RNA editing enzyme ADAR2 regulates P-glycoprotein expression in murine breast cancer cells through the circRNA-miRNA pathway.

Among the various RNA modifications, adenosine-to-inosine RNA editing, catalyzed by adenosine deaminase acting on RNA (ADAR) family, ADAR1 and ADAR2, is the most common nucleotide conversion in mammalian cells. The pathological relevance of ADAR expression has been highlighted in recent human genetic studies. Low expression of the ADAR2 gene is correlated with a poor prognosis in breast cancer patients, but the underlying mechanism remains enigmatic. In this study, we constructed Adar2-knockdown (Adar2-KD) murine breast cancer 4T1 cells and observed their reduced susceptibility to chemotherapeutic drug doxorubicin. Downregulation of ADAR2 induced the expression of P-glycoprotein (P-gp), leading to a reduction in the intracellular accumulation of doxorubicin. The upregulation of P-gp occurred at the post-transcriptional level due to the decreased miR-195a-3p function. The search for the underlying cause of the induction of P-gp expression in Adar2-KD 4T1 cells led to the identification of circular RNA (circRNA) circHif1a as a sponge for miR-195a-3p. The enhanced expression of circHif1a inhibited miR-195a-3p function, resulting in the upregulation of P-gp expression. These results suggest that ADAR2 acts as a suppressor of circHif1a biogenesis and then allows miR-195a-3p to interfere with P-gp translation. Our findings may help to improve drug efficacy by clarifying the mechanism of chemoresistance in breast cancer.

RNA editing enzyme ADAR1 controls miR-381-3p-mediated expression of multidrug resistance protein MRP4 via regulation of circRNA in human renal cells.

Multidrug resistance-associated protein 4 (MRP4), a member of the C subfamily of ATP-binding cassette transporters, is highly expressed in the kidneys of mammals and is responsible for renal elimination of numerous drugs. Adenosine deaminase acting on RNA 1 (ADAR1) has been reported to regulate gene expression by catalyzing adenosine-to-inosine RNA editing reactions; however, potential roles of ADAR1 in the regulation of MRP4 expression have not been investigated. In this study, we found that downregulation of ADAR1 increased the expression of MRP4 in human renal cells at the posttranscriptional level. Luciferase reporter assays and microarray analysis revealed that downregulation of ADAR1 reduced the levels of microRNA miR-381-3p, which led to the corresponding upregulation of MPR4 expression. Circular RNAs (circRNAs) are a type of closed-loop endogenous noncoding RNAs that play an essential role in gene expression by acting as miRNA sponges. We demonstrate that ADAR1 repressed the biogenesis of circRNA circHIPK3 through its adenosine-to-inosine RNA editing activity, which altered the secondary structure of the precursor of circHIPK3. Furthermore, in silico analysis suggested that circHIPK3 acts as a sponge of miR-381-3p. Indeed, we found overexpression of circHIPK3 induced the expression of MRP4 through its interference with miR-381-3p. Taken together, our study provides novel insights into regulation of the expression of xenobiotic transporters through circRNA expression by the RNA editing enzyme ADAR1.

RNA editing enzyme ADAR1 governs the circadian expression of P-glycoprotein in human renal cells by regulating alternative splicing of the ABCB1 gene.

The expression and function of some xenobiotic transporters vary according to the time of the day, causing the dosing time-dependent changes in drug disposition and toxicity. P-glycoprotein (P-gp), encoded by the ABCB1 gene, is highly expressed in the kidneys and functions in the renal elimination of various drugs. The elimination of several P-gp substrates was demonstrated to vary depending on administration time, but the underlying mechanism remains unclear. We found that adenosine deaminase acting on RNA (ADAR1) was involved in the circadian regulation of P-gp expression in human renal proximal tubular epithelial cells (RPTECs). After synchronization of the cellular circadian clock by dexamethasone treatment, the expression of P-gp exhibited a significant 24-h oscillation in RPTECs, but this oscillation was disrupted by the downregulation of ADAR1. Although ADAR1 catalyzes adenosine-to-inosine (A-to-I) RNA editing in double-stranded RNA substrates, no significant ADAR1-regulated editing sites were detected in the human ABCB1 transcripts in RPTECs. On the other hand, downregulation of ADAR1 induced alternative splicing in intron 27 of the human ABCB1 gene, resulting in the production of retained intron transcripts. The aberrant spliced transcript was sensitive to nonsense-mediated mRNA decay, leading to the decreased stability of ABCB1 mRNA and prevention of the 24-h oscillation of P-gp expression. These findings support the notion that ADAR1-mediated regulation of alternative splicing of the ABCB1 gene is a key mechanism of circadian expression of P-gp in RPTECs, and the regulatory mechanism may underlie the dosing time-dependent variations in the renal elimination of P-gp substrates.

Estradiol regulation of P-glycoprotein expression in mouse kidney and human tubular epithelial cells, implication for renal clearance of drugs.

P-glycoprotein (P-gp/ABCB1) is an ATP-binding cassette drug efflux transporter expressed in a variety of tissues that affects the pharmacokinetic disposition of many drugs. Although several studies have reported gender-dependent differences in the expression of P-gp, the role of sex hormones in regulating the expression of P-gp and its transport activity has not been well understood. In this study, we demonstrated that 17ß-estradiol has the ability to induce the expression of P-pg in mouse kidneys and cultured human renal proximal tubular epithelial cells. After intravenous injection of a typical P-gp substrate, digoxin, renal clearance in female mice was approximately 2-fold higher than that in male mice. The expression of murine P-gp and its mRNA (Abcb1a and Abcb1b) were also higher in female mice than in male mice. The expression of P-gp in cultured renal tissues prepared from female and male mice was significantly increased by 17ß-estradiol, but not testosterone. Similar 17ß-estradiol-induced expression of P-gp was also detected in cultured human tubular epithelial cells, accompanied by the enhancement of its transport activity of digoxin. The present findings suggest the contribution of estradiol to female-predominant expression of P-gp in renal cells, which is associated with sex-related disparities in the renal elimination of digoxin.