Specific and reliable detection of Myosin 1C isoform A by RTqPCR in prostate cancer cellsстатьяЭлектронная публикация
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Дата последнего поиска статьи во внешних источниках: 16 января 2019 г.
Аннотация: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.