Abstract
Non-coding RNAs are important regulators of protein-coding genes. The current study characterized an antisense long non-coding RNA, ATP1A1-AS1, which is located on the opposite strand of the Na/K-ATPase α1 gene. Our results show that four splice variants are expressed in human adult kidney cells (HK2 cells) and embryonic kidney cells (HEK293 cells). These variants can be detected in both cytosol and nuclear fractions. We also found that the inhibition of DNA methylation has a differential effect on the expression of ATP1A1-AS1 and its sense gene. To investigate the physiological role of this antisense gene, we overexpressed the ATP1A1-AS1 transcripts, and examined their effect on Na/K-ATPase expression and related signaling function in human kidney cells. The results showed that overexpression of the ATP1A1-AS1-203 transcript in HK2 cells reduced the Na/K-ATPase α1 (ATP1A1) gene expression by approximately 20% (p < 0.05), while reducing the Na/K-ATPase α1 protein synthesis by approximately 22% (p < 0.05). Importantly, overexpression of the antisense RNA transcript attenuated ouabain-induced Src activation in HK2 cells. It also inhibited the cell proliferation and potentiated ouabain-induced cell death. These results demonstrate that the ATP1A1-AS1 gene is a moderate negative regulator of Na/K-ATPase α1, and can modulate Na/K-ATPase-related signaling pathways in human kidney cells.
Highlights
Recent advances in genome-wide sequencing has led to the transcription of the majority of the human genome, revealing that only a small portion of the transcribed RNAs codes proteins [1,2,3]
The RNAs lacking protein coding information were defined as non-coding RNAs, which, depending on their length, were further divided into short non-coding RNAs and long non-coding RNAs [4,5,6]. lncRNAs have structures of introns and exons like most messenger RNAs. lncRNAs could have polyA tail and splice variants [3,7]
Our previous study showed that reduction of Na/K-ATPase α1 by 50–60% through siRNA transfection only partially blocked ouabain-induced Src activation in pig kidney proximal tubule cells [25,31]
Summary
Recent advances in genome-wide sequencing has led to the transcription of the majority of the human genome, revealing that only a small portion of the transcribed RNAs codes proteins [1,2,3]. Our previous study showed that reduction of Na/K-ATPase α1 by 50–60% through siRNA transfection only partially blocked ouabain-induced Src activation in pig kidney proximal tubule cells [25,31] These results suggest that other effects in addition to the Na/K-ATPase reduction may exist and contribute to the Src kinase regulation and cell proliferation when ATP1A1-AS1 was overexpressed. Our data showed that the alternative splicing of antisense gene exist in human kidney cells, which may play a role in regulating the sense gene expression. Identifying these mechanisms in the future will provide more effective tools to manipulate the Na/K-ATPase α1 expression, and protect normal renal and cardiac function in humans. It merits further studies to fully understand the physiological role of this antisense RNA
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