miR-27b-3p modulates liver sinusoidal endothelium dedifferentiation in chronic liver disease

Abstract

Background: During chronic liver diseases, LSECs undergo a dedifferentiation process contributing to the development of hepatic microvascular dysfunction. Although microRNAs (miRNAs) have been associated with chronic liver disease, their role as modulators of liver endothelial phenotype is mostly unknown. Therefore, the aim of this study was to analyze miRNAs as regulators of hepatic sinusoidal endothelial dysfunction in chronic liver disease to suggest novel and translatable therapeutic options for cirrhosis.

Methods: Global expression of miRNAs was determined in primary LSECs from healthy and cirrhotic patients (alcohol abuse) and rats (CCl4 inhalation). LSECs were transfected with the mimetic or inhibitor of dysregulated miRNAs or with quantum dot nano-complexes containing miR-27b-3p or negative control, and endothelial phenotype was analyzed by RNA sequencing, quantitative PCR, and western blot. Endothelial or mesenchymal phenotypes were analyzed in LSEC by RNA sequencing, followed by pathway analyses and gene deconvolution.

Results: In all, 30 and 69 dysregulated miRNAs were identified in human and rat cirrhosis, respectively, of which 6 miRNAs were commonly dysregulated. Specific exogenous downregulation of miR-27b-3p was associated with the upregulation of target genes, suggesting a correlation between loss of miR-27b-3p and LSEC dedifferentiation. Finally, the expression of miR-27b-3p was efficiently and physiologically re-established in cirrhotic LSECs using nano-miR-27b-3p, leading to modulation of 1055 genes compared with the negative control, ultimately leading to inhibition of the endothelial-to-mesenchymal transition process observed in cirrhosis.

Conclusions: Loss of miR-27b-3p expression contributes to LSECs dedifferentiation in cirrhosis. The use of nano-miR-27b-3p represents a new therapeutic option for hepatic diseases coursing with endothelial dysfunction.

Keywords: LSEC; cirrhosis; endothelial-to-mesenchymal transition; hepatic microcirculation; quantum dots.

Graphical abstract

FIGURE 1

miRNAs profile in cirrhotic LSECs. (A) miRNA sequencing was performed in primary LSECs from healthy and cirrhotic humans and rats. (B) Heatmaps show 30 dysregulated miRNAs in cirrhotic human LSECs and (C) 69 in cirrhotic rat LSECs compared with healthy cells. (D) The set plot depicts common dysregulated miRNAs in both human and rat LSECs. (E) Expression of the 6 common dysregulated miRNAs in cirrhotic human (top) and rat (bottom) LSECs. N=6 independent LSECs isolations per group. Significantly dysregulated miRNAs were selected with fc>1.5 and p<0.05 (B, C) or p<0.1 (D, E). Abbreviation: DE miRNAs, differentially expressed microRNAs.

FIGURE 2

Dysregulated miRNAs characterization. (A) Predicted interactions of downregulated and upregulated miRNAs with differentially expressed mRNAs in cirrhotic rat LSECs. (B) miRNAs expression in an in vitro capillarization model after 24 and 48 hours of culture. (C) miRNA or negative control transfection in primary healthy LSECs methodology. (D, E) miRNAs expression after 24 and 48 hours of transfection with the mimic or inhibitor of the miRNA in an in vitro capillarization model. (F, G) Lamb1 and End1 expression after 48 hours of transfection with the mimic or inhibitor of the miRNA in an in vitro capillarization model. N=3–6 independent experiments. Data (mean±SEM) were analyzed by one-way ANOVA or by Student t test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 versus freshly isolated LSECs (0 h); ^# p<0.05, ^## p<0.01, and ^#### p<0.0001 versus the corresponding negative control group. Abbreviation: miRNAs, microRNAs.

FIGURE 3

miR-27b-3p characterization in LSECs. (A) miR-27b-3p expression in primary cirrhotic rat LSECs. (B) Gene expression of miR-27b-3p target genes analyzed by mRNA sequencing in cirrhotic rat LSECs. (C) Validation of miR-27b-3p target genes by quantitative PCR and (D) in the in vitro capillarization model. N=11 and 7 for healthy and cirrhotic LSECs, respectively, in (A), N=6 in (B, C), and N=3 in (D). Data (mean±SEM) were analyzed by Student t test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 versus healthy LSECs or 24 hours of culture.

FIGURE 4

Nano-miR-27b-3p treatment in cirrhotic LSECs. (A) miR-27b-3p expression after mimic transfection in cirrhotic rat LSECs using lipofectamine. (B) Delivery of quantum dots nano-complexes containing miR-27b-3p to cirrhotic LSECs methodology. (C) miR-27b-3p expression after nano-miR-27b-3p treatment for 48 hours. (D) Volcano plot illustrating differently expressed genes. Blue dots: significantly lower expression in nano-miR-27b-3p; green dots: significantly higher expression in nano-miR-27b-3p. (E) Heatmap of top 40 DEGs after nano-miR-27b-3p treatment. (F) Canonical pathways involved in DEGs after nano-miR-27b-3p treatment analyzed by IPA software. N=6–8 independent experiments. Data (mean±SEM) were analyzed by Student t test: *p<0.05, ***p<0.001 versus negative control group. Significant DEGs were selected with fc>1.5 and p<0.05. Abbreviations: DEGs, differentially expressed genes; IPA, Ingenuity Pathway Analysis; miRNAs, microRNAs.

FIGURE 5

miR-27b-3p is involved in the EndMT process. (A) Heatmap of genes involved in the EndMT process illustrating the mesenchymal phenotype of cirrhotic LSECs. (B) Heatmap showing regression of EndMT process after restoration of miR-27b-3p in cirrhotic LSECs. (C) Western blot and corresponding quantification of Snail in cirrhotic LSECs treated with nano-mir-27b-3p or negative control. (D) LSEC phenotype classification by gene deconvolution depending on gene expression of endothelial or mesenchymal profiles in cells from panels A and B. N=5–6 independent experiments. Data (mean±SEM) were analyzed by Student t test: ^# p<0.01 versus healthy LSECs and *p<0.05 versus the negative control group. Abbreviation: EndMT, endothelial-to-mesenchymal transition.