High­throughput screening identification of a small­molecule compound that induces ferroptosis and attenuates the invasion and migration of hepatocellular carcinoma cells by targeting the STAT3/GPX4 axis.

Hepatocellular carcinoma (HCC) is a lethal malignancy. Although considerable efforts have been made in recent years regarding treatments, effective therapeutic drugs for HCC remain insufficient. In the present study, polyphyllin VI was identified as a potential therapeutic drug for HCC by screening natural herbal compounds. The therapeutic effects of polyphyllin VI were assessed using Cell Counting Kit­8, lactate dehydrogenase release and colony formation assays. The occurrence of ferroptosis was determined by assessing lipid peroxidation by reactive oxygen species, malondialdehyde levels, intracellular ferrous iron levels, and the mRNA and protein levels of glutathione peroxidase 4 (GPX4). The migratory and invasive abilities of HCC cells were examined using wound healing and Transwell assays. The results revealed that polyphyllin VI inhibited the proliferation, invasion and metastasis of HCC cells (HCCLM3 and Huh7 cells) by inducing ferroptosis. In addition, through a network pharmacology­based approach and molecular docking analyses, it was found that polyphyllin VI may target the signal transducer and activator of transcription 3 (STAT3). HCC cells were treated with polyphyllin VI or a STAT3 inhibitor (Stattic), both of which exerted similar inhibitory effects on protein expression. Furthermore, immunofluorescence staining revealed that polyphyllin VI significantly inhibited the nuclear translocation of p­STAT3 in HCC cells. Mechanistically, by the overexpression of STAT3, it was confirmed that STAT3 binds to GPX4 and promotes its protein expression and transcription, whereas polyphyllin VI induces ferroptosis by inhibiting the STAT3/GPX4 axis. Subsequently, in vivo experiments revealed that polyphyllin VI inhibited the growth of subcutaneously transplanted tumors. On the whole, findings of the present study suggest that polyphyllin VI inhibits STAT3 phosphorylation, which inhibits GPX4 expression and induces the ferroptosis of HCC cells, eventually inhibiting their invasion and metastasis. These data suggest that polyphyllin VI may be a candidate for the prevention and treatment of HCC.

RNA-seq profiling reveals PBMC RNA as a potential biomarker for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and has extremely high morbidity and mortality. Although many existing studies have focused on the identification of biomarkers, little information has been uncovered regarding the PBMC RNA profile of HCC. We attempted to create a profile throughout using expression of peripheral blood mononuclear cell (PBMC) RNA using RNA-seq technology and compared the transcriptome between HCC patients and healthy controls. Seventeen patients and 17 matched healthy controls were included in this study, and PBMC RNA was sequenced from all samples. Sequencing data were analyzed using bioinformatics tools, and quantitative reverse transcription PCR (qRT-PCR) was used for selected validation of DEGs. A total of 1,578 dysregulated genes were found in the PBMC samples, including 1,334 upregulated genes and 244 downregulated genes. GO enrichment and KEGG studies revealed that HCC is closely linked to differentially expressed genes (DEGs) implicated in the immune response. Expression of 6 selected genes (SELENBP1, SLC4A1, SLC26A8, HSPA8P4, CALM1, and RPL7p24) was confirmed by qRT-PCR, and higher sensitivity and specificity were obtained by ROC analysis of the 6 genes. CALM1 was found to gradually decrease as tumors enlarged. Nearly the opposite expression modes were obtained when compared to tumor sequencing data. Immune cell populations exhibited significant differences between HCC and controls. These findings suggest a potential biomarker for the diagnosis of HCC. This study provides new perspectives for liver cancer development and possible future successful clinical diagnosis.

CircRNA expression profiling of PBMCs from patients with hepatocellular carcinoma by RNA-sequencing.

Circular RNAs (circRNAs) are differentially expressed in various cancer types. The present study aimed to investigate the expression and clinical implication of circRNAs in hepatocellular carcinoma (HCC) and to evaluate the potential of circRNAs as diagnostic biomarkers for HCC. CircRNA expression was profiled in 19 patients with HCC and 19 normal controls using ribosomal RNA-depleted RNAs. Differentially expressed circRNAs (DE-circRNAs) between HCC and controls were identified using CIRI2 and distinct circRNA expression signatures were screened. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were used to predict the potential functions of these DE-circRNAs and the circRNA-miRNA-mRNA regulatory networks were then constructed. Several DE-circRNAs were selected and confirmed by RT-qPCR. A total of 40 DE-circRNAs (27 upregulated and 13 downregulated) were identified between patients with HCC and controls. Functional annotation indicated that these DE-circRNAs were involved in cellular components, molecular functions and cancer-associated pathways related to HCC. These included pathways in cancer, TNF signaling pathway, hepatitis B, hepatitis C and hepatocyte differentiation. The circRNA-miRNA-mRNA regulatory network was generated based on 11 candidate circRNAs. Receiver operating characteristic curve analysis indicated that Homo sapiens (hsa)_circ_0073239, hsa_circ_007090, hsa_circ_0008304, hsa_circ_0017586, hsa_circ_0000369 and hsa_circ_0001181 may serve as potential biomarkers for HCC. Results from Cell Counting Kit-8 assay suggested that small interfering RNA targeting hsa_circ_0001181 reduced the proliferation of HepG2 cells, which implicated it as a potential therapeutic target for HCC. Therefore, in the present study, the differential expression pattern and important role of circRNAs in HCC were determined. The present results highlight the diagnostic potential of circRNAs in HCC and provide novel insight into the development of and treatment approaches for HCC.

Efficacy and safety of a chinese herbal formula (invigorating kidney and strengthening spleen) in chronic hepatitis B virus carrier: results from a multicenter, randomized, double-blind, and placebo-controlled trial.

A Chinese Herbal Formula (CHF) has acquired a certain therapeutic effect on chronic HBV infection. To assess the efficacy and safety of CHF on HBV replication in chronic HBV carriers, we performed a randomized, double-blind, and placebo-controlled trial involving patients from 16 centers. A total of 300 confirmed chronic HBV carriers were randomized at baseline in a ratio of 2 : 1 to receive either CHF or placebo for 52 weeks. The results showed that a greater proportion of CHF than placebo treated patients achieved virological response at week 52; the mean decline of serum HBsAg levels in the CHF group dropped more obviously than that in the control group at all stages of the treatment; however, the rates of HBeAg loss and seroconversion had no difference between the two groups. Meanwhile, were presented significant increases in IFN- γ ; IL-2 levels and reductions in IL-4 and IL-10 levels in the treatment group compared to the control group at week 52. There were no drug-related serious adverse events. In conclusion, the treatment with 52-week CHF is safe and effective in inhibiting HBV replication in chronic HBV carriers. The ability of the compound to modulate host immune function probably contributed to this effect.