Specific and reliable detection of myosin 1c isoform a by RTqPCR in prostate cancer cells

Aleena A. Saidova, Daria M. Potashnikova, Anna V. Tvorogova, Ivan V. Maly, Wilma A. Hofmann, Ivan Vorobyev

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Article numbere5970
JournalPeerJ
Volume2018
Issue number11
DOIs
Publication statusPublished - Jan 1 2018

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prostatic neoplasms
Myosins
myosin
Prostatic Neoplasms
Protein Isoforms
Cells
cell lines
neoplasms
RNA Isoforms
Genes
Prostate
Cell Line
Messenger RNA
Neoplasms
cells
Staining and Labeling
genes
Flow cytometry
Biopsy
neoplasm cells

Keywords

  • Myosin 1C A isoform
  • Prostate cancer
  • RT qPCR

ASJC Scopus subject areas

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Saidova, A. A., Potashnikova, D. M., Tvorogova, A. V., Maly, I. V., Hofmann, W. A., & Vorobyev, I. (2018). Specific and reliable detection of myosin 1c isoform a by RTqPCR in prostate cancer cells. PeerJ, 2018(11), [e5970]. https://doi.org/10.7717/peerj.5970

Specific and reliable detection of myosin 1c isoform a by RTqPCR in prostate cancer cells. / Saidova, Aleena A.; Potashnikova, Daria M.; Tvorogova, Anna V.; Maly, Ivan V.; Hofmann, Wilma A.; Vorobyev, Ivan.

In: PeerJ, Vol. 2018, No. 11, e5970, 01.01.2018.

Research output: Contribution to journalArticle

Saidova, AA, Potashnikova, DM, Tvorogova, AV, Maly, IV, Hofmann, WA & Vorobyev, I 2018, 'Specific and reliable detection of myosin 1c isoform a by RTqPCR in prostate cancer cells', PeerJ, vol. 2018, no. 11, e5970. https://doi.org/10.7717/peerj.5970
Saidova AA, Potashnikova DM, Tvorogova AV, Maly IV, Hofmann WA, Vorobyev I. Specific and reliable detection of myosin 1c isoform a by RTqPCR in prostate cancer cells. PeerJ. 2018 Jan 1;2018(11). e5970. https://doi.org/10.7717/peerj.5970
Saidova, Aleena A. ; Potashnikova, Daria M. ; Tvorogova, Anna V. ; Maly, Ivan V. ; Hofmann, Wilma A. ; Vorobyev, Ivan. / Specific and reliable detection of myosin 1c isoform a by RTqPCR in prostate cancer cells. In: PeerJ. 2018 ; Vol. 2018, No. 11.
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abstract = "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.",
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AB - 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.

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