Introduction
Hepatic fibrosis is a common wound-healing response to diverse chronic liver diseases. The pathogenesis of hepatic fibrosis is mostly featured by excessive accumulation of extracellular matrix (ECM) in the liver [
1], which is mainly derived from activated hepatic stellate cell (HSC). Upon different kinds of hepatic injuries, quiescent HSCs are activated and transformed into proliferative, fibrogenic and contractile myofibroblasts, which are characterized by expressing high level of
Acta2 (smooth muscle actin alpha 2). HSC activation is recognized as a pivotal event during liver fibrogenesis [
2,
3].
Single-cell transcriptomic analysis is widely used in basic scientific and clinical researches, and has reshaped our understanding of many complex biological processes. Compared with traditional sequencing methods, single-cell RNA sequencing (scRNA-seq) can offer unprecedented insight into HSC heterogeneity and identify genes highly related to HSC activation at single-cell resolution. Recently, HSC zonation has been proposed as a determinant of the liver fibrogenesis response [
4]. The subsets of HSC and myofibroblast during hepatic fibrosis have been identified by single-cell transcriptomic analysis [
5]. However, the differentiation trajectory of HSC-to-myofibroblast transition and the switching genes during this process remains not well understood.
In the present study, we in silico reconstructed a single-lineage pseudotime trajectory of HSC activation in vitro based on the scRNA-seq data. Intriguingly, we identified a novel cell state during HSC transdifferentiation, which may be relevant to cancer-associated fibroblasts (CAFs). In addition, we dissected key switching genes contributing to HSC activation, then, we established a four-gene combination based on the top switching genes and evaluated its diagnostic accuracy in predicting advanced liver fibrosis in humans.
Discussion
Abnormal activation, proliferation, and migration of HSCs cause hepatic fibrosis and cirrhosis [
34]. HSC-to-myofibroblast transition is a key event during liver fibrogenesis induced by diverse chronic liver injury. The scRNA-seq data (GSE132662) obtained from the HSCs cultured in vitro and harvested at day 0, 1, 3, 7, and 9 were analyzed in this study. Our results revealed a novel cell state during HSC activation, which may be relevant to CAFs. Moreover, we identified some switching genes contributing to HSC activation, and established a four-gene combination to predict advance hepatic fibrosis in patients with NAFLD or HBV.
That primary HSCs isolated from healthy mouse were cultivated for up to 7 days on uncoated plastic dishes is the classic model of HSC-to-myofibroblast transition [
35]. In the present study, we in silico reconstructed a pseudotime trajectory of HSC transdifferentiation based on the single-cell transcriptomic data of the culture-activated HSC in vitro, and identified a novel state of HSC. Compared with classic activated HSC, the cells belonging to the new state express high HSC activation markers as well as relatively low collagen genes, but express high levels of genes enriched for the regulation of signal transduction by p53 class mediator, some of which were involved in the biological pathways related to cancer. For example, it has been demonstrated that
Rack1 is highly expressed by activated HSC and upregulated in HCC [
36], moreover,
Rack1 can promote self-renewal of cancer stem cells in patients with HCC [
22].
Npm1 was identified in diverse cellular processes such as ribosome biogenesis, cell proliferation, and regulation of tumor suppressors p53/TP53 [
37]. It’s associated with HCC [
21]. The expression of
Mif and
Cstb in liver tissue were also verified to be involved in liver carcinogenesis [
23,
24].
Liver fibrosis is a wound healing and scar repair response to liver injury. Tumors are considered as “wounds that never heal”, and more than 80% of HCC develop from cirrhosis caused by chronic liver diseases such as viral hepatitis and NAFLD [
38,
39]. Activated HSCs had been found in the stroma of HCC [
40], and activated peritumoral HSCs were demonstrated to be associated with tumor recurrence and mortality [
41]. CAFs, a key player in hepatocarcinogenesis, probably mainly originate from HSC and may play an essential role in the pathogenesis of HCC and CCA [
17,
42,
43]. TME is defined as the tumor cell population in a complex mixture of surrounding stroma, including CAFs, endothelial cells, immune cells, and ECM [
44]. Recently, it has been proposed that the premalignant microenvironment (PME) should be differentiated between the TME in HCC. PME characterized by chronic liver injury, inflammation, and fibrosis, precedes tumor development [
43]. We supposed that the HSCs belonging to the novel state may be an important component in PME and have the potential to convert into CAFs in TME. Further studies on the fate tracing of this specific state of HSC and regulation mechanisms of HSC-to-CAF transition may contribute to approach the goal of targeting myofibroblasts in the PME or TME for tumor prevention or therapy.
By GeneSwitches, we identified a lot of switching genes along the pseudotime trajectory of HSC transdifferentiation.
Xist, a long noncoding RNA, can regulate HSC activation by enhancing ethanol-induced HSC autophagy [
45]. Interestingly, other switching genes
Hmox1,
Hspb1,
Tnfrsf12a,
Cdkn1a, and
Thbs1 also have been regarded as modulators of autophagy [
46‐
50]. We assumed that autophagy in HSC may strongly contribute to HSC activation at an early stage. On the other hand, the expression of
Angptl6, a regulator of the chemotactic activity of endothelial cells and inducer of neovascularization [
51] were downregulated during HSC activation. We inferred that
Angptl6 may be related to HSC functionality in healthy liver and HSC-to-myofibroblast transition in liver injury. Moreover, GeneSwitches identified some distinct switching genes for the new state of HSC and the classic culture-activated HSC, respectively. The identification of these switching genes that are specific to each trajectory (state) along with their timings may facilitate further experiments to reveal the determinants of these bifurcations.
Using Monocle2 and GeneSwitches approaches on the HSC population resolved in pseudotime, we identified top switching genes correlated with HSC activation. Our results showed that genes mirroring HSC transdifferentiation trajectory have potential diagnostic value in the prediction of advanced hepatic fibrosis from biopsy gene expression data in patients with NAFLD or HBV. Particularly, the four-gene combination,
AEBP1,
ITGAV,
LOXL2, and
TAGLN, exhibited high accuracy in predicting fibrosis severity.
AEBP1 encoded protein plays a role in adipogenesis and smooth muscle cell differentiation. Importantly, its expression upregulates with worsening fibrosis in liver biopsies from patients with nonalcoholic steatohepatitis [
52].
LOXL2 is essential to the biogenesis of connective tissue and mediates collagen crosslinking. It was strongly expressed in fibrotic liver in mice, moreover, inhibition of
LOXL2 attenuates thioacetamide-induced hepatic fibrosis [
53]. Integrin αV, encoded by
ITGAV gene, is closely associated with fibrosis in several organs [
54].
TAGLN is an early marker of smooth muscle differentiation and regarded as an important target in anti-HBV-positive HCC [
55]. According to COMPARTMENTS Subcellular localization database (
http://compartments.jensenlab.org) [
56], we found that
AEBP1 and
LOXL2 encode secreted proteins while
ITGAV and
TAGLN are located in cytosol or extracellular. Our findings indicated that
ITGAV and
TAGLN expression were mainly in the cytoplasm of activated human HSC. These four genes have the potential to serve as predictive markers of advanced fibrosis in patients.
There are several limitations in this study. First, the results analyzed using bulk RNA-seq data of HSCs isolated from human liver showed to some extent differences from the findings obtained from scRNA-seq data of HSCs isolated from mouse liver. Besides species difference, that the sample size of HSCs isolated from human is too small may contributed to the differences in results. In the further study, we plan to obtain scRNA-seq data of HSCs isolated from human liver including quiescent and culture-activated HSC and perform analyses to validate our findings derived from mouse HSCs. Second, before using the four-gene combination (A-I-L-T) to identify advanced liver fibrosis among patients with chronic liver diseases, the serum levels of transcripts or proteins of these four genes want to be evaluated in healthy and patients.
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