Functional genetic variants of the disulfidptosis-related INF2 gene predict survival of hepatitis B virus-related hepatocellular carcinoma.

Disulfidptosis is a novel form of programmed cell death involved in migration and invasion of cancer cells, but few studies investigated the roles of genetic variants in disulfidptosis-related genes in survival of patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). We used Cox proportional hazards regression analyses, Kaplan-Meier curves and receiver operating characteristic curves to assess effects of genetic variants in 14 disulfidptosis-related genes on overall survival of 866 HBV-HCC patients. The Bayesian false discovery probability was used for multiple testing corrections. We also investigated biological mechanisms of the significant variants through expression quantitative trait loci analyses using the data from publicly available databases, luciferase reporter assays and differential expression analyses. As a result, we identified two independently functional single nucleotide polymorphisms (SNPs) (INF2 rs4072285 G > A and INF2 rs4444271 A > T) that predicted overall survival of HBV-HCC patients, with adjusted hazard ratios of 1.60 (95% CI = 1.22-2.11, P = 0.001) and 1.50 (95% CI = 1.80-1.90, P < 0.001), respectively, after multiple testing correction. Luciferase reporter assays indicated that both INF2 rs4072285 A and INF2 rs4444271 T alleles increased INF2 mRNA expression levels (P < 0.001) that were also higher in HCC tumor tissues than in adjacent normal tissues (P < 0.001); such elevated INF2 expression levels were associated with a poorer survival of HBV-HCC patients (P < 0.001) in the TCGA database. In summary, this study supported that INF2 rs4072285 and INF2 rs4444271 may be novel biomarkers for survival of HBV-HCC patients.

Transcriptome sequencing and metabolome analysis reveal the metabolic reprogramming of partial hepatectomy and extended hepatectomy.

Surgical resection remains a critical treatment option for many patients with primary and secondary hepatic neoplasms. Extended hepatectomy (eHx) may be required for some patients with large tumors, which may cause liver failure and death. Partial hepatectomy (pHx) and eHx mouse models were constructed, liver tissues were sampled at 18, 36, and 72 h posthepatectomy. Transcriptome and metabolome analyses were employed to explore the different potential mechanisms in regeneration and injury between pHx and eHx. The results showed that eHx was associated with more severe liver injury and lower survival rates than pHx. Transcriptomics data showed there were 1842, 2129, and 1277 differentially expressed genes (DEGs) in eHx and 962, 1305, and 732 DEGs in pHx at 18, 36, and 72 h posthepatectomy, respectively, compared with the those in the sham groups. Compared with pHx, the number of DEGs in the eHx group reached a maximum of 230 at 18 h after surgery and decreased sequentially to 87 and 43 at 36 and 72 h. Metabolomics analysis identified a total of 1399 metabolites, and 48 significant differentially produced metabolites (DPMs) were screened between eHx and pHx. Combined analysis of DEGs and DPMs indicated that cholesterol metabolism and insulin resistance may be two important pathways for liver regeneration and mouse survival postextended hepatectomy. Our results showed the global influence of pHx and eHx on the transcriptome and metabolome in mouse liver, and revealed cholesterol metabolism and insulin resistance pathways might be involved in regeneration post-pHx and -eHx.

Complement inhibition alleviates donor brain death-induced liver injury and posttransplant cascade injury by regulating phosphoinositide 3-kinase signaling.

Brain death (BD) donors are the primary source of donor organs for liver transplantation. However, the effects of BD on donor livers and outcomes after liver transplantation remain unclear. Here, we explored the role of complement and the therapeutic effect of complement inhibition in BD-induced liver injury and posttransplantation injury in a mouse BD and liver transplantation model. For complement inhibition, we used complement receptor 2 (CR2)-Crry, a murine inhibitor of C3 activation that specifically targets sites of complement activation. In the mouse model, BD resulted in complement activation and liver injury in donor livers and a cascade liver injury posttransplantation, mediated in part through the C3a-C3aR (C3a receptor) signaling pathway, which was ameliorated by treatment with CR2-Crry. Treatment of BD donors with CR2-Crry improved graft survival, which was further improved when recipients received an additional dose of CR2-Crry posttransplantation. Mechanistically, we determined that complement inhibition alleviated BD-induced donor liver injury and posttransplant cascade injury by regulating phosphoinositide 3-kinase (PI3K) signaling pathways. Together, BD induced donor liver injury and cascade injury post-transplantation, which was mediated by complement activation products acting on PI3K signaling pathways. Our study provides an experimental basis for developing strategies to improve the survival of BD donor grafts in liver transplantation.

Callispheres® drug-eluting beads transarterial chemoembolization might be an efficient and safety down-staging therapy in unresectable liver cancer patients.

PURPOSE: The purpose was to explore the effect of drug-eluting beads transarterial chemoembolization (DEB-TACE) on down-staging in unresectable liver cancer patients. METHODS: A total of 180 patients with PHC treated by TACE were retrospectively analyzed. These included 80 cases in the DEB-TACE group and 100 cases in the cTACE group. Of these, 56 had complete clinical data (DEB-TACE: 24, cTACE: 32), and 23 patients received hepatectomy after TACE as a down-staging therapy (DEB-TACE: 15, cTACE: 8). Data (including clinical characteristics, clinical efficacy, tumor response, tumor diameters, residual liver volume, and liver function indexes before and after TACE, RFS, OS, and complications were collected and compared. Treatment response was evaluated at 1 month after TACE. Tumor diameter was evaluated by abdominal computed tomography scan. The residual liver volume was evaluated by IQQA liver system, and relapse-free survival (RFS) and overall survival (OS) were calculated by Kaplan-Meier curves. RESULTS: The conversion rate in DEB-TACE group was higher than cTACE group (18.8% vs 8%, p = 0.032). In DEB-TACE group, 17 patients achieved objective response rate (ORR) which was higher than cTACE group (70.8% vs 34.4%, p = 0.007). The tumor necrosis rate was higher in DEB-TACE group, but there was no significant difference between the two groups (p = 0.053). Tumor diameter was decreased after TACE compared to before TACE (DEB-TACE: 9.4 ± 3.3 vs. 5.4 ± 3.5 cm, p = 0.003; cTACE: 9.7 ± 2.6 vs. 6.9 ± 2.2, p = 0.036). As to residual liver volume, it was increased after TACE compared to before TACE (1066.2 cm3 vs. 1180.3 cm3, p = 0.007) in DEB-TACE group, while there was no significant difference in cTACE group (1046.4 cm3 vs. 1170 cm3, p = 0.339) compared by paired-sample t-test, but there was no significant difference before and after TACE when compared by unpaired-sample t-test (p > 0.05). After TACE at 1 month, the AFP level in the DEB-TACE group was significantly lower than that in the cTACE group (p = 0.003). For survival, the median RFS was 26.0 months in DEB-TACE group and 15 months in cTACE group; there was significant difference between the two groups (p = 0.0465). As to OS, the median OS in DEB-TACE group was higher than that in cTACE group, but there was no significant difference between the two groups (p = 0.165). For safety profiles, in terms of liver function and adverse events, there was no significant difference between the two groups. CONCLUSION: Compared with cTACE, DEB-TACE might be a more efficient and safety down-staging treatment in unresectable liver cancer patients.

Comparison of surgical outcomes among open, laparoscopic, and robotic pancreatoduodenectomy: a single-center retrospective study.

BACKGROUND: There is no general consensus on the feasibility and safety of robotic pancreatoduodenectomy (RPD) and whether it increases surgical risks. The purpose of this study was to assess the safety, feasibility, and rationality of RPD by comparing perioperative data among open pancreatoduodenectomy (OPD), laparoscopic pancreatoduodenectomy (LPD), and RPD performed in our center in recent years. METHODS: Clinical data of patients had undergone RPD (n = 32), LPD (n = 21), and OPD (n = 86) in The First Affiliated Hospital of Guangxi Medical University between January 2016 and June 2020 were retrospectively collected and analyzed. RESULTS: RPD required more time for operation (537.2 min vs. 441.5 min, p < 0.001) than OPD did, but less time to remove abdominal drainage tube (12.5 d vs. 17.3 d, p = 0.001). The differences between the RPD group and LPD group were interesting, as the two groups had similar operation time (537.2 min vs. 592.9 min, p = 1.000) and blood loss (482.8 ml vs. 559.5 ml, p > 0.05), but the RPD group had a higher activity of daily living score on postoperative day 3 (35.8 vs. 25.7, p = 0.0017) and a lower rate of conversion to OPD (6.5% vs. 38.1%, p = 0.011). Regarding complications, such as the postoperative pancreatic fistula, abdominal hemorrhage, intra-abdominal infection, bile leakage, reoperation, and perioperative mortality, there were no significant differences among the three groups. CONCLUSIONS: Not only is RPD feasible and reliable, it also offers significant advantages in that it improves postoperative recovery of skills needed for everyday life, has a low conversion rate to open surgery, and does not increase surgical risks.

m6A demethylase ALKBH5 inhibits pancreatic cancer tumorigenesis by decreasing WIF-1 RNA methylation and mediating Wnt signaling.

BACKGROUND: Pancreatic cancer is one of the most lethal types of cancer with extremely poor diagnosis and prognosis, and chemo-resistance remains a major challenge. The dynamic and reversible N6-methyladenosine (m6A) RNA modification has emerged as a new layer of epigenetic gene regulation. METHODS: qRT-PCR and IHC were applied to examine ALKBH5 levels in normal and pancreatic cancer tissues. Cancer cell proliferation and chemo-resistance were evaluated by clonogenic formation, chemosensitivity detection, and Western blotting assays. m6A-seq was performed to identify target genes. We evaluated the inhibitory effect of ALKBH5 in both in vivo and in vitro models. RESULTS: Here, we show that m6A demethylase ALKBH5 is downregulated in gemcitabine-treated patient-derived xenograft (PDX) model and its overexpression sensitized pancreatic ductal adenocarcinoma (PDAC) cells to chemotherapy. Decreased ALKBH5 levels predicts poor clinical outcome in PDAC and multiple other cancers. Furthermore, silencing ALKBH5 remarkably increases PDAC cell proliferation, migration, and invasion both in vitro and in vivo, whereas its overexpression causes the opposite effects. Global m6A profile revealed altered expression of certain ALKBH5 target genes, including Wnt inhibitory factor 1 (WIF-1), which is correlated with WIF-1 transactivation and mediation of the Wnt pathway. CONCLUSIONS: Our work uncovers the tumor suppressive and chemo-sensitizing function for ALKBH5, which provides insight into critical roles of m6A methylation in PDAC.

Tetramethylpyrazine ameliorates hepatic fibrosis through autophagy-mediated inflammation.

BACKGROUND: Imbalanced immune response and hepatic fibrosis are key factors related to the progression of chronic liver diseases. Tetramethylpyrazine (TMP), a natural alkaloid, has been widely used for treating liver injury. In this study, we explored the effect of TMP on hepatic fibrosis and the related mechanisms regulating autophagy. METHODS: A rat model of hepatic fibrosis and a model using an hepatic stellate cell line (HSC-T6) were created using CCl4 and platelet-derived growth factor (PDGF). Staining with haematoxylin and eosin (HE), Masson's stain, and TUNEL were performed for pathological diagnosis. ELISA, Western blotting, and immunofluorescence analyses were conducted to determine the expression levels of the specific markers for fibrosis, autophagy, inflammation, and signalling pathways. RESULTS: TMP treatment significantly rescued pathological injury and hepatic fibrosis. It also alleviated imbalances in the immune system, accumulation of extracellular matrix, and autophagy signals in hepatic fibrosis. At the same time, we found that application of the autophagy inducer rapamycin enhanced the therapeutic effect of TMP, whereas the autophagy inhibitor 3-methyladenine, PI3K pathway inhibitor LY294002, and AKT pathway agonist SC79 did the opposite. CONCLUSIONS: TMP exerts therapeutic effects in hepatic fibrosis mainly through promoting autophagy to ameliorate inflammation by inhibiting the AKT-mTOR signalling pathway, providing a new perspective for the treatment of chronic liver diseases.

MicroRNA-329-mediated PTTG1 downregulation inactivates the MAPK signaling pathway to suppress cell proliferation and tumor growth in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a severe malignancy usually producing a poor prognosis and high mortality rate. MicroRNAs (miRNAs) have been reported in association with CCA; however, the role miR-329 plays in the CCA condition still remains unclear. Therefore, this study was conducted to explore the underlying mechanism of which miR-329 is influencing the progression of CCA. This work studied the differential analysis of the expression chips of CCA obtained from the Gene Expression Omnibus database. Next, to determine both the expression and role of pituitary tumor transforming gene-1 (PTTG1) in CCA, the miRNAs regulating PTTG1 were predicted. In the CCA cells that had been intervened with miR-329 upregulation or inhibition, along with PTTG1 silencing, expression of miR-329, PTTG1, p-p38/p38, p-ERK5/ERK5, proliferating cell nuclear antigen (PCNA), Cyclin D1, Bcl-2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and caspase-3 were determined. The effects of both miR-329 and PTTG1 on cell proliferation, cell-cycle distribution, and apoptosis were also assayed. The miR-329 was likely to affect the CCA development through regulation of the PTTG1-mediated mitogen-activated protein kinase (MAPK) signaling pathway. The miR-329 targeted PTTG1, leading to inactivation of the MAPK signaling pathway. Upregulation of miR-329 and silencing of PTTG1 inhibited the CCA cell proliferation, induced cell-cycle arrest, and subsequently promoted apoptosis with elevations in Bax, cleaved caspase-3, and total caspase-3, but showed declines in PCNA, Cyclin D1, and Bcl-2. Moreover, miR-329 was also found to suppress the tumor growth by downregulation of PTTG1. To summarize, miR-329 inhibited the expression of PTTG1 to inactivate the MAPK signaling pathway, thus suppressing the CCA progression, thereby providing a therapeutic basis for the CCA treatment.

Natural immunoglobulin M initiates an inflammatory response important for both hepatic ischemia reperfusion injury and regeneration in mice.

Complement plays a role in both hepatic ischemia reperfusion (IR) injury (IRI) and liver regeneration, but it is not clear how complement is activated in either process. We investigated the role of self-reactive immunoglobulin M (IgM) antibodies in activating complement after hepatic IR and liver resection. Natural IgM antibodies that recognize danger-associated molecular patterns (neoepitopes) activate complement following both hepatic IR and liver resection. Antibody-deficient Rag1-/- mice were protected from hepatic IRI, but had increased hepatic injury and an impaired regenerative response after 70% partial hepatectomy (PHx). We identified two IgM monoclonal antibodies (mAbs) that specifically reversed the effect of Rag1 deficiency in both models; B4 (recognizes Annexin IV) and C2 (recognizes subset of phospholipids). Focusing on the B4-specific response, we demonstrated sinusoidal colocalization of IgM and C3d in Rag1-/- mice that were reconstituted with B4 mAb, and furthermore that the Annexin IV neoepitope is specifically and similarly expressed after both hepatic IR and PHx in wild-type (WT) mice. A single-chain antibody construct (scFv) derived from B4 mAb blocked IgM binding and reduced injury post-IR in WT mice, although, interestingly, B4scFv did not alter regeneration post-PHx, indicating that anti-Annexin IV antibodies are sufficient, but not necessary, for the regenerative response in the context of an entire natural antibody repertoire. We also demonstrated expression of the B4 neoepitope in postischemic human liver samples obtained posttransplantation and a corollary depletion in IgM recognizing the B4 and C2 neoepitopes in patient sera following liver transplantation. Conclusion: These data indicate an important role for IgM in hepatic IRI and regeneration, with a similar cross-species injury-specific recognition system that has implications for the design of neoepitope targeted therapeutics. (Hepatology 2018;67:721-735).

The diagnostic performance of gadoxetic acid disodium-enhanced magnetic resonance imaging and contrast-enhanced multi-detector computed tomography in detecting hepatocellular carcinoma: a meta-analysis of eight prospective studies.

AIM: The purpose of this study was to determine the relative diagnostic benefit of gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) over contrast-enhanced multi-detector computed tomography (CEMDCT) for the detection of hepatocellular carcinoma (HCC). METHODS: Two investigators searched multiple databases from inception to January 8, 2019, for studies comparing Gd-EOB-DTPA-enhanced MRI with CEMDCT in adults suspected of HCC. Two reviewers independently selected studies and extracted data. RESULTS: Eight studies were included enrolling 498 patients. MRI showed significantly higher sensitivity than CT (0.85 vs. 0.68). There was no significant difference in the specificity of MRI and CT (0.94 vs. 0.93). The negative likelihood ratio and positive likelihood ratio of MRI and CT were not significantly different (0.16 vs. 0.15 and 14.7 vs. 11.2, respectively). The summary receiver operating characteristics (SROC) of MRI was higher than that of CT at 0.96 vs. 0.91. In the subgroup analysis with a lesion diameter below 2 cm, the sensitivity of MRI was significantly higher than that of CT (0.79 vs. 0.46). CONCLUSION: Gd-EOB-DTPA-enhanced MRI showed higher sensitivity and overall diagnostic accuracy than CEMDCT especially for hepatocellular carcinoma lesions smaller than 2 cm. KEY POINTS: • Gd-EOB-DTPA-enhanced MRI can detect small lesions of hepatocellular carcinoma. • Gd-EOB-DTPA-enhanced MRI showed higher sensitivity and overall diagnostic accuracy than CEMDCT in patients with hepatocellular carcinoma. • Eight prospective studies showed that Gd-EOB-DTPA-enhanced MRI provides greater diagnostic confidence.

Cpg-ODN, a TLR9 Agonist, Aggravates Myocardial Ischemia/Reperfusion Injury by Activation of TLR9-P38 MAPK Signaling.

BACKGROUND/AIMS: Toll-like receptors (TLRs) have been implicated in myocardial ischemia/ reperfusion (I/R) injury. We examined the effect of CpG-oligodeoxynucleotide (ODN) on myocardial I/R injury. METHODS: Male Sprague-Dawley rats were treated with either CpG-ODN or control ODN 1 h prior to myocardial ischemia (30 min) followed by reperfusion. Rats treated with phosphate-buffered saline (PBS) served as I/R controls (n = 8/group). Infarct size was determined by 2,3,5-triphenyltetrazolium chloride and Evans blue straining. Cardiac function was examined by echocardiography before and up to 14 days after myocardial I/R. RESULTS: CpG-ODN administration significantly increased infarct size and reduced cardiac function and survival rate after myocardial I/R, compared to the PBS-treated I/R group. Control-ODN did not alter I/R-induced myocardial infarct size, cardiac dysfunction, and survival rate. Additionally, CpG-ODN promoted I/R-induced myocardial apoptosis and cleaved caspase-3 levels in the myocardium. CpG-ODN increased TLR9 activation and p38 phosphorylation in the myocardium. In vitro data also suggested that CpG-ODN treatment induced TLR9 activation and p38 phosphorylation. Importantly, p38 mitogen-activated protein kinase (MAPK) inhibition abolished CpG-ODN-induced cardiac injury. CONCLUSION: CpG-ODN, the TLR9 ligand, accelerates myocardial I/R injury. The mechanisms involve activation of the TLR9-p38 MAPK signaling pathway.

Erythropoietin promoted the proliferation of hepatocellular carcinoma through hypoxia induced translocation of its specific receptor.

BACKGROUND: Erythropoietin (EPO) is a hypoxia-inducible stimulator of erythropoiesis. Besides its traditional application in anemia therapy, it offers an effective treatment in the cancer patients, especially those who receive chemotherapy. Several reports indicated that it could promote the tumor cell proliferation through its specific receptor (EPOR). Unfortunately, the role of EPO/EPOR in hepatocellular carcinoma (HCC) progressing is still uncertain. METHODS: Protein in tumor tissue from HCC patients or H22 tumor-bearing mice was detected with immunohistochemistry. Cells were cultured under 1% oxygen to establish hypoxia. RT-PCR and western blotting were used to measure mRNA and protein of EPO/EPOR, respectively. MTT, flow cytometry and PCNA staining were used to detect cell proliferation. Immunofluorescence staining was applied to study the expression and location of cellular EPOR. The EPOR binding studies were performed with 125I-EPO radiolabeling assay. RESULTS: EPO and EPOR protein were up-regulated in HCC tissue of patients and H22-bearing mice. These were positively correlated with hypoxia-inducible factor -1 α and ki-67. Hypoxia up-regulated the expression of EPO and EPOR in HepG2 cells. It also induced the proliferation and increased the percentage of divided cells after 24, 48 and 72 h treatment. These were inhibited in cells pre-treated with 0.5 µg/mL soluble-EPOR. Immunofluorescence staining presented that EPOR was obviously translocated from nucleus to cytoplasm and membrane under hypoxia. EPOR binding activity was also increased after exposure to hypoxia. Recombinant human erythropoietin obviously elevated cell proliferation rate and the percentage of divided under hypoxia but not normoxia, which were also inhibited by soluble-EPOR. CONCLUSIONS: Our result indicated for the first time that EPO promoted the proliferation of HCC cells through hypoxia induced translocation of it specific receptor. Trial registration TJC20141113, retrospectively registered.

Overexpression of Yes-associated protein and its association with clinicopathological features of hepatocellular carcinoma: A meta-analysis.

BACKGROUND: Yes-associated protein (YAP) overexpression is reported to be associated with risk of hepatocellular carcinoma (HCC) but current studies have not explored the relationship between YAP expression with HCC clinicopathological features. METHODS: To assess these associations, a meta-analysis was performed which included four eligible studies including 391 HCC cases and 334 controls. There were eight eligible studies to investigate the association between YAP expression in HCC and clinicopathological features of liver cancer patients. Literature was obtained from PubMed, Embase, Wangfang and China National Knowledge Infrastructure. RESULTS: Analysis indicated that YAP expression in HCC was greater than in adjacent non-tumour tissue (odds ratio [OR], 15.80, 95% confidence interval [CI], 10.53-23.70, P<.00001; heterogeneity=.30). YAP overexpression in HCC was significantly associated with vascular invasion (OR, 2.21, 95% CI, 11.64-2.97, P<.00001, heterogeneity=.10), less cellular differentiation (OR, 2.38, 95% CI, 1.61-3.51, P<.00001, heterogeneity=.333), tumours larger than 5 cm (OR, 2.52, 95% CI, 1.75-3.62, P<.00001; heterogeneity=.17) and TNM tumour stage I + II (OR, 0.44, 95% CI, 0.28-0.75, P=.00003, heterogeneity=.12). CONCLUSIONS: Overexpression of YAP contributes to HCC formation, and its overexpression is associated with vascular invasion, low cellular differentiation tumours larger than 5 cm and TNM tumour stage III + IV.

HOXA7 plays a critical role in metastasis of liver cancer associated with activation of Snail.

BACKGROUND: Liver cancer is one of the main causes of cancer-related death in human. HOXA7 has been proved to be related with several cancers. METHOD: The expression levels of HOXA7 were examined by Western blot, qRT-PCR or ICH. MTT was used to detect the proliferative rate of liver cancer cells. The invasive abilities were examined by matrigel and transwell assay. The metastatic abilities of liver cancer cells were revealed in BALB/c nude mice. RESULTS: In this report, we revealed that HOXA7 promoted metastasis of HCC patients. First, increased levels of HOXA7 were examined in liver cancer especially in metastatic liver cancer. Moreover, higher expression level of HOXA7 was associated with poorer prognosis of liver cancer patients. Overexpression of HOXA7 significantly enhanced proliferation, migration, invasion in vitro and tumor growth and metastasis in vivo meanwhile silencing HOXA7 significantly inhibited the aboves abilities of liver cancer cells. In this research, we identified that HOXA7 performed its oncogenic characteristics through activating Snail. CONCLUSION: Collectively, we identify the critical role and possible mechanism of HOXA7 in metastasis of liver cancer which suggest that HOXA7 may be a potential therapeutic target of liver cancer patients.

Dihydroceramide-desaturase-1-mediated caspase 9 activation through ceramide plays a pivotal role in palmitic acid-induced HepG2 cell apoptosis.

In this study, results showed that the inhibition of PA-induced HepG2 cell growth takes place in a time- and concentration-dependent manner, that activation of caspase 9 is necessary for PA-induced HepG2 cell apoptosis, that dihydroceramide desaturase 1 (DES1) plays a key role in PA-mediated caspase 9 and caspase 3 activation, and that palmitoleic acid (POA), an omega-7 monounsaturated fatty acid, reverses PA-induced apoptosis through DES1 â†’ Ceramide â†’ Caspase 9 â†’ Caspase 3 signaling.