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Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation
Published on: February 1, 2019
Binase cleaves cellular noncoding RNAs and affects coding mRNAs
Vladimir A Mitkevich1, Nickolai A Tchurikov, Pavel V Zelenikhin
1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
Abstract:
Bacterial RNases are promising tools for the development of anticancer drugs. Neoplastic transformation leads to enhanced accumulation of rRNA and tRNA, and altered expression of regulatory noncoding RNAs. Cleavage of RNA in cancer cells is the main reason for the cytotoxic effects of exogenic RNases. We have shown that binase, a cytotoxic ribonuclease from Bacillus intermedius, affects the total amount of intracellular RNA and the expression of proapoptotic and antiapoptotic mRNAs. For four cell lines, we visualized cellular RNA by fluorescence microscopy, and determined RNA levels, viability and apoptosis by flow cytometry. We found that the level of cellular RNA was decreased in cells that were sensitive to the cytotoxic effects of binase. The RNA level was lowered by 44% in HEK cells transfected with the hSK4 gene of the Ca(2+)-activated potassium channels (HEKhSK4) and by 20% in kit-transformed myeloid progenitor FDC-P1iR1171 cells. The most significant decrease in RNA levels was registered in the subpopulations of apoptotic cells. However, the binase-induced RNA decrease did not correlate with apoptosis. Kit-transformed cells with binase-induced RNA decrease retained viability if the interleukin-dependent proliferation pathway was activated. Using quantitative RT-PCR with RNA samples isolated from the binase-treated HEKhSK4 cells, we found that the amount of mRNA of the antiapoptotic bcl-2 gene in vivo was reduced about two-fold. In contrast, expression of the proapoptotic genes p53 and hSK4 was increased 1.5-fold and 4.3-fold, respectively. These results show that binase is a regulator of RNA-dependent processes of cell proliferation and apoptosis.
Insights
Binase, a bacterial ribonuclease, reduces intracellular RNA levels in cancer cells, impacting cell proliferation and apoptosis. This cytotoxic effect offers potential for novel anticancer drug development targeting RNA regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Bacterial RNases show potential as anticancer agents by targeting RNA accumulation in neoplastic cells.
- Cancer cells exhibit altered RNA expression, including rRNA, tRNA, and regulatory noncoding RNAs.
- Exogenic RNases induce cytotoxicity in cancer cells primarily through RNA cleavage.
Purpose of the Study:
- To investigate the effects of binase, a cytotoxic ribonuclease from Bacillus intermedius, on intracellular RNA levels and gene expression in cancer cells.
- To determine the correlation between binase-induced RNA reduction, cell viability, and apoptosis.
- To explore binase's role in regulating RNA-dependent processes of cell proliferation and apoptosis.
Main Methods:
- Utilized fluorescence microscopy to visualize cellular RNA in four cell lines.
- Employed flow cytometry to quantify RNA levels, cell viability, and apoptosis.
- Conducted quantitative RT-PCR to analyze mRNA expression of key apoptotic and antiapoptotic genes.
Main Results:
- Binase treatment significantly decreased intracellular RNA levels in sensitive cell lines (e.g., 44% in HEKhSK4 cells).
- The most substantial RNA decrease was observed in apoptotic cell subpopulations, though not directly correlated with apoptosis.
- Binase altered the expression of apoptosis-related genes, reducing bcl-2 mRNA and increasing p53 and hSK4 mRNA.
Conclusions:
- Binase effectively reduces intracellular RNA levels and modulates the expression of proapoptotic and antiapoptotic genes.
- Binase's impact on RNA levels and gene expression suggests its potential as a regulator of cancer cell proliferation and apoptosis.
- The study highlights binase as a promising candidate for developing novel RNA-targeting anticancer therapies.
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