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Purpose Development of cataract is associated with apoptotic death of the lens epithelial cells. The purpose of this investigation was to examine whether this could be explained by enhancement in the expression of certain pro-apoptotic microRNAs (miRs), known to induce apoptosis by hybridizing with target mRNAs, with the consequence of gene silencing. In addition, it was intended to investigate if such expression could be antagonized by reactive oxygen species (ROS) scavengers. Methods CD-1 mice weighing about 20 g were divided into three groups and fed diets, respectively, as follows: Control diet, 25% galactose diet, and 25% galactose diet containing 1% sodium pyruvate. After eight days of such a regimen, the mice were euthanized, their lenses promptly isolated and frozen in liquid nitrogen, and RNAs isolated by extraction with standard methods and converted to cDNAs. The miR-specific cDNAs were then quantified by polymerase chain reaction (PCR) using a 96-well microRNA array cassette using an ABI 7900 HT PCR machine. The results were then analyzed using Bioscience software. Results The lens samples were positive for all of the 84 miRs expected, on the basis of the specific sequences used for amplification. However, as would be apparent from the microarray plot for the normal and galactosemic lenses, expression of at least 24 apoptotic miRs was upregulated. Six apoptotic miRs were downregulated. In the lenses of animals where the galactose diet was fortified with sodium pyruvate, the expression of 12 miRs was completely prevented. The upregulation observed in the other 14 miRs was also significantly downregulated. A comparison of the galactose and galactose plus pyruvate group clearly indicated that pyruvate inhibits the transcription of apoptotic miRS. Conclusions The prevention of galactose-induced enhancement in the expression of apoptotic miRs by pyruvate, a compound well known to scavenge reactive oxygen species as well as to inhibit their formation, strongly suggests that the upregulation of miRs in galactosemic animals is due to generation of reactive oxygen species. This is in conformity with our previous studies showing that pyruvate and other ROS scavengers inhibit apoptosis as well as cataract formation. |
