Up-regulation of miR-let7a-5p Leads to Decreased Expression of ABCC2 in Obstructive Cholestasis

Abstract

Adenosine triphosphate-binding cassette subfamily C member 2 (ABCC2/Abcc2) is critically important to biliary excretion of many endobiotic and xenobiotic compounds, and is a major driving force for bile acid-independent bile flow. Abcc2 expression is reduced at the messenger RNA (mRNA) and protein levels in various forms of experimental cholestasis. In a microRNA (miRNA) screen of mouse liver after biliary obstruction, we found that miRNA let7a-5p was significantly up-regulated approximately 4-fold. Similarly, ABCC2 mRNA was depleted and miRNA let7a-5p was elevated over 4-fold in livers of children with biliary atresia compared with normal livers. In silico analysis predicted that let7a-5p would target the 3' untranslated region (3' UTR) of ABCC2/Abcc2 RNA. The objective of this study was to determine whether let7a-5p contributes to the depletion of ABCC2/Abcc2 in cholestasis. To demonstrate the functional importance of miRNA let7a-5p in regulating the expression of ABCC2, co-transfection of a let7a-5p mimic and an ABCC2-3' UTR luciferase construct into Huh-7 cells led to a marked inhibition of luciferase activity by about 60%-70% compared with controls, which was reversed by a let7a-5p mimic inhibitor. Expression of this mimic led to a significant decrease in endogenous ABCC2 mRNA and protein levels in a Huh-7 liver cell line, which could be blocked by expression of a let7a-5p mimic inhibitor. Injection of a lentivirus let7a-5p inhibitor into normal mouse liver or into mouse liver after common bile duct ligation led to a significant increase in endogenous Abcc2 mRNA and protein levels and a depletion of let7a-5p mRNA levels compared with untreated, saline-injected livers or livers treated with an inactive lentivirus control. Conclusion: These studies demonstrate that miR-let7a-5p is involved in regulating ABCC2/Abcc2 expression, and is aberrantly up-regulated in obstructive cholestasis.

Figure 1

The effect of CBDL on expression of let7a‐5p. Three days of CBDL ligation led to an over 4‐fold increase in let7a‐5p miRNA levels compared with livers from sham‐operated mice on RT‐PCR analysis. *P < 0.01.

Figure 2

Predicted binding sites for the seed sequence of let7a‐5p in the 3′ UTR of Abcc2 (mouse) and ABCC2 (human) mRNAs using the bioinformatic tools.

Figure 3

Effect of 3 days of CBDL in the mouse on Abcc2 mRNA and protein expression. There was significant depletion of Abcc2 mRNA (Fig. 1A) and protein levels (Fig. 1B) on RT‐PCR and western blot analysis, respectively, in CBDL versus sham‐operated mice. Experiments with CBDL mice were repeated 3 times. *P < 0.01.

Figure 4

Expression of ABCC2 mRNA and let7a‐5p in infants with biliary atresia. Decreased expression of ABCC2 mRNA (Fig. 4A) and up‐regulation of let7a‐5p (Fig. 4B) was found in livers of 3 children with biliary atresia versus livers of 3 healthy children on RT‐PCR analysis. *P < 0.01.

Figure 5

Effect of a let7a‐5p mimic and a mimic inhibitor on expression of an ABCC2‐3′ UTR‐luciferase construct expressed in Huh7 cells 48 hours after transfection. (A) Expression of a let7a‐5p mimic significantly repressed luciferase activity. The activity of the mimic was completely reversed by a mimic inhibitor. (B) The effect of the let7a‐5p mimic on luciferase activity was completely abrogated by mutation of the putative let7a‐5p binding site in the ABCC2‐3′ UTR. The miR199a‐5p (C) and miR223‐3p (D) mimics had no effect on the expression of an ABCC2‐3′ UTR‐luciferase construct expressed in Huh7 cells. Each panel represents the mean + SEM of three separate experiments. ^# P < 0.01.

Figure 6

Effect of a let7a‐5p mimic on endogenous ABCC2 mRNA in Huh‐7 cells. Overexpression of the let7a‐5p mimic but not the miR 223‐3p and miR199b‐3p mimics led to a significant decrease in ABCC2 mRNA levels. In contrast, expression of the miR 223‐3p and miR199b‐3p mimics had no effect on ABCC2 levels. Expression of a let7a‐5p mimic and mimic inhibitor led to an increase in endogenous ABCC2 mRNA levels (Fig. 6B), whereas expression of the miR 223‐3p and miR199b‐3p mimics and inhibitors had no effect on ABCC2 mRNA levels. *P < 0.01.

Figure 7

Western blot showing the effect of a let7a‐5p mimic on endogenous ABCC2 protein levels in Huh‐7 cells (Fig. 7A). Transfection of a let7a‐5p mimic into Huh‐7 cells led to a significant decrease in ABCC2 protein levels which could be blocked by expression of a let7a‐5p mimic inhibitor. Expression of let7a‐5p had no effect on the amounts of BSEP, NTCP, or FXR proteins in Huh‐7 cells (Fig. 7B). Quantification of blots using the Bio‐Rad ChemiDoc System. *Mean + SEM of three separate experiments. *P < 0.01.

Figure 8

The effect of a lentivirus let7a‐5p inhibitor on endogenous let7a‐5p and ABCC2 expression in Huh‐7 cells. Cells were infected with a lentivirus let‐7a‐5p inhibitor construct and analyzed after 48 hours. This construct led to a significant decrease in let7a‐5p mRNA (A) and a marked increase in ABCC2 mRNA (B) and protein levels (C,D) in these cells compared with controls. *P < 0.001.

Figure 9

Effect of a lentivirus let7a‐5p inhibitor on endogenous let7a‐5p and Abcc2 expression in normal mouse liver. In normal mouse livers, injection of the lentivirus‐let7a‐5p inhibitor construct led to a significant depletion of let7a‐5p mRNA levels and an increase in endogenous Abcc2 mRNA and protein levels compared with untreated mouse liver or liver treated with an inactive lentivirus control. *P < 0.01.

Figure 10

Effect of a lentivirus let7a‐5p inhibitor on let7a‐5p and Abcc2 expression in mouse liver after CBDL. After CBDL, injection of a lentivirus‐let7a‐5p inhibitor led to a significant decrease in let7a‐5p mRNA levels (10A) and an increase in endogenous Abcc2 mRNA (10B) and protein levels (10C and 10D) compared with untreated mouse liver or liver treated with an inactive lentivirus control. *P < 0.01.