The Role of LncRNA H19 in Estrogen‐Induced Cholestatic Injury
Authors: Li Xiaojiaoyang , Liu Runping , Zhao Derrick , Pandak William M , Hylemon Phillip B , Zhou Huiping 
Published: November 2023

IntroductionThe mechanisms underlying estrogen‐related cholestasis are still not fully identified. The mdr2 knockout (mdr2−/−) mouse is a well‐established model of cholestatic liver disease. It has been shown that female mdr2−/− mice develop more severe hepatobiliary damage, which is correlated with a higher proportion of taurocholate (TCA) in bile. The long non‐coding RNA (lncRNA) H19 is an imprinted and maternally expressed gene and has been identified as an estrogen‐targeted gene. A recent study reported that H19 expression is significantly induced in human fibrotic/cirrhotic liver and bile duct ligated (BDL) mouse liver. However, whether H19 expression is correlated to differential severity of cholestatic injury related to gender difference remains unknown. The aim of the current study is to determine the expression of H19 during disease progression in both male and female mdr2−/− mice and further examine the potential role of H19 in estrogen‐mediated cholestatic injury.MethodsAge and gender‐matched wild type (WT) and mdr2−/− mice were used. At the age of 60 or 100 days, mice were sacrificed. Blood was collected for measuring serum levels of ALT, AST, ALP, total bile acids (TBA) and hydroxyproline using commercial kits. Liver samples were processed for HE and Masson's trichrome staining as well as immunohistochemical analysis. Primary hepatocytes, Kupffer cells and cholangiocytes from 100 days old WT and mdr2−/− mice were used to examine the expression of H19. An adenoviral shRNA of H19 was used to knock down the expression of H19 in cultured cholangiocytes and in mdr2−/− mice. The expression level of H19 and the mRNA levels of other target genes were measured by real‐time RT‐PCR. The protein levels of Bcl2, small heterodimer partner (SHP), p‐ERK1/2 and total‐ERK1/2 were determined by Western blot analysis.ResultsAt the age of 100 days, both male and female mdr2−/− mice developed significant cholestatic injury. However, the female mdr2−/− mice had more severe hepatic injury and fibrosis. H19 expression was induced by ~200‐fold in 100 days old mdr2−/− female mouse liver, while hepatic H19 level in 60 days old mdr2−/− mice or 100 days old mdr2−/− male mice was non‐detectable or only slightly increased. Hepatic H19 was mainly expressed in cholangiocytes not hepatocytes and Kupffer cells. Both estrogen and TCA induced H19 expression. Knocking down H19 using an adenoviral shRNA not only significantly reduced TCA/estrogen‐induced expression of fibrotic genes and sphingosine 1‐phosphate receptor 2 (S1PR2) in cholangiocytes, but also markedly reduced cholestatic injury in female mdr2−/− mice. Furthermore, the expression of SHP was substantially inhibited at the advanced stage of liver fibrosis, which was reversed by H19 shRNA in female mdr2−/− mice.ConclusionEstrogen‐induced accumulation of TCA and subsequent induction of H19 plays a critical role in promoting cholestatic liver injury. It represents a key factor that causes gender disparity of cholestatic liver injury in mdr2−/− mice.Support or Funding InformationThis work was supported by National Institutes of Health Grant R01 DK104893 (to HZ and PBH), R01DK‐057543‐11 (to PBH and HZ), VA Merit Award I01BX001390 (to HZ); Massey Cancer Center pilot grant (to HZ and PBH). Microscopy was performed at the VCU Microscopy Facility, supported in part by funding from NIH‐NCI Cancer Center Grant P30 CA016059.