Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many research areas. This concern is also true for quantifying coding and noncoding RNAs, with the remarkable increase in publications reporting RNA profiling and sequencing studies. To address the problem, we propose the following recommendations: (a) accurate documentation of experimental procedures in Materials and methods (and not only in the supplementary information, as many journals have a strict mandate for making Materials and methods as visible as possible in the main text); (b) submission of RT-qPCR raw data for all experiments reported; and (c) adoption of a unified, simple format for submitted RT-qPCR raw data. The Real-time PCR Data Essential Spreadsheet Format (RDES) was created for this purpose.
- Klíčová slova
- RNA, RT-qPCR, accuracy, quantification,
- MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- lidé MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- reprodukovatelnost výsledků MeSH
- RNA * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- RNA * MeSH
BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic. METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, γ-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients. RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients. CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors.
- Klíčová slova
- Aneuploidy, MSS, Noncoding RNA, Tumorigenesis,
- MeSH
- aurora kinasa B metabolismus MeSH
- azoxymethan toxicita MeSH
- chemorezistence genetika MeSH
- chromozomální nestabilita * MeSH
- cytogenetické vyšetření MeSH
- dextrany toxicita MeSH
- experimentální nádory chemicky indukované genetika patologie MeSH
- genový knockdown MeSH
- karcinogeneze genetika MeSH
- kolon cytologie patologie MeSH
- kolorektální nádory chemicky indukované genetika patologie MeSH
- lidé MeSH
- myši transgenní MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- organoidy MeSH
- primární buněčná kultura MeSH
- proteiny vázající RNA genetika metabolismus MeSH
- protokoly antitumorózní kombinované chemoterapie farmakologie terapeutické užití MeSH
- protoonkogenní proteiny c-myc metabolismus MeSH
- regulace genové exprese u nádorů MeSH
- RNA dlouhá nekódující genetika metabolismus MeSH
- signální transdukce genetika MeSH
- střevní sliznice cytologie patologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Názvy látek
- AURKB protein, human MeSH Prohlížeč
- aurora kinasa B MeSH
- azoxymethan MeSH
- BOP1 protein, human MeSH Prohlížeč
- dextrany MeSH
- long non-coding RNA CCAT2, human MeSH Prohlížeč
- MYC protein, human MeSH Prohlížeč
- proteiny vázající RNA MeSH
- protoonkogenní proteiny c-myc MeSH
- RNA dlouhá nekódující MeSH
