Background/Aims: We performed this study to determine the role of IL-17 in the immune microenvironment of hepatitis B virus- (HBV-) related hepatocellular carcinoma (HCC). Methods: HepG2 cells were treated with IL-17, STAT3 inhibitor S31-201 or IL-6 neutralizing monoclonal antibody (IL-6 mAb). Cell proliferation and migration were compared using the Cell Counting kit-8 (CCK-8) and Transwell assays, respectively. Real-time quantitative PCR (RT-qPCR), Western Blot, ELISA, immunofluorescence and histological staining were used for determining the expression levels of IL-17, IL-6, MCP-1, CCL5, VEGF, STAT3 and p-STAT3. HCC xenograft models were constructed in wild type and IL-17 knockout mice to clarify the effects of IL-17 on HCC in vivo. Results: Exogenous IL-17 enhanced the proliferation and migration of HepG2 cells, and it activated the phosphorylation of STAT3. RT-qPCR and ELISA showed that IL-17 promoted the expression of IL-6. The CCK-8 and Transwell assays showed that S31-201 or IL-6 mAb remarkably reversed the promotion effects of proliferation and migration by exogenous IL-17 in HepG2 cells. Additionally, IL-6 could promote the phosphorylation of STAT3, while IL-6 mAb acted as an inhibitor, and exogenous IL-17 could neutralize the inhibitory effects of IL-6 mAb. In vivo, compared to the wild type mice, the tumor volume, weight, density and size were decreased in IL-17 knockout mice. Additionally, the expression levels of p-STAT3, IL-6, MCP-1, CCL5 and VEGF decreased in IL-17 knockout mice. Conclusions: IL-17 can enhance the proliferation of HepG2 cells in vitro and in vivo via activating the IL-6/STAT3 pathway. Therefore, the IL-17/IL-6/STAT3 signaling pathway is a potential therapeutic target for HBV-related HCC.

Background Circular RNAs (circRNAs) can be encapsulated into exosomes to participate in intercellular communication, affecting the malignant progression of a variety of tumors. Dysfunction of CD8 + T cells is the main factor in immune escape from hepatocellular carcinoma (HCC). Nevertheless, the effect of exosome-derived circRNAs on CD8 + T-cell dysfunction needs further exploration. Methods The effect of circCCAR1 on the tumorigenesis and metastasis of HCC was assessed by in vitro and in vivo functional experiments. The function of circCCAR1 in CD8 + T-cell dysfunction was measured by enzyme-linked immunosorbent assay (ELISA), western blotting and flow cytometry. Chromatin immunoprecipitation, biotinylated RNA pull-down, RNA immunoprecipitation, and MS2 pull-down assays were used to the exploration of mechanism. A mouse model with reconstituted human immune system components (huNSG mice) was constructed to explore the role of exosomal circCCAR1 in the resistance to anti-PD1 therapy in HCC. Results Increased circCCAR1 levels existed in tumor tissues and exosomes in the plasma of HCC patients, in the culture supernatant and HCC cells. CircCCAR1 accelerated the growth and metastasis of HCC in vitro and in vivo. E1A binding protein p300 (EP300) and eukaryotic translation initiation factor 4A3 (EIF4A3) promoted the biogenesis of circCCAR1, and Wilms tumor 1-associated protein (WTAP)-mediated m6A modification enhanced circCCAR1 stability by binding insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3). CircCCAR1 acted as a sponge for miR-127-5p to upregulate its target WTAP and a feedback loop comprising circCCAR1/miR-127-5p/WTAP axis was formed. CircCCAR1 is secreted by HCC cells in a heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1)-dependent manner. Exosomal circCCAR1 was taken in by CD8 + T cells and caused dysfunction of CD8 + T cells by stabilizing the PD-1 protein. CircCCAR1 promoted resistance to anti-PD1 immunotherapy. Furthermore, increased cell division cycle and apoptosis regulator 1 (CCAR1) induced by EP300 promoted the binding of CCAR1 and β-catenin protein, which further enhanced the transcription of PD-L1. Conclusions The circCCAR1/miR-127-5p/WTAP feedback loop enhances the growth and metastasis of HCC. Exosomal circCCAR1 released by HCC cells contributes to immunosuppression by facilitating CD8 + T-cell dysfunction in HCC. CircCCAR1 induces resistance to anti-PD1 immunotherapy, providing a potential therapeutic strategy for HCC patients.

Background: Most patients with hepatitis B virus (HBV) infection will develop hepatocellular carcinoma (HCC). This study aimed to explore the potential mechanism of miR-142-3p in HCC caused by HBV infection.Methods: HepG2 cells and M1 macrophages were cocultured and then infected with HBV to establish an in vitro model. MicroRNA (miRNA) and messenger RNA (mRNA) expression was analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. The protein expressions of COX2, ACSL4, PTGS2, GPX4, and NOX1 were analyzed by Western blot. Flow cytometry and TUNEL assays were used to assess cell reactive oxygen species (ROS) and ferroptosis, respectively. Cell invasion and migration were measured by Transwell assay. To evaluate the ferroptosis of M1-type macrophages, glutathione (GSH), malondialdehyde (MDA), and Fe 2+ content was detected by corresponding kits. Dual luciferase reporter gene detection verified the targeting relationship between miR-142-3p and SLC3A2.Results: MiR-142-3p was highly expressed in HBV-infected HCC patients and HBV-infected M1-type macrophages. Inhibition of miR-142-3p or overexpression of SLC3A2 reversed ferroptosis and inhibited the proliferation, migration, and invasion of HCC cells.Conclusions: Our findings indicated that miR-142-3p promoted HBV-infected M1-type macrophage ferroptosis through SLC3A2, affecting the production of GSH, MDA, and Fe 2+ and accelerating the development of HCC. The regulation of miR-142-3p and its target genes will help to clarify the pathogenesis of HCC induced by HBV infection and provide new theoretical foundations and therapeutic targets.

Objective. To establish a standardized animal model for liver fibrosis with the same assessment criteria for liver fibrosis studies that have been established on a unified platform. Methods. The standardized liver fibrosis model was established using Sprague-Dawley (SD) rats that either received an intraperitoneal injection of carbon tetrachloride (CCl4) in small dosages or ingested an ethanol solution. Results. The definite corresponding rules among modeling of different weeks and corresponding serology indices as well as different pathological staging can be observed by modeling with small dosages and slow, individualized, and combined administrations. Conclusion. This method can be used for the standardized establishment of a liver fibrosis model in rats across 5 pathological stages, ranging from S0 to S4, with a high success rate (89.33%) and low death rate (17.3%) because of the application of multiple hypotoxic chemicals for modeling. We refer to the criteria of Histological Grading and Staging of Chronic Hepatitis for Fibrosis established by the 10th World Digestive Disease Academic Conference in Los Angeles in September 1994 (revised in November 2000).