysis of MYO 1C A using appropriate reference genes is sufficient for PC detection even in low-abundance cancer specimens. Competing Interests: The authors declare there are no competing interests.
Databáze:	MEDLINE
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