Specific and reliable detection of Myosin 1C isoform A by RTqPCR in prostate cancer cells
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...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
PeerJ Inc.
2018-11-01
|
| Series: | PeerJ |
| Subjects: | |
| Online Access: | https://peerj.com/articles/5970.pdf |
| _version_ | 1797418976598294528 |
|---|---|
| author | Aleena A. Saidova Daria M. Potashnikova Anna V. Tvorogova Ivan V. Maly Wilma A. Hofmann Ivan A. Vorobjev |
| author_facet | Aleena A. Saidova Daria M. Potashnikova Anna V. Tvorogova Ivan V. Maly Wilma A. Hofmann Ivan A. Vorobjev |
| author_sort | Aleena A. Saidova |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T06:40:47Z |
| format | Article |
| id | doaj.art-efdffc1808a648eb995d8edda46d1bd5 |
| institution | Directory Open Access Journal |
| issn | 2167-8359 |
| language | English |
| last_indexed | 2024-03-09T06:40:47Z |
| publishDate | 2018-11-01 |
| publisher | PeerJ Inc. |
| record_format | Article |
| series | PeerJ |
| spelling | doaj.art-efdffc1808a648eb995d8edda46d1bd52023-12-03T10:51:20ZengPeerJ Inc.PeerJ2167-83592018-11-016e597010.7717/peerj.5970Specific and reliable detection of Myosin 1C isoform A by RTqPCR in prostate cancer cellsAleena A. Saidova0Daria M. Potashnikova1Anna V. Tvorogova2Ivan V. Maly3Wilma A. Hofmann4Ivan A. Vorobjev5Biological Faculty, M.V. Lomonosov Moscow State University, Moscow, RussiaBiological Faculty, M.V. Lomonosov Moscow State University, Moscow, RussiaA.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Moscow, RussiaDepartment of Physiology and Biophysics, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, United States of AmericaDepartment of Physiology and Biophysics, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, United States of AmericaBiological Faculty, M.V. Lomonosov Moscow State University, Moscow, RussiaBackground 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.https://peerj.com/articles/5970.pdfProstate cancerMyosin 1C A isoformRT qPCR |
| spellingShingle | Aleena A. Saidova Daria M. Potashnikova Anna V. Tvorogova Ivan V. Maly Wilma A. Hofmann Ivan A. Vorobjev Specific and reliable detection of Myosin 1C isoform A by RTqPCR in prostate cancer cells PeerJ Prostate cancer Myosin 1C A isoform RT qPCR |
| title | Specific and reliable detection of Myosin 1C isoform A by RTqPCR in prostate cancer cells |
| title_full | Specific and reliable detection of Myosin 1C isoform A by RTqPCR in prostate cancer cells |
| title_fullStr | Specific and reliable detection of Myosin 1C isoform A by RTqPCR in prostate cancer cells |
| title_full_unstemmed | Specific and reliable detection of Myosin 1C isoform A by RTqPCR in prostate cancer cells |
| title_short | Specific and reliable detection of Myosin 1C isoform A by RTqPCR in prostate cancer cells |
| title_sort | specific and reliable detection of myosin 1c isoform a by rtqpcr in prostate cancer cells |
| topic | Prostate cancer Myosin 1C A isoform RT qPCR |
| url | https://peerj.com/articles/5970.pdf |
| work_keys_str_mv | AT aleenaasaidova specificandreliabledetectionofmyosin1cisoformabyrtqpcrinprostatecancercells AT dariampotashnikova specificandreliabledetectionofmyosin1cisoformabyrtqpcrinprostatecancercells AT annavtvorogova specificandreliabledetectionofmyosin1cisoformabyrtqpcrinprostatecancercells AT ivanvmaly specificandreliabledetectionofmyosin1cisoformabyrtqpcrinprostatecancercells AT wilmaahofmann specificandreliabledetectionofmyosin1cisoformabyrtqpcrinprostatecancercells AT ivanavorobjev specificandreliabledetectionofmyosin1cisoformabyrtqpcrinprostatecancercells |