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

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.