miR-122-3p targets UBE2I to regulate the immunosuppression of liver cancer and the intervention of Liujunzi formula.

ETHNOPHARMACOLOGICAL RELEVANCE: Liujunzi formula has been used to treat liver cancer in China for many years, but its underlying mechanism remains unclear. We previously found that decreased expression of miR-122-3p was associated with liver cancer. In this study, we aimed to explore the target of miR-122-3p and the effect of the Liujunzi formula on miR-122-3p and its downstream events in liver cancer. MATERIAL AND METHODS: Bioinformatics pinpointed potential targets of miR-122-3p. The actual target was confirmed by miRNA mimic/inhibitor transfections and a dual-luciferase reporter assay. RNA-seq looked at downstream genes impacted by this target. Flow cytometry checked for changes in T cell apoptosis levels after exposing them to liver cancer cells. Gene expression was measured by RT-qPCR, western blotting, and immunofluorescence staining. RESULTS: Cell experiments found the Liujunzi extract (LJZ) upregulated miR-122-3p and in a dose-dependent manner. Bioinformatics analysis found UBE2I was a potential target of miR-122-3p, which was validated through experiments using miRNA mimics/inhibitors and a dual-luciferase reporter assay. RNA-seq data implicated the NF-κB pathway as being downstream of the miR-122-3p/UBE2I axis, further confirmed by forcing overexpression of UBE2I. Bioinformatic evidence suggested a link between UBE2I and T cell infiltration in liver cancer. Given that the NF-κB pathway drives PD-L1 expression, which can inhibit T cell infiltration, we investigated whether PD-L1 is a downstream effector of miR-122-3p/UBE2I. This was corroborated through mining public databases, UBE2I overexpression studies, and tumor-T cell co-culture assays. In addition, we also confirmed that LJZ downregulates UBE2I and NF-κB/PD-L1 pathways through miR-122-3p. LJZ also suppressed SUMOylation in liver cancer cells and protected PD-1+ T cells from apoptosis induced by co-culture with tumor cells. Strikingly, a miR-122-3p inhibitor abrogated LJZ's effects on UBE2I and PD-L1, and UBE2I overexpression rescued the LJZ-mediated effects on NF-κB and PD-L1. CONCLUSIONS: miR-122-3p targets UBE2I, thereby suppressing the NF-κB signaling cascade and downregulating PD-L1 expression, which potentiates anti-tumor immune responses. LJZ bolsters anti-tumor immunity by modulating the miR-122-3p/UBE2I/NF-κB/PD-L1 axis in liver cancer cells.

[Bupleuri Radix-Paeoniae Radix Alba medicated plasma exerts effects on HepG2 hepatoma cells by regulating miR-1297/PTEN signaling axis].

The present study aimed to investigate the effect and mechanism of Bupleuri Radix-Paeoniae Radix Alba medicated plasma on HepG2 hepatoma cells by regulating the microRNA-1297(miR-1297)/phosphatase and tensin homologue deleted on chromosome 10(PTEN) signaling axis. Real-time quantitative PCR(RT-qPCR) was carried out to determine the mRNA levels of miR-1297 and PTEN in different hepatoma cell lines. The dual luciferase reporter assay was employed to verify the targeted interaction between miR-1297 and PTEN. The cell counting kit-8(CCK-8) was used to detect cell proliferation, and the optimal concentration and intervention time of the medicated plasma were determined. The cell invasion and migration were examined by Transwell assay and wound healing assay. Cell cycle distribution was detected by PI staining, and the apoptosis of cells was detected by Annexin V-FITC/PI double staining. The mRNA levels of miR-1297, PTEN, protein kinase B(Akt), and phosphatidylinositol 3-kinase(PI3K) were determined by RT-qPCR. Western blot was employed to determine the protein levels of PTEN, Akt, p-Akt, caspase-3, caspase-9, B-cell lymphoma-2(Bcl-2), and Bcl-2-associated X protein(Bax). The results showed that HepG2 cells were the best cell line for subsequent experiments. The dual luciferase reporter assay confirmed that miR-1297 could bind to the 3'-untranslated region(3'UTR) in the mRNA of PTEN. The medicated plasma inhibited the proliferation of HepG2 cells, and the optimal intervention concentration and time were 20% and 72 h. Compared with the blank plasma, the Bupleuri Radix-Paeoniae Radix Alba medicated plasma, miR-1297 inhibitor, miR-1297 inhibitor + medicated plasma all inhibited the proliferation, invasion, and migration of HepG2 cells, increased the proportion of cells in the G_0/G_1 phase, decreased the proportion of cells in the S phase, and increased the apoptosis rate. The medicated plasma down-regulated the mRNA levels of miR-1297, PI3K, and Akt and up-regulated the mRNA level of PTEN. In addition, it up-regulated the protein levels of PTEN, Bax, caspase-3, and caspsae-9 and down-regulated the protein levels of p-Akt, p-PI3K, and Bcl-2. In conclusion, Bupleuri Radix-Paeoniae Radix Alba medicated plasma can inhibit the expression of miR-1297 in HepG2 hepatoma cells, promote the expression of PTEN, and negatively regulate PI3K/Akt signaling pathway, thereby inhibiting the proliferation and inducing the apoptosis of HepG2 cells.

[Study on mechanism of icariin-induced ferroptosis in HepG2 hepatoma carcinoma cells through PPARG/FABP4/GPX4 pathway].

The aim of this study was to explore the mechanism of icaritin-induced ferroptosis in hepatoma HepG2 cells. By bioinformatics screening, the target of icariin's intervention in liver cancer ferroptosis was selected, the protein-protein interaction(PPI) network was constructed, the related pathways were focused, the binding ability of icariin and target protein was evaluated by molecular docking, and the impact on patients' survival prognosis was predicted and the clinical prediction model was built. CCK-8, EdU, and clonal formation assays were used to detect cell viability and cell proliferation; colorimetric method and BODIPY 581/591 C1 fluorescent probe were used to detect the levels of Fe~(2+), MDA and GSH in cells, and the ability of icariin to induce HCC cell ferroptosis was evaluated; RT-qPCR and Western blot detection were used to verify the mRNA and protein levels of GPX4, xCT, PPARG, and FABP4 to determine the expression changes of these ferroptosis-related genes in response to icariin. Six intervention targets(AR, AURKA, PPARG, AKR1C3, ALB, NQO1) identified through bioinformatic analysis were used to establish a risk scoring system that aids in estimating the survival prognosis of HCC patients. In conjunction with patient age and TNM staging, a comprehensive Nomogram clinical prediction model was developed to forecast the 1-, 3-, and 5-year survival of HCC patients. Experimental results revealed that icariin effectively inhibited the activity and proliferation of HCC cells HepG2, significantly modulating levels of Fe~(2+), MDA, and lipid peroxidation ROS while reducing GSH levels, hence revealing its potential to induce ferroptosis in HCC cells. Icariin was found to diminish the expression of GPX4 and xCT(P<0.01), inducing ferroptosis in HCC cells, potentially in relation to inhibition of PPARG and FABP4(P<0.01). In summary, icariin induces ferroptosis in HCC cells via the PPARG/FABP4/GPX4 pathway, providing an experimental foundation for utilizing the traditional Chinese medicine icariin in the prevention or treatment of HCC.

[Baicalin induces ferroptosis in HepG2 cells by inhibiting ROS-mediated PI3K/Akt/FoxO3a signaling pathway].

This study aims to investigate whether baicalin induces ferroptosis in HepG2 cells and decipher the underlying mechanisms based on network pharmacology and cell experiments. HepG2 cells were cultured in vitro and the cell viability was detected by the cell counting kit-8(CCK-8). The transcriptome data of hepatocellular carcinoma were obtained from the Cancer Genome Atlas(TCGA), and the ferroptosis gene data from FerrDb V2. The DEG2 package was used to screen the differentially expressed genes(DEGs), and the common genes between DEGs and ferroptosis genes were selected as the target genes that mediate ferroptosis to regulate hepatocellular carcinoma progression. The functions and structures of the target genes were analyzed by Gene Ontology(GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment with the thresholds of P<0.05 and |log_2(fold change)|>0.5. DCFH-DA probe was used to detect the changes in the levels of cellular reactive oxygen species(ROS) in each group. The reduced glutathione(GSH) assay kit was used to measure the cellular GSH level, and Fe~(2+) assay kit to determine the Fe~(2+) level. Real-time quantitative PCR(RT-PCR) was employed to measure the mRNA levels of glutathione peroxidase 4(GPX4) and solute carrier family 7 member 11(SLC7A11) in each group. Western blot was employed to determine the protein levels of GPX4, SLC7A11, phosphatidylinositol 3-kinase(PI3K), p-PI3K, protein kinase B(Akt), p-Akt, forkhead box protein O3a(FoxO3a), and p-FoxO3a in each group. The results showed that treatment with 200 µmol·L~(-1) baicalin for 48 h significantly inhibited the viability of HepG2 cells. Ferroptosis in hepatocellular carcinoma could be regulated via the PI3K/Akt signaling pathway. The cell experiments showed that baicalin down-regulated the expression of SLC7A11 and GPX4, lowered the GSH level, and increased ROS accumulation and Fe~(2+) production in HepG2 cells. However, ferrostatin-1, an ferroptosis inhibitor, reduced baicalin-induced ROS accumulation, up-regulated the expression of SLC7A11 and GPX4, elevated the GSH level, and decreased PI3K, Akt, and FoxO3a phosphorylation. In summary, baicalin can induce ferroptosis in HepG2 cells by inhibiting the ROS-mediated PI3K/Akt/FoxO3a pathway.

Sporoderm-broken spores of Ganoderma lucidum modulate hepatoblastoma malignancy by regulating RACK1-mediated autophagy and tumour immunity.

Hepatoblastoma (HB), a primary liver tumour, is notorious for its high metastatic potential and poor prognosis. Ganoderma lucidum, an edible mushroom species utilized in traditional Chinese medicine for addressing various tumour types, presents an intriguing avenue for HB treatment. However, the effectiveness of G. lucidum in managing HB and its underlying molecular mechanism necessitates further exploration. Standard in vitro assays were conducted to evaluate the impact of sporoderm-broken spores of G. lucidum (SBSGL) on the malignant characteristics of HB cells. The mechanism of SBSGL in treating HB and its tumour immunomodulatory effects were explored and validated by various experiments, including immunoprecipitation, Western blotting, mRFP-GFP-LC3 adenovirus transfection and co-localization analysis, as well as verified with in vivo experiments in this regard. The results showed that SBSGL effectively inhibited the malignant traits of HB cells and suppressed the O-GlcNAcylation of RACK1, thereby reducing its expression. In addition, SBSGL inhibited immune checkpoints and regulated cytokines. In conclusion, SBSGL had immunomodulatory effects and regulated the malignancy and autophagy of HB by regulating the O-GlcNAcylation of RACK1. These findings suggest that SBSGL holds promise as a potential anticancer drug for HB treatment.

A Strategy based on Bioinformatics and Machine Learning Algorithms Reveals Potential Mechanisms of Shelian Capsule against Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a prevalent and life-threatening form of cancer, with Shelian Capsule (SLC), a traditional Chinese medicine (TCM) formulation, being recommended for clinical treatment. However, the mechanisms underlying its efficacy remain elusive. This study sought to uncover the potential mechanisms of SLC in HCC treatment using bioinformatics methods. METHODS: Bioactive components of SLC were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and HCC-related microarray chip data were sourced from the Gene Expression Omnibus (GEO) database. The selection criteria for components included OB ≧ 30% and DL ≧ 0.18. By integrating the results of differential expression analysis and weighted gene co-expression network analysis (WGCNA), disease-related genes were identified. Therapeutic targets were determined as shared items between candidate targets and disease genes. Protein-protein interaction (PPI) network analysis was conducted for concatenated genes, with core protein clusters identified using the MCODE plugin. Machine learning algorithms were applied to identify signature genes within therapeutic targets. Subsequently, immune cell infiltration analysis, single-cell RNA sequencing (sc-RNA seq) analysis, molecular docking, and ADME analysis were performed for the screened genes. RESULTS: A total of 153 SLC ingredients and 170 candidate targets were identified, along with 494 HCCrelated disease genes. Overlapping items between disease genes and drug candidates represented therapeutic genes, and PPI network analysis was conducted using concatenated genes. MCODE1 and MCODE2 cluster genes underwent Disease Ontology (DO), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Four signature genes (TOP2A, CYP1A2, CYP2B6, and IGFBP3) were identified from 28 therapeutic genes using 3 machine learning algorithms, with ROC curves plotted. Molecular docking validated the interaction modes and binding abilities between signature genes and corresponding compounds, with free binding energy all <-7 kcal/mol. Finally, ADME analysis revealed similarities between certain SLC components and the clinical drugs Sorafenib and Lenvatinib. CONCLUSION: In summary, our study revealed that the mechanism underlying the anti-HCC effects of SLC involves interactions at three levels: components (quercetin, beta-sitosterol, kaempferol, baicalein, stigmasterol, and luteolin), pathways (PI3K-Akt signaling pathway, TNF signaling pathway, and IL-17 signaling pathway), and targets (TOP2A, CYP1A2, CYP2B6, and IGFBP3). This study provides preliminary insights into the potential pharmacological mechanisms of SLC in HCC treatment, aiming to support its clinical application and serve as a reference for future laboratory investigations.

A codelivery system loaded with PDL1-siRNA and sorafenib enhances the therapeutic effect of sorafenib on hepatocellular carcinoma via TAT-poly-SS-lysine modified chitosan.

Sorafenib (SF) is a first-line drug for the treatment of hepatocellular carcinoma (HCC) in clinical practice. However, acquired drug resistance tremendously limits the clinical efficacy of sorafenib in treating HCC, which has attracted great attention. PDL1 plays a crucial role in the drug resistance of HCC. Here, a codelivery system based on poly-SS-lysine modified chitosan (TAT-C-SS-P) was established and was applied to deliver sorafenib and PDL1-siRNA for synergetic HCC therapy. The successful synthesis of TAT-C-SS-P was confirmed by 1H NMR. Additionally, sorafenib and PDL1-siRNA were successfully transported into the cells as the decreased expression of VEGF and PD-L1 by administrated with TAT-C-SS-P@SF@ PDL1-siRNA. Simultaneously, the expression of pro-apoptosis proteins cyt-c and Bax was prominently augmented, whereas the expression of anti-apoptosis protein Bcl-2 was decreased. The reduced expression of PDL1 resulted in the downregulation of P-GP and MRP1, which contributed to more sorafenib aggregation in tumor cells. Moreover, TAT-C-SS-P@PDL1-siRNA@SF efficiently promotes apoptosis of HepG2-SI cells, as the apoptosis rate rised to 73 %. A sorafenib-insensitive model was established to evaluate in vivo antitumor effect of TAT-C-SS-P@PDL1-siRNA@SF. TAT-C-SS-P@PDL1-siRNA@SF showed a tumor inhibition rate of 90.2 ± 3.5 % and no significant decrease in body weight. Taken together, our study provided compelling evidence that TAT-C-SS-P@PDL1-siRNA@SF has great potential application in the treatment of HCC clinically.

[Trametenolic acid inhibits migration and invasion of hepatocellular carcinoma HepG2.2.15 cells via RhoC/ROCK1 pathway].

This study investigated the effect of trametenolic acid(TA) on the migration and invasion of human hepatocellular carcinoma HepG2.2.15 cells by using Ras homolog gene family member C(RhoC) as the target and probed into the mechanism, aiming to provide a basis for the utilization of TA. The methyl thiazolyl tetrazolium(MTT) assay was employed to examine the proliferation of HepG2.2.15 cells exposed to TA, and scratch and Transwell assays to examine the cell migration and invasion. The pull down assay was employed to determine the impact of TA on RhoC GTPase activity. Western blot was employed to measure the effect of TA on the transport of RhoC from cytoplasm to cell membrane and the expression of RhoC/Rho-associated kinase 1(ROCK1)/myosin light chain(MLC)/matrix metalloprotease 2(MMP2)/MMP9 pathway-related proteins. RhoC was over-expressed by transient transfection of pcDNA3.1-RhoC. The changes of F-actin in the cytoskeleton were detected by Laser confocal microscopy. In addition, the changes of cell migration and invasion, expression of proteins in the RhoC/ROCK1/MLC/MMP2/MMP9 pathway, and RhoC GTPase activity were detected. The subcutaneously transplanted tumor model of BALB/c nude mice and the low-, medium-, and high-dose(40, 80, and 120 mg·kg~(-1), respectively) TA groups were established and sorafenib(20 mg·kg~(-1)) was used as the positive control. The tumor volume and weight in each group were measured, and the expression of related proteins in the tumor tissue was determined by Western blot. The results showed that TA inhibited the proliferation of HepG2.2.15 cells in a concentration-dependent manner, with the IC_(50) of 66.65 and 23.09 µmol·L~(-1) at the time points of 24 and 48 h, respectively. The drug administration groups had small tumors with low mass. The tumor inhibition rates of sorafenib and low-, medium-and high-dose TA were 62.23%, 26.48%, 55.45%, and 62.36%, respectively. TA reduced migrating and invading cells and inhibited RhoC protein expression and RhoC GTPase activity in a concentration-dependent manner, dramatically reducing RhoC and membrane-bound RhoC GTPase. The expression of ROCK1, MLC, p-MLC, MMP2, and MMP9 downstream of RhoC can be significantly inhibited by TA, as confirmed in both in vitro and in vivo experiments. After HepG2.2.15 cells were transfected with pcDNA3.1-RhoC to overexpress RhoC, TA down-regulated the protein levels of RhoC, ROCK1, MLC, p-MLC, MMP2, and MMP9 and decreased the activity of RhoC GTPase, with the inhibition level comparable to that before overexpression. In summary, TA can inhibit the migration and invasion of HepG2.2.15 cells. It can inhibit the RhoC/ROCK1/MLC/MMP2/MMP9 signaling pathway by suppressing RhoC GTPase activity and down-regulating RhoC expression. This study provides a new idea for the development of autophagy modulators targeting HSP90α to block the proliferation and inhibit the invasion and migration of hepatocellular carcinoma cells via multiple targets of active components in traditional Chinese medicines.

N6-Methyladenosine Reader YTHDF1 Promotes Stemness and Therapeutic Resistance in Hepatocellular Carcinoma by Enhancing NOTCH1 Expression.

N6-methyladenosine (m6A) RNA modification is the most common and conserved epigenetic modification in mRNA and has been shown to play important roles in cancer biology. As the m6A reader YTHDF1 has been reported to promote progression of hepatocellular carcinoma (HCC), it represents a potential therapeutic target. In this study, we evaluated the clinical significance of YTHDF1 using human HCC samples and found that YTHDF1 was significantly upregulated in HCCs with high stemness scores and was positively associated with recurrence and poor prognosis. Analysis of HCC spheroids revealed that YTHDF1 was highly expressed in liver cancer stem cells (CSC). Stem cell-specific conditional Ythdf1 knockin (CKI) mice treated with diethylnitrosamine showed elevated tumor burden as compared with wild-type mice. YTHDF1 promoted CSCs renewal and resistance to the multiple tyrosine kinase inhibitors lenvatinib and sorafenib in patient-derived organoids and HCC cell lines, which could be abolished by catalytically inactive mutant YTHDF1. Multiomic analysis, including RNA immunoprecipitation sequencing, m6A methylated RNA immunoprecipitation sequencing, ribosome profiling, and RNA sequencing identified NOTCH1 as a direct downstream of YTHDF1. YTHDF1 bound to m6A modified NOTCH1 mRNA to enhance its stability and translation, which led to increased NOTCH1 target genes expression. NOTCH1 overexpression rescued HCC stemness in YTHDF1-deficient cells in vitro and in vivo. Lipid nanoparticles targeting YTHDF1 significantly enhanced the efficacy of lenvatinib and sorafenib in HCC in vivo. Taken together, YTHDF1 drives HCC stemness and drug resistance through an YTHDF1-m6A-NOTCH1 epitranscriptomic axis, and YTHDF1 is a potential therapeutic target for treating HCC. SIGNIFICANCE: Inhibition of YTHDF1 expression suppresses stemness of hepatocellular carcinoma cells and enhances sensitivity to targeted therapies, indicating that targeting YTHDF1 may be a promising therapeutic strategy for liver cancer.

Combinatorial Gene Expression Profiling of Serum HULC, HOTAIR, and UCA1 lncRNAs to Differentiate Hepatocellular Carcinoma from Liver Diseases: A Systematic Review and Meta-Analysis.

Hepatocellular carcinoma (HCC) presents a significant global health challenge due to limited early detection methods, primarily relying on conventional approaches like imaging and alpha-fetoprotein (AFP). Although non-coding RNAs (ncRNAs) show promise as potential biomarkers in HCC, their true utility remains uncertain. We conducted a comprehensive review of 76 articles, analyzing 88 circulating lncRNAs in 6426 HCC patients. However, the lack of a standardized workflow protocol has hampered holistic comparisons across the literature. Consequently, we herein confined our meta-analysis to only a subset of these lncRNAs. The combined analysis of serum highly upregulated in liver cancer (HULC) gene expression with homeobox transcript antisense intergenic RNA (HOTAIR) and urothelial carcinoma-associated 1 (UCA1) demonstrated markedly enhanced sensitivity and specificity in diagnostic capability compared to traditional biomarkers or other ncRNAs. These findings could have substantial implications for the early diagnosis and tailored treatment of HCC.

Comprehensive Cancer Genomic Profiling of Liver Metastasis Led to the Unexpected Identification of Colorectal Cancer.

Comprehensive genomic profiling (CGP) of a metastatic liver tumor biopsy specimen suggested that the patient, who was initially diagnosed with cholangiocarcinoma, had colorectal cancer. The identification of both FBXW7 and APC mutations is deemed characteristic of colorectal cancer. Indeed, subsequent colonoscopy revealed sigmoid colon carcinoma that led to tumor resection followed by systemic chemotherapy. CGP is principally used to identify agents that might potentially benefit the patient. However, results must be interpreted carefully to ensure consistency with the initial diagnosis.

Nrf2 knockout attenuates the astragaloside IV therapeutic effect on kidney fibrosis from liver cancer by regulating pSmad3C/3L pathways.

Fibrotic kidney injury from hepatocarcinogenesis seriously impacts treatment effect. Astragaloside IV (AS-IV), an extract of Astragalus membranaceus, has several pharmacological activities, which are useful in the treatment of edema and fibrosis. Nrf2/HO-1 is a key antioxidant stress pathway and help treatment of kidney injury. Smad3 phosphorylation is implicated in hepatocarcinogenesis. Our previous study clarified that Smad3 is differentially regulated by different phosphorylated forms of Smad3 on hepatocarcinogenesis. Therefore, we investigated the contribution of AS-IV on the therapy of kidney fibrosis from hepatocarcinogenesis. And the focus was on whether the phosphorylation of Smad3 and the regulation of Nrf2/HO-1 pathway were involved during AS-IV therapy and whether there is an effect of Nrf2 knockout on the phosphorylation of Smad3. We performed TGF-ß1 stimulation on HK-2 cells and intervened with AS-IV. Furtherly, we investigated renal injury of AS-IV on Nrf2 knockout mice during hepatocarcinogenesis and its mechanism of action. On the one hand, in vitro results showed that AS-IV reduced the ROS and α-SMA expression of HK-2 by promoting the expression pSmad3C/p21 of and Nrf2/HO-1 and suppressed the expression of pSmad3L/PAI-1. On the other hand, the in vivo results of histopathological features, serological biomarkers, and oxidative damage indicators showed that Nrf2 knockout aggravated renal injury. Besides, Nrf2 deletion decreased the nephroprotective effect of AS-IV by suppressing the pSmad3C/p21 pathway and promoting the pSmad3L/PAI-1 pathway. The experimental results were as we suspected. And we identify for the first time that Nrf2 deficiency increases renal fibrosis from hepatocarcinogenesis and attenuates the therapeutic effects of AS-IV via regulating pSmad3C/3L signal pathway.

Multiomics identifies metabolic subtypes based on fatty acid degradation allocating personalized treatment in hepatocellular carcinoma.

BACKGROUND AND AIMS: Molecular classification is a promising tool for prognosis prediction and optimizing precision therapy for HCC. Here, we aimed to develop a molecular classification of HCC based on the fatty acid degradation (FAD) pathway, fully characterize it, and evaluate its ability in guiding personalized therapy. APPROACH AND RESULTS: We performed RNA sequencing (RNA-seq), PCR-array, lipidomics, metabolomics, and proteomics analysis of 41 patients with HCC, in which 17 patients received anti-programmed cell death-1 (PD-1) therapy. Single-cell RNA sequencing (scRNA-seq) was performed to explore the tumor microenvironment. Nearly, 60 publicly available multiomics data sets were analyzed. The associations between FAD subtypes and response to sorafenib, transarterial chemoembolization (TACE), immune checkpoint inhibitor (ICI) were assessed in patient cohorts, patient-derived xenograft (PDX), and spontaneous mouse model ls. A novel molecular classification named F subtype (F1, F2, and F3) was identified based on the FAD pathway, distinguished by clinical, mutational, epigenetic, metabolic, and immunological characteristics. F1 subtypes exhibited high infiltration with immunosuppressive microenvironment. Subtype-specific therapeutic strategies were identified, in which F1 subtypes with the lowest FAD activities represent responders to compounds YM-155 and Alisertib, sorafenib, anti-PD1, anti-PD-L1, and atezolizumab plus bevacizumab (T + A) treatment, while F3 subtypes with the highest FAD activities are responders to TACE. F2 subtypes, the intermediate status between F1 and F3, are potential responders to T + A combinations. We provide preliminary evidence that the FAD subtypes can be diagnosed based on liquid biopsies. CONCLUSIONS: We identified 3 FAD subtypes with unique clinical and biological characteristics, which could optimize individual cancer patient therapy and help clinical decision-making.

Identification of formononetin as the active compound of CR-SR in hepatocellular carcinoma treatment: An integrated approach combining network pharmacology and weighted gene co-expression networks.

Hepatocellular carcinoma (HCC) is a life-threatening disease for which there is no cure. Traditional Chinese medicine is a treasure trove of Medicinals that has been used for thousands of years. In China, the traditional herb pair, Curcumae Rhizoma and Sparganii Rhizoma (CR-SR) represent a classic herbal combination used for the treatment of HCC. However, the drug targets and pharmacological mechanism of action of CR-SR in the treatment of HCC are unclear. To address this, we screened the active components and drug targets of CR-SR from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and a high-throughput experiment- and reference-guided database of traditional Chinese medicines (HERB database). Combined with the weighted co-expression network analysis of dataset GSE76427, we constructed an active component-target-disease regulatory network. It was found that CR-SR's active components for HCC treatment included trans-gondoic acid, beta-sitosterol, stigmasterol, hederagenin, and formononetin. These compounds specifically targeted the genes Estrogen Receptor 1 (ESR1), Cyclin A2 (CCNA2), Checkpoint Kinase 1 (CHEK1), and Nuclear Receptor Coactivator 2 (NCOA2). ESR1, CCNA2, and CHEK1 genes showed significant differences in survival prognosis, expression levels, and statistical significance during the pathological stage. Moreover, their high affinity for formononetin was determined through molecular docking analysis. Cell assays and high-throughput sequencing were performed to reveal that the inhibitory effect of formononetin on HepG2 cell proliferation was related to hepatocyte metabolism and cell cycle regulation-related pathways. This study provides insights into potential HCC treatments.

Andrographolide anti-proliferation and metastasis of hepatocellular carcinoma through LncRNA MIR22HG regulation.

Hepatocellular carcinoma (HCC) treatment is a major challenge. Although andrographolide (Andro) has an anti-proliferation effect on HCC, its underlying mechanism is not yet elucidated, and whether Andro can inhibit HCC metastasis has not been reported. The present study aimed to clarify whether Andro inhibits SK-Hep-1 cell proliferation and HCC metastasis, and the mechanisms. The results showed that Andro significantly reduced the survival of HCC cells and tumor weight and volume in tumor-bearing nude mice. Andro also triggered apoptosis of HCC cells and upregulated MIR22HG, Cleaved Caspase 9/7/3 expression levels, and downregulated BCL-2 mRNA, BCL-2 expression levels. Knockdown of MIR22HG or overexpression of HuR attenuated the effects of Andro on the signal transduction of mitochondrial apoptotic pathway and proliferation ability in HCC cells. Moreover, Andro significantly reduced the invasive ability of the cells and the level of HCC cell lung metastasis, upregulated miR-22-3p expression level and downregulated HMGB1 and MMP-9 expression levels. MIR22HG or miR-22-3p knockdown attenuated the effects of Andro on the signaling of HMGB1/MMP-9 pathway and invasive ability in HCC cells, while the overexpression of HMGB1 attenuated the inhibitory effects of Andro on the MMP-9 expression level and invasive ability in HCC cells. Thus, the regulation of MIR22HG-HuR/BCL-2 and MIR22HG/HMGB1 signaling pathways is involved in the anti-HCC proliferation and metastasis effects of Andro. This study provided a new pharmacological basis for Andro in HCC treatment and, for the first time, identified a natural product molecule capable of positively regulating MIR22HG, which has a robust biological function.

Artemisia carvifolia Buch silver nanoparticles downregulate the Rap2A gene in liver cancer.

Liver cancer is the second main reason of death globally. In the current study, Rap2A protein a member of Ras Gtpase was selected as a drug target for liver cancer which has been identified as an oncogene in different types of tumors. The present study aimed to evaluate Artemisia carvifolia Buch extract and its silver nanoparticles against liver cancer targeting the Rap2A gene. The synthesized silver nanoparticles showed an absorbance peak at 450 nm by a UV-Vis spectrophotometer. SEM revealed that polyhedral silver nanoparticles had a size ranging from 80 ± 6 nm. Furthermore, amines, aldehydes, ketones and alcohols of Artemisia carvifolia were found involved in the reduction and stabilization of nanoparticles by FTIR. Moreover, XRD and EDX confirmed the cubic crystalline nature and particle elemental composition, respectively. Furthermore, the cytotoxicity against HePG2 cancer cell lines was also found significant with an IC50 value of 2.57 µM for silver nanoparticles and 11.57 µM for plant extract. The gene expression and protein level of Rap2A were also decreased in plant extract and nanoparticle-treated cells compared to control groups. The apoptotic potential of extract and nanoparticles was also determined by evaluating the apoptotic pathway genes and protein including BAX, caspase 3, 8 and 9. Significantly elevated levels of expression of these genes by real-time qPCR along with increased protein levels by ELISA were found. This is the first-ever report describing the synthesis and efficacy of silver nanoparticles of Artemisia carvifolia Buch against liver cancer.

URI alleviates tyrosine kinase inhibitors-induced ferroptosis by reprogramming lipid metabolism in p53 wild-type liver cancers.

The clinical benefit of tyrosine kinase inhibitors (TKIs)-based systemic therapy for advanced hepatocellular carcinoma (HCC) is limited due to drug resistance. Here, we uncover that lipid metabolism reprogramming mediated by unconventional prefoldin RPB5 interactor (URI) endows HCC with resistance to TKIs-induced ferroptosis. Mechanistically, URI directly interacts with TRIM28 and promotes p53 ubiquitination and degradation in a TRIM28-MDM2 dependent manner. Importantly, p53 binds to the promoter of stearoyl-CoA desaturase 1 (SCD1) and represses its transcription. High expression of URI is correlated with high level of SCD1 and their synergetic expression predicts poor prognosis and TKIs resistance in HCC. The combination of SCD1 inhibitor aramchol and deuterated sorafenib derivative donafenib displays promising anti-tumor effects in p53-wild type HCC patient-derived organoids and xenografted tumors. This combination therapy has potential clinical benefits for the patients with advanced HCC who have wild-type p53 and high levels of URI/SCD1.

[Inhibitory effect and molecular mechanism of sinomenine on human hepatocellular carcinoma HepG2 and SK-HEP-1 cells].

This study aimed to investigate the effect and molecular mechanism of sinomenine on proliferation, apoptosis, metastasis, and combination with inhibitors in human hepatocellular carcinoma HepG2 cells and SK-HEP-1 cells. The effect of sinomenine on the growth ability of HepG2 and SK-HEP-1 cells were investigated by CCK-8 assay, colony formation assay, and BeyoClick~(TM) EdU-488 staining. The effect of sinomenine on DNA damage was detected by immunofluorescence assay, and the effect of sinomenine on apoptosis of human hepatocellular carcinoma cells was clarified by Hoechst 33258 staining and CellEvent~(TM) Cystein-3/7Green ReadyProbes~(TM) reagent assay. Cell invasion assay and 3D tumor cell spheroid invasion assay were performed to investigate the effect of sinomenine on the invasion ability of human hepatocellular carcinoma cells in vitro. The effect of sinomenine on the regulation of protein expression related to the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription 3(STAT3) signaling pathway in HepG2 and SK-HEP-1 cells was examined by Western blot. Molecular docking was used to evaluate the strength of affinity of sinomenine to the target cysteinyl aspartate specific proteinase-3(caspase-3) and STAT3, and combined with CCK-8 assay to detect the changes in cell viability after combination with STAT3 inhibitor JSI-124 in combination with CCK-8 assay. The results showed that sinomenine could significantly reduce the cell viability of human hepatocellular carcinoma cells in a concentration-and time-dependent manner, significantly inhibit the clonogenic ability of human hepatocellular carcinoma cells, and weaken the invasive ability of human hepatocellular carcinoma cells in vitro. In addition, sinomenine could up-regulate the cleaved level of poly ADP-ribose polymerase(PARP), a marker of apoptosis, and down-regulate the protein levels of p-Akt, p-mTOR, and p-STAT3 in human hepatocellular carcinoma cells. Molecular docking results showed that sinomenine had good affinity with the targets caspase-3 and STAT3, and the sensitivity of sinomenine to hepatocellular carcinoma cells was diminished after STAT3 was inhibited. Therefore, sinomenine can inhibit the proliferation and invasion of human hepatocellular carcinoma cells and induce apoptosis, and the mechanism may be attributed to the activation of caspase-3 signaling and inhibition of the Akt/mTOR/STAT3 pathway. This study can provide a new reference for the in-depth research and clinical application of sinomenine and is of great significance to further promote the scientific development and utilization of sinomenine.

FANCD2 as a novel prognostic biomarker correlated with immune and drug therapy in Hepatitis B-related hepatocellular carcinoma.

BACKGROUND: Ferroptosis is related to the immunosuppression of tumors and plays a critical role in cancer progression. Fanconi anemia complementation group D2 (FANCD2) is a vital gene that regulates ferroptosis. However, the mechanism of action of FANCD2 in Hepatitis B-related hepatocellular carcinoma (HCC) remains unknown. In this study, we investigated the prognostic significance and mechanism of action of FANCD2 in Hepatitis B-related HCC. METHODS: The expression of FANCD2 in Hepatitis B-related HCC was explored using The Cancer Genome Atlas (TCGA) and validated using the Gene Expression Omnibus (GEO) database. Univariate and multivariate Cox regression analyses and Kaplan-Meier survival curves were used to analyze the relationship between FANCD2 expression and the overall survival of patients with Hepatitis B-related HCC. Protein-protein interaction networks for FANCD2 were built using the STRING website. In addition, correlations between FANCD2 expression and the dryness index, tumor mutational burden, microsatellite instability (MSI), immune pathways, genes involved in iron metabolism, and sorafenib chemotherapeutic response were analyzed. RESULTS: Our results indicated that FANCD2 was significantly overexpressed in Hepatitis B-related HCC and demonstrated a strong predictive ability for diagnosis (Area Under Curve, 0.903) and prognosis of the disease. High FANCD2 expression was associated with poor prognosis, high-grade tumors, high expression of PDL-1, high MSI scores, and low sorafenib IC50 in Hepatitis B-related HCC. BRCA1, BRCA2, FAN1, and FANCC were vital proteins interacting with FANCD2. The expression level of FANCD2 significantly correlated with the infiltration levels of Treg cells, B cells, CD8 + T cells, CD4 + T cells, neutrophils, macrophages, myeloid dendritic cells, and NK cells in Hepatitis B-related HCC. FANCD2 was positively correlated with the tumor proliferation signature pathway, DNA repair, and cellular response to hypoxia. CONCLUSION: Our study indicated that FANCD2 was a potential novel biomarker and immunotherapeutic target against Hepatitis B-related HCC, which might be related to the chemotherapeutic response to sorafenib.

Sequencing Systemic Therapy in Hepatocellular Carcinoma.

OPINION STATEMENT: Multiple treatment options are now approved for unresectable hepatocellular carcinoma (HCC). An immune checkpoint inhibitor (ICI)-containing regimen should be highly considered as the first-line treatment when there is no contraindication, especially in those with hepatitis virus-related HCC, due to proven superior overall survival (OS) compared to sorafenib. Atezolizumab plus bevacizumab and durvalumab plus tremelimumab remain the treatment of choice among all ICI-containing regimens, unless contraindications to either of the medications exist. Although sorafenib is still the only medication currently approved for select patients with Child-Pugh B (CP) HCC in the first-line setting, atezolizumab plus bevacizumab is being studied in this patient population. Moreover, patients with post-liver transplantation recurrence may benefit from tyrosine kinase inhibitors (TKIs), while more studies are still needed to determine the safety of ICIs in this setting. Interestingly, multiple potential biomarkers, including tumor mutational burden (TMB), microsatellite instability (MSI) status, and PD-L1 expression level, have inconsistently predicted response to ICIs in patients with HCC. Limited evidence is available to guide treatment choice in later-line settings after progressing on ICIs, and decisions should be based on the safety profile of the treatment regimen and patient preference. Multiple trials are ongoing to elucidate the optimal treatment sequence. Of note, we believe that TKIs (e.g., cabozantinib, regorafenib, lenvatinib, and sorafenib) could be more beneficial in later-line settings to broaden inhibition of other pathways apart from vascular endothelial growth factor (VEGF). When conventional treatment options are exhausted, tissue biopsy may be helpful to reveal rare targetable mutations, such as RET gene fusions.