Autophagy-mediated reduction of miR-345 contributes to hepatic cystogenesis in polycystic liver disease

JHEP Rep. 2021 Aug 5;3(5):100345. doi: 10.1016/j.jhepr.2021.100345. eCollection 2021 Oct.

Abstract

Background & aims: Polycystic liver disease (PLD) is characterised by increased autophagy and reduced miRNA levels in cholangiocytes. Given that autophagy has been implicated in miRNA regulation, we tested the hypothesis that increased autophagy accounts for miRNA reduction in PLD cholangiocytes (PLDCs) and accelerated hepatic cystogenesis.

Methods: We assessed miRNA levels in cultured normal human cholangiocytes (NHCs), PLDCs, and isolated PLDC autophagosomes by miRNA-sequencing (miRNA-seq), and miRNA targets by mRNA-seq. Levels of miR-345 and miR-345-targeted proteins in livers of animals and humans with PLD, in NHCs and PLDCs, and in PLDCs transfected with pre-miR-345 were assessed by in situ hybridisation (ISH), quantitative PCR, western blotting, and fluorescence confocal microscopy. We also assessed cell proliferation and cyst growth in vitro, and hepatic cystogenesis in vivo.

Results: In total, 81% of miRNAs were decreased in PLDCs, with levels of 10 miRNAs reduced by more than 10 times; miR-345 was the most-reduced miRNA. In silico analysis and luciferase reporter assays showed that miR-345 targets included cell-cycle and cell-proliferation-related genes [i.e. cell division cycle 25A (CDC25A), cyclin-dependent kinase 6 (CDK6), E2F2, and proliferating cell nuclear antigen (PCNA)]; levels of 4 studied miR-345 targets were increased in PLDCs at both the mRNA and protein levels. Transfection of PLDCs with pre-miR-345 increased miR-345 and decreased the expression of miR-345-targeted proteins, cell proliferation, and cyst growth in vitro. MiR-345 accumulated in autophagosomes in PLDCs but not NHCs. Inhibition of autophagy increased miR-345 levels, decreased the expression of miR-345-targeted proteins, and reduced hepatic cystogenesis in vitro and in vivo.

Conclusion: Autophagy-mediated reduction of miR-345 in PLDCs (i.e. miRNAutophagy) accelerates hepatic cystogenesis. Inhibition of autophagy restores miR-345 levels, decreases cyst growth, and is beneficial for PLD.

Lay summary: Polycystic liver disease (PLD) is an incurable genetic disorder characterised by the progressive growth of hepatic cysts. We found that hepatic cystogenesis is increased when the levels of miR-345 in PLD cholangiocytes (PLDCs) are reduced by autophagy. Restoration of miR-345 in PLDCs via inhibition of autophagy decreases hepatic cystogenesis and thus, is beneficial for PLD.

Keywords: ADPKD, autosomal dominant polycystic kidney disease; ADPLD, autosomal dominant polycystic liver disease; AGO2, Argonaute 2; ALG8, alpha-1,3-glucosyltransferase; ALG9, alpha-1,2-mannosyltransferase; ARPKD, autosomal recessive polycystic kidney disease; CDC25A, cell division cycle 25A; CDK6, cyclin-dependent kinase 6; Cell cycle-related proteins; Cholangiocyte proliferation; Cholangiocytes; DNAJB11, DnaJ heat shock protein family (Hsp40) member B11; DZIP1L, DAZ interacting zinc finger protein 1 like; FDR, false discovery rate; GANAB, glucosidase II alpha subunit; GO, Gene Ontology; Genetic liver diseases; HCQ, hydroxychloroquine; ISH, in situ hybridisation; KEGG, Kyoto Encyclopedia of Genes and Genomes; LRP5, low-density lipoprotein receptor-related protein 5; NHC, normal human cholangiocyte; NRC, normal rat cholangiocyte; PCK, polycystic kidney; PCKC, polycystic kidney rat cholangiocyte; PCNA, proliferating cell nuclear antigen; PKD1/2, polycystic kidney disease 1/2; PKHD1, polycystic kidney and hepatic disease 1; PLD treatment; PLD, polycystic liver disease; PLDC, polycystic liver disease cholangiocyte; PRKCSH, protein kinase C substrate 80K-H; RPM, reads per million; SEC61B, SEC61 translocon subunit beta; SEC63, SEC63 homolog, protein translocation regulator; WT, wild type; mTOR, mammalian target of rapamycin; miRISC, RNA-induced silencing complex; miRNA-seq, miRNA-sequencing; snRNA, small nuclear RNA.