ABSTRACT
Objective: To analyze and compare the differentially expressed genes (DEGs) of Yes-associated protein (YAP)-positive and negative hepatocytes and further understand the preliminary functional characteristics of YAP-positive hepatocytes in an early mouse model of nonalcoholic steatohepatitis (NASH) with transcriptome sequence (RNA-Seq). Methods: C57BL/6 mice were fed with methionine-choline deficiency (MCD) diet for 2 weeks to establish an early NASH model, and the control group was fed with normal diet. Liver tissue was stained with hematoxylin-eosin (HE) and Sirius red, and the pathological score was recorded. The expression of YAP and P-YAP were determined by immunohistochemistry (IHC) in liver tissues. Primary hepatocytes with viability greater than 90% were isolated and purified by collagenase perfusion combined with Percoll density gradient centrifugation. YAP-positive and negative hepatocytes were assessed by YAP antibody, flow cytometry and RNA-Seq analyses. Sequencing results were screened by GO, KEGG and interaction network analysis methods. RT-PCR was used to verify the expression levels of YAP and some DEGs in liver tissue model group. Two samples mean was compared by independent samples t-test. Results: Compared with the control group, the HE-stained liver tissue of MCD-induced mice at 2 weeks showed steatosis (pathological score 1.07±0.21), accompanied by lobular inflammation (pathological score 1.13±0.32) and ballooned hepatocyte (pathological score 0.80) ±0.20). Sirius red staining showed non-significant liver fibrosis (pathological score 0.40±0.40). IHC showed partial YAP-positive hepatocytes expression in an early stage of NASH. RNA-Seq analysis showed that clean reads of YAP-positive and negative hepatocytes were 49 310 604 and 5 4820 036, respectively. Compared with YAP-negative hepatocytes, YAP-positive hepatocytes had differential expression of 5 565 genes, including 1 662 up-regulated genes and 3 903 down-regulated genes. GO analysis of up-regulated genes showed that the metabolic processes related to mitochondrial functions, such as purine nucleoside triphosphate and nucleoside triphosphate were significantly enriched in biological processes (BP), while down-regulated gene analysis showed that olfactory-related receptor were significantly enriched in BP. KEGG analysis showed that DEGs were enriched in 292 pathways, and oxidative phosphorylation (OXPHOS) pathway was significantly enriched in signaling pathway. RT-PCR validated that inflammatory factors (interleukin-1ß, interleukin-6), YAP and its target genes (Cyr61, Ankrd1), and Cox5b and Sdhc genes were significantly up-regulated in the OXPHOS pathway, which was consistent with the sequencing results. In addition, eight key genes with interaction network analysis were predicted. Conclusion: Changes in hepatocyte metabolic levels may be associated with increased YAP activity in an early stage of NASH.
Subject(s)
Non-alcoholic Fatty Liver Disease , Animals , Disease Models, Animal , Hepatocytes/metabolism , Liver/pathology , Methionine/genetics , Methionine/metabolism , Mice , Mice, Inbred C57BL , Non-alcoholic Fatty Liver Disease/pathology , Sequence Analysis , TranscriptomeABSTRACT
The transcriptional coactivator Yes-associated protein (YAP) is involved in the regulation of various cellular physiological activities and plays an important role in controlling the size of tissues and organs for maintaining the homeostasis. YAP not only affects the transcriptional expression of its downstream target genes, but also interacts with multiple signaling pathways to form complex regulatory networks to perform specific biological functions. In addition to Hippo signaling pathway mediated protein phosphorylation, the regulatory mechanisms of YAP activity also takes part in multiple fields. YAP activity alteration can affect the morphological characteristics and physiological functions of liver tissue, and participate in the occurrence and development of liver diseases, such as liver fibrosis and primary liver cancer. This paper mainly discusses the close relationship between YAP and liver pathophysiology, in order to provide reference for further exploring its potential application in molecular targeted therapy of liver diseases.
