Abstract
Potent RNase activities were found in the serum of mammals but the physiological function of the RNases was never well illustrated, largely due to the caveats in methods of RNase activity measurement. None of the existing methods can distinguish between RNases with different target specificities. A systematic study was recently carried out in our lab to investigate the site-specificity of serum RNases on double-stranded RNA substrates, and found that serum RNases cleave double-stranded RNAs predominantly at 5′-U/A-3′ and 5′-C/A-3′ dinucleotide sites, in a manner closely resembling RNase A. Based on this finding, a FRET assay was developed in the current study to measure this site-specific serum RNase activity in human samples using a double stranded RNA substrate. We demonstrated that the method has a dynamic range of 10−5 mg/ml- 10−1 mg/ml using serial dilution of RNase A. The sera of 303 cancer patients were subjected to comparison with 128 healthy controls, and it was found that serum RNase activities visualized with this site-specific double stranded probe were found to be significantly reduced in patients with gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, ovary cancer, cervical cancer, bladder cancer, kidney cancer and lung cancer, while only minor changes were found in breast and colon cancer patients. This is the first report using double stranded RNA as probe to quantify site-specific activities of RNase A in a serum. The results illustrated that RNase A might be further evaluated to determine if it can serve as a new class of biomarkers for certain cancer types.
Highlights
RNase A is an endoribonuclease with functions in RNA metabolism and regulation of gene expression
The fast degradation processes make it too hard to monitor the degradation process in real-time. This situation further leads to the difficulties in establishing quantitative measurements of RNase activity
We speculated that double-stranded RNAs might be used as a substrate for measuring serum RNase activity
Summary
RNase A is an endoribonuclease with functions in RNA metabolism and regulation of gene expression. An antitumor activity was reported for an RNase A with cytotoxic and cytostatic properties [5,6]. As a unique evolutionary conserved protein family, the members of RNase A superfamily are normally composed of 124 amino acids [8]. The members of RNase A superfamily are widely expressed and present in serum and tissues of mammals[9]. RNase A is the predominant endoribonuclease component in human organs and tissues [9]. In addition to function in mRNA and 18S rRNA degradation, RNase 4 and RNase 5 were reported to have an angiogenesis function, whereas RNase 5 in particular could promote the growth of blood vessel [1]
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