Aim: Cholangiocarcinoma (CCA) is a highly malignant tumor found in the bile duct epithelial cells, and the second most common primary tumor of the liver. However, the pivotal roles of molecular biomarkers in oncogenesis of CCA are unclear. Therefore, we aim to explore the underlying mechanisms of progression and screen for novel prognostic biomarkers and treatment targets.Method: The data of mRNA sequencing and clinical information of CCA patients in The Cancer Genome Atlas was analyzed by weighted gene coexpression network analysis (WGCNA). Modules and clinical traits were constructed according to Pearson's correlation analysis, and Gene Ontology and pathway enrichment analysis were applied. Hub genes of these modules were screened by intramodule analysis; Cytoscape with Search Tool for the Retrieval of Interacting Genes was utilized to visualize protein–protein interaction of these modules; hub genes of these modules were validated afterwards. Furthermore, the significance of these genes was confirmed by survival analysis.Results: Genes MRPS18A, CST1, and SCP2 were identified as candidate genes in the module, which was associated with clinical traits including pathological stage, histological grade, and liver function and which also affected overall survival of CCA patients. Nineteen hub genes were analyzed together and were associated with progression and prognosis of CCA. Survival analyses found that several of the multiple genes could serve as biomarkers to stratify CCA patients into low‐ and high‐risk groups.Conclusion: These candidate genes could be involved in progression of CCA, which could serve as novel prognostic markers and treatment targets. Moreover, most of them were first reported in CCA and deserve further research.

Background and Aims: Metabolic dysfunction–associated steatotic liver disease (MASLD) can progress to severe forms such as metabolic dysfunction–associated steatohepatitis (MASH). Effective treatments for MASH are urgently needed. This study aimed to evaluate the efficacy and safety of chiglitazar, a PPAR pan-agonist, in MASLD with hypertriglyceridemia and insulin resistance. Approach and Results: In this phase II multicenter, randomized, double-blind and placebo-controlled study, 104 patients with MASLD with hypertriglyceridemia and insulin resistance were randomized 2:2:1 to receive 48 mg, 64 mg of chiglitazar, or placebo once daily for 18 weeks. The primary endpoint was the percentage change in liver fat content measured by magnetic resonance imaging proton density fat fraction (MRI-PDFF) at week 18. Chiglitazar significantly reduced liver fat content, with percentage change from baseline at week 18 of −28.1% (95% CI −37.5 to −18.7) in the 48 mg group and −39.5% (95% CI −49.0 to −30.0) in the 64 mg group, compared with −3.2% (95% CI −16.8 to 10.4) in placebo group. The differences compared with placebo were −24.9% ( p <0.05) for the 48 mg group and −36.3% ( p <0.001) for the 64 mg group. Chiglitazar also significantly improved liver injury-related biomarkers such as ALT, AST, and γ-GT. Liver fibrosis indicators, lipid parameters, insulin resistance, and metabolic syndrome showed an improved trend. Both doses of chiglitazar were well tolerated, with most adverse events being mild to moderate. Conclusions: Chiglitazar significantly reduced liver fat content in MASLD with hypertriglyceridemia and insulin resistance, with a dose-dependent effect and a favorable safety profile.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are considered to be a potential therapy for end-stage liver disease. However, the therapeutic mechanism of BM-MSCs remains unclear. The aim of the current study was to investigate the role of paracrine signaling in BM‑MSCs in liver cirrhosis in vitro. Human BM‑MSCs and hepatic stellate cells (HSCs) were cultured using a vertical double cell co‑culture system. Groups were divided into HSCs alone (control group) and the co‑culture system of BM‑MSCs with HSCs (experimental group). HSC morphology was observed by inverted phase contrast microscopy. The proliferative capacity of HSCs was measured with the MTT assay and flow cytometry. Hoechst staining was performed to examine the apoptosis of HSCs. Transforming growth factor (TGF)‑β1 and Smad7 mRNA expression were detected by reverse transcription‑quantitative polymerase chain reaction and western blotting. BM‑MSCs did not inhibit the proliferation of HSCs at 24 h, however significantly inhibited the proliferation of HSCs at 48 and 72 h. BM‑MSCs additionally induced the apoptosis of HSCs at 48 h. The concentration of TGF‑β1 in the supernatant at 24 h and 48 h in the co‑cultured system was observed to be significantly lower than in the control group (P<0.05). The level of TGF‑β1 mRNA in the experimental group at 48 h was significantly lower than the control group, however Smad7 mRNA levels were significantly greater than in the control group. Additionally, TGF‑β1 protein levels were significantly lower than in the control group, however levels of Smad7 were greater than the control group. It was concluded that BM‑MSCs are able to inhibit the proliferation and promote the apoptosis of HSCs. In addition, the mechanism may be associated with inhibition of the TGF-β1/Smad pathway in HSCs.

It has been confirmed that the inhibitors of poly ADP-ribose polymerF(^9ase‑1 (PARP‑1) can inhibit the proliferation, apoptosis and invasion of tumor cells. However, the effects of inhibitors of PARP‑1 on hepatocellular carcinoma remain to be elucidated. The aim of the present study was to investigate the effect of three types of PARP‑1 inhibitor on the proliferation, apoptosis and migration of hepatocellular carcinoma in vitro. An MTT assay was performed to detect the proliferation of HepG2 cells following treatment with the PARP‑1 inhibitors, AG014699, BSI‑201 and AZD‑2281. Flow cytometry was used to detect the apoptosis of HepG2 cells, Western blot analysis was used to detect the protein expression of Casepase‑3, Casepase‑8, B‑cell lymphoma 2 (Bcl‑2)‑associated X protein (Bax), Bcl‑2, phosphatase and tensin homolog (PTEN), tissue inhibitor of metalloproteinase (TIMP) 3 and matrix metalloprotease (MMP) 3. A Transwell assay was performed to detect the migration of HepG2 cells. The results showed that AG014699, BSI‑201 and AZD‑2281 had an inhibitory effect on the proliferation of HepG2 cells in a time‑ and concentration‑dependent manner. AG014699 at concentrations of 10, 30 and 50 µmol/l, and BSI‑201 at concentrations of 20, 40 and 60 µmol/l induced the apoptosis of HepG2 cells, and the apoptotic rates were particularly high at 48 h (31, vs. 0.01%; P<0.01 and 24.12, vs. 0.03%, respectively; P<0.01). The protein expression levels of Caspase 3, Caspase 8, Bax, PTEN and TIMP 3 increased with increasing drug concentrations, whereas the protein levels of Bcl‑2 and MMP3 decreased with increasing drug concentrations, and were significantly different compared with those in the control group (P<0.01). In conclusion, AG014699, BSI‑201 and AZD‑2281 inhibitors of PARP‑1 significantly inhibited the proliferation of HepG2 cells, however, AG014699 and BSI‑201 demonstrated more sensitivity, induced apoptosis and inhibited migration of the hepatocellular carcinoma cells, which may be associated with alterations of the apoptosis signaling pathway and the expression of proteins associated with migration.

GLS4 is a first-in-class hepatitis B virus (HBV) capsid assembly modulator that inhibits HBV replication by interfering with assembly and disassembly of the virus nucleocapsid, this prospective, open-label, comparative, phase 2b trial evaluated the antiviral activity and safety of GLS4/ritonavir (RTV) combined with entecavir in hepatitis B e antigen-positive patients. 250 CHB patients were enrolled, including treatment-naïve patients and those interrupted anti-HBV drugs for ≥ 6 months (Part A, n=125), and patients who had taken ETV for ≥1 year and had achieved viral suppression (Part B, n=125). Patients were randomly allocated to receive 120mg GLS4/100mg RTV plus 0.5mg ETV or 0.5mg ETV monotherapy for 96 weeks. In the mid-term, in Part A (n=122), greater least-squares mean (LSM) changes from baseline were observed in the GLS4/RTV plus ETV cohort than in ETV monotherapy cohort in HBV DNA (-6.28 vs -5.72 log10 IU/ml, p=0.0005), HBsAg (-0.87 vs -0.65 log10 IU/ml, p=0.0653), HBV pgRNA (-3.83 vs -1.91 log10 copies/ml, p<0.0001); The proportions of both HBV DNA and pgRNA negative patients were 17.3% (13/75, GLS4/RTV plus ETV) and 0% (0/30, ETV monotherapy). In Part B (n=123), greater mean LSM reductions in HBsAg (-0.17 vs -0.06 log10 IU/ml, p=0.0013), HBV pgRNA (-1.61 vs -0.28 log10 copies/ml, p<0.0001) were also observed in the GLS4/RTV+ETV cohort. the proportions of both HBV DNA and pgRNA-negative patients were 71.6% (48/67, GLS4/RTV plus ETV) and 18.9% (7/37, ETV monotherapy), respectively. No patients achieved HBsAg loss at week 48. GLS4/RTV + ETV were well tolerated, the most common adverse events were elevated alanine aminotransferase levels and hypertriglyceridemia, which were reversed by temporary GLS4/RTV discontinuation. The primary analysis at week 48 showed that the antiviral efficacy of GLS4/RTV with ETV was clearly superior to that of ETV monotherapy. GLS4/RTV with ETV was well tolerated; further studies evaluating its safety and efficacy are ongoing. (clinical trial identifier: NCT04147208).