Introduction
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, which accounts for 70-90% of all primary liver cancer cases worldwide [
1]. Risk factors for HCC vary by race and environment, and in Southeast Asia, about 80% of HCC cases are caused by hepatitis B virus (HBV) infection [
2]. Although intensive hepatitis B vaccination efforts and the use of nucleoside antiviral drugs in this region have contributed to the prevention of HCC, for most patients, diagnosis is made at the middle or late disease stage when progression from chronic HBV infection to HCC occurs, resulting in poor prognosis [
3]. The treatment of HCC includes surgery, chemotherapy, targeted drugs, liver transplantation, intervention and et al. Current clinical guidelines recommend comprehensive intervention, among which early diagnosis, early detection and early surgical resection are the key to improve the long-term therapeutic effect of HCC [
4]. Therefore, the research of early diagnosis biomarkers of HCC, especially the disease caused by HBVs infection, is of great significance for improving the clinical management of HCC.
Chronic HBV infection progresses to HCC via an important intermediate pathological state, liver fibrosis/cirrhosis [
5]. More than 80% of HCC occurs in fibrotic or cirrhotic livers, suggesting that liver fibrosis creates a precancerous environment in the liver, which promote HCC development [
5,
6]. Fibroblasts, as essential stromal components of HCC, play an important role in the initiation, accumulation, and progression of liver fibrosis [
6]. In addition, in HCC, fibroblasts can be transformed into cancer-associated fibroblasts (CAFs), a distinctive feature of the tumor microenvironment (TME), which has been extensively reported to modulate HCC progression [
7]. As important stromal components of TME, CAFs mediate tumor-extracellular matrix communication, and are involved in extravascular formation, immune escape, and exosome molecule secretion outside tumors [
7]. As a result, CAFs have been investigated for their potential role in HCC pathogenesis and as targets in HCC treatment [
8]. Thus, a better understanding of the unique pathological functions of CAFs in the progression of HCC will provide a basis for development of precise therapy of HCC.
Pathologically, CAFs are considered to be highly heterogeneous: genetic changes and dysregulation of gene expression in normal fibroblasts generate CAFs [
9]. Immunologically, CAFs interact with the TME network inside tumors in two ways: CAFs recruit immune cells, such as neutrophils and dendritic cells to promote immune escape and inhibit immune cells, such as B cells and T cells, to achieve immune tolerance [
10]. Currently, single cell sequencing is valuable technology for investigating the communication between cancer cells and tumor microenvironment represented by CAFs, especially CAFs’ role in gene expression and immune cell regulation [
11]. Here, using systematic data analysis, we accurately screened HCC for CAFs and grouped them into subclasses to evaluate their biological function, developmental trajectory and clinical correlation. Cell communication, signal pathway weight and key gene analysis, and in vitro experiments revealed the role of
PTN expressed in CAFs in HCC, and the regulatory role of B cells and CD8 + T cells in
PTN-mediated HBV cirrhosis-HCC progression.
Discussion
The pathogenesis of HCC is closely related to liver fibrosis caused by chronic hepatitis virus infection. Therefore, HCC mostly co-occurs with liver fibrosis or cirrhosis, suggesting that hepatic fibrosis plays an important role in the formation of precancerous lesions of the liver [
15]. CAFs is considered one of the most abundant stromal cells in HCC, that produce large amounts of collagens, leading to the formation of fibrosis and cancer development [
16]. Hence, CAF particates in the pathogenesis of HCC, especially HBV cirrhosis-HCC progression, and can thus be an ideal target for the design of agents for inhibiting liver fibrosis [
7]. We exploited the precision of single-cell sequencing in determining the cellular and molecular heterogeneity of tumor samples and integrated bioinformatics analyses to study the molecular typing, clinical features, cell communication, and signaling pathways of CAF in HCC. The study found and verified that
PTN was an important biomarker of CAF in HCC and could be an significant mediator for HBV cirrhosis-HCC progression.
With the progress of HCC treatment technology, much progress has been made in the clinical management of HCC. In particular, the recent five years of clinical application of targeted drugs for HCC have brought huge benefits to patients [
4]. However, the five-year survival rate of HCC has not been substantially improved. Factors such as tumor metastasis, recurrence and drug resistance of targeted drugs are obstacles limiting the further improvement of the diagnosis and treatment level of HCC [
4,
17]. Therefore, early detection, early diagnosis and early surgical resection are the most important methods to effectively improve the quality of life of patients [
17]. Thus, the discovery of biomarkers for the early diagnosis of HCC is still the focus of researchers [
18]. The HBV infection is the starting factor, HCC is the final result, and liver cirrhosis is the intermediate stage. Liver cirrhosis plays a bridging role in HBV fibrosis -HCC progression [
15]. Inhibiting HBV invasion and reducing the progression of liver cirrhosis may be an important means to prevent the occurrence of HCC during HBV infection [
19]. However, HBV fibrosis/cirrhosis of HCC progression lacks specific biomarkers [
18]. The
PTN found in this study may be a CAF specific biomarker and an important molecular mediator mediating HBV infection-related liver fibrosis/ cirrhosis and HCC. To further study on
PTN, specially, the research of liquid biopsy technology based on
PTN is helpful to improve the diagnosis and treatment level of HCC.
Different from second-generation sequencing, here, based on single-cell sequencing data, we used Cell cluster analysis and Cell type annotation methods to accurately mine high-purity fibroblast cells and target genes. Single-cell sequencing data can be used to infer and discover cell types in an unbiased manner, while analyzing the genome, transcriptome, and epigenome of individual cells. Therefore, we obtained fibroblasts with subset 2 and 8. The trajectory analysis further clarified that fibroblasts appear early in HCC, indicating the role of fibroblasts in HCC pathogenesis. Then, the correlation analysis between fibroblasts and second-generation sequencing clinical information was carried out using multi-system biostatistics method, which could not only overcome the small number of single cell samples and the lack of clinical data, but also avoid the deviation of second-generation sequencing data. Finally, we use Cell communication analysis based on CellChat algorithm to carry out cell communication. cells communicated weight analysis of signaling pathways, the target gene
PTN of CAF and B cells and CD8 + T cells mediating cirrhosis-HCC progression were obtained. The precise inference, systematic quantification and comparative analysis of complex cell communication networks by CellChat algorithm can be used to investigate cell communication from different molecular levels [
12]. Thus, these analyses focused on cell population-fibroblast -CAF-
PTN, which was a layer by layer analysis process of tissues-cells-genes. The above research methods provided support for excavating the regulatory genes of cirrhosis-HCC progression. However, some limitations should be identified. First, the research method lacked comprehensive verification and needed more data support. Secondly and inevitably, the method of this study was non-specific, incomplete and universal, which required the verification of biomarkers in cell and animal experiments. Thirdly, the role of
PTN in mediating CAF in cirrhosis-HCC progression need further exploration.Data has shown that CAFs regulate many wide-range of genes and transcription factors to activate signals which trigger fibroblasts to promote carcinogenesis [
16]. Previous study showed that the genes upregulated in tumor subtypes were also prominently expressed in fibroblasts, which is the main cell type in the stroma [
20]. In this study, we found novel CAFs-derived gene signatures that play an important role in the development of HCC. We found that
PTN was a novel mediator of CAF in HBV cirrhosis-HCC progression. PTN (encoding by the
PTN gene) is a small cationic protein with potent mitogenic and angiogenic activity, and has been associated with a wide range of important biological events, including tumorigenesis [
21]. Fibroblast growth factor and signaling proteins are known to increase
PTN expression level, resulting in activation of fibroblasts which then aggressively promote cancer progression [
22,
23]. Thus,
PTN may be a promising therapeutic target for cancer treatment [
24]. Previous research showed that
PTN enhances the growth of hepatocytes and its expression is increased after severe liver injury, such as partial hepatectomy and hepatitis, and promotes liver regeneration [
25]. However,
PTN has been associated with hepatic fibrosis and hepatocarcinogenesis. For example, Kohashi et al. found that
PTN expression was increased in carbon tetrachloride-induced fibrotic liver [
26]; Park et al. found that
PTN was expressed in HSCs, Kupffer cells, and hepatocytes from fibrotic liver. It inhibited the TGFb1-induced apoptosis thereby promoting liver fibrogenesis and carcinogenesis [
27]. In this study, we uncovered
PTN to be an important mediator of HBV cirrhosis-HCC progression through integrated bioinformatics analysis and validated its clinical significance in cirrhosis-HCC progression patients. Further investigations into the role of
PTN in liver fibrogenesis and carcinogenesis may lead to the development of
PTN-targeted therapies for HCC.
Here, we found strong interaction of CAF2 with B cells and CD8 + T cells, suggesting that CAF may contribute to the remodeling of immune microenvironment and the interaction of CAF2-B cells and CAF2-CD8 + T cells may mediate the regulatory role of
PTN. Close functional links between inflammation and fibrosis have been reported, which involve several key players such as B cells and T cells that contribute to liver fibrosis and HCC [
28]. Several proinflammatory mediators, including IL-1, IL-6, and TNF-α are derived from inflammatory cells such as B cells and T cells within the chronically injured liver and can contribute to hepatocarcinogenesis [
29]. These findings are consistent with our results that the ability of CAFs to modulate the immune system and the interaction between CAF2-B cells and CAF2-CD8 + T cells is mediated by
PTN. [
30] Analysis of the relationship between
PTN expression and immune of TME uncovered
PTN was associated with B cells memory and CD8 + T cells. Though CD8 + T cells contributed to anti-tumor responses, previous research found that T cells are mainly restricted to stromal zones, including fibroblasts, and CAFs may induce antigen-specific deletion of CD8 + T Cells to protect tumor cells [
31]. Goplen et al. revealed CD8 + T cells accumulate in the lungs did not have the ability to fight infection, but are involved in the development and maintenance of lung inflammation and fibrosis [
32]. We speculated that T cells CD8 + lost its protective properties in the microenvironment of liver fibrosis, or loses its anti-fibrosis function due to depletion. We also found that the interaction of
PTN-
SDC1 and
PTN-
NCL may mediate the effect of B cells and CD8 + T cells on cancer-associated fibroblast-related HCC.
PTN have been implicated in numerous inflammatory conditions such as chronic hepatitis. Hepatic stellate cells regulate the hepatic immune cells such as macrophages, B cells, T cells, and natural killer cell, by releasing a wide variety of chemokines to promote fibrogenesis [
33,
34]. Thus,
PTN may be involved in the regulation of hepatic immune system by modulating hepatic stellate cells, although further research is necessary to validate this phenomenon.
HBV infection has been linked to the development of liver fibrosis and HCC [
5]. In our study, we found that
PTN derived from CAF had a much greater effect on HBV-related HCC than non-HBV-related HCC. Transfection of HBV plasmid into non-HBV-related HCC cells significantly promoted the activity of
PTN in tumor cells. Clinical analysis also revealed that
PTN was associated with liver cirrhosis and HCC, which further indicated that
PTN may be an important target of cirrhosis and HCC induced by HBV. Although
PTN has been reported to play a role in fibrogenesis [
26] and tumorigenesis [
21], the role of
PTN in hepatitis B virus cirrhosis-HCC progression has not been sufficiently studied.
PTN is regulated by miRNA and hepatitis B virus X protein is associated with the pathogenesis of HBV-related HCC [
35,
36]. However, few studies have explored the mechanism by which
PTN contribute to fibrosis in HCC. The aim of our research was to identify novel biomarkers associated with fibrosis/cirrhosis-HCC progression due to HBV infection. Since
PTN was previously linked to the occurrence of liver fibrogenesis [
26] and tumor development [
21], we hypothesized that
PTN may promote the progression of liver fibrosis/cirrhosis to HCC. Therefore, integrated analysis of big data from clinical samples was conducted to explore the clinical value of
PTN in cirrhotic and HCC. Collectively, we found that
PTN derived from CAFs acted as a novel mediator of the CAFs’s effects on HBV cirrhosis-HCC progression.
In conclusion, this study found that PTN derived from CAFs participated in hepatocarcinogenesis by regulating fibrosis, and that PTN may mediated the effects of CAF on HBV infection associated with liver fibrosis in HCC. These findings suggest the role of PTN in HBV fibrosis/cirrhosis-HCC progression and its value into the clinical management of HBV fibrosis and HCC.
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