Nuclear respiratory factor 1 regulates super enhancer-controlled SPIDR to protect hepatocellular carcinoma cells from oxidative stress.

BACKGROUND: Cellular response to oxidative stress plays significant roles in hepatocellular carcinoma (HCC) development, yet the exact mechanism by which HCC cells respond to oxidative stress remains poorly understood. This study aimed to investigate the role and mechanism of super enhancer (SE)-controlled genes in oxidative stress response of HCC cells. METHODS: The GSE112221 dataset was used to identify SEs by HOMER. Functional enrichment of SE-controlled genes was performed by Metascape. Transcription factors were predicted using HOMER. Prognosis analysis was conducted using the Kaplan-Meier Plotter website. Expression correlation analysis was performed using the Tumor Immune Estimation Resource web server. NRF1 and SPIDR expression in HCC and normal liver tissues was analyzed based on the TCGA-LIHC dataset. ChIP-qPCR was used to detect acetylation of lysine 27 on histone 3 (H3K27ac) levels of SE regions of genes, and the binding of NRF1 to the SE of SPIDR. To mimic oxidative stress, HepG2 and Hep3B cells were stimulated with H2O2. The effects of NRF1 and SPIDR on the oxidative stress response of HCC cells were determined by the functional assays. RESULTS: A total of 318 HCC-specific SE-controlled genes were identified. The functions of these genes was significant association with oxidative stress response. SPIDR and RHOB were enriched in the "response to oxidative stress" term and were chosen for validation. SE regions of SPIDR and RHOB exhibited strong H3K27ac modification, which was significantly inhibited by JQ1. JQ1 treatment suppressed the expression of SPIDR and RHOB, and increased reactive oxygen species (ROS) levels in HCC cells. TEAD2, TEAD3, NRF1, HINFP and TCFL5 were identified as potential transcription factors for HCC-specific SE-controlled genes related to oxidative stress response. The five transcription factors were positively correlated with SPIDR expression, with the highest correlation coefficient for NRF1. NRF1 and SPIDR expression was up-regulated in HCC tissues and cells. NRF1 activated SPIDR transcription by binding to its SE. Silencing SPIDR or NRF1 significantly promoted ROS accumulation in HCC cells. Under oxidative stress, silencing SPIDR or NRF1 increased ROS, malondialdehyde (MDA) and γH2AX levels, and decreased superoxide dismutase (SOD) levels and cell proliferation of HCC cells. Furthermore, overexpression of SPIDR partially offset the effects of NRF1 silencing on ROS, MDA, SOD, γH2AX levels and cell proliferation of HCC cells. CONCLUSION: NRF1 driven SPIDR transcription by occupying its SE, protecting HCC cells from oxidative stress-induced damage. NRF1 and SPIDR are promising biomarkers for targeting oxidative stress in the treatment of HCC.

GANT61, an inhibitor of Gli1, inhibits the proliferation and migration of hepatocellular carcinoma cells.

Constitutive activation of Hedgehog (Hh) signaling has been implicated in many cancers including hepatocellular carcinoma (HCC). Among them, the terminal glioma-associated oncogene homolog 1 (Gli1) regulates the expression of critical genes in the Hh pathway. The current study aims to evaluate the anti-HCC effect of the Gli1 inhibitor, GANT61. In vitro analysis including cell counting kit-8 (CCK-8) assay, flow cytometry, and migration and invasion assay were adopted to evaluate the effect of GANT61 on HCC cell lines. In vivo, xenograft studies were also performed to verify the effect of GANT61 on HCC. By CCK-8 assay, we found that GANT61 could significantly reduce the growth of HCC cell lines Huh7 and hemophagocytic lymphohistiocytosis (HLE), and their IC50 concentrations were 4.481 and 6.734 µM, respectively. Flow cytometry shows that GANT61 induced cell cycle arrest in the G2/M phase and accelerated apoptosis of both HLE and Huh7 cells. While migration and invasion assay shows that GANT61 weakens cells' migration and invasion ability. Besides that, GANT61 inhibits the expression of Gli1, FoxM1, CyclinD1, and Bcl-2, upregulates the level of Bax protein, and also reverses the epithelial-mesenchymal transition program by downregulating the expression of Vimentin and N-Cadherin and upregulating the expression of epithelial E-Cadherin expression. Furthermore, GANT61 inhibits the growth of subcutaneous xenografts of Huh7 cells in nude mice. Overall, this study suggests that Gli1 is a potential target for therapy and GANT61 shows promising therapeutic potential for future treatment in HCC.

RNA-Seq and Immune Repertoire Analysis of Normal and Hepatocellular Carcinoma Relapse After Liver Transplantation.

Background: Hepatocellular carcinoma (HCC) relapse is the main reason for the poor prognosis of HCC after Liver transplantation (LT). This study aimed to explore the molecular mechanisms and immune repertoire profiles of HCC relapse. Material and Methods: RNA-seq of blood samples from patients with normal (n=12) and HCC relapse (n=6) after LT was performed to identify differentially expressed genes (DEGs) and key signalling pathways. The DEGs and immune genes were further analyzed by bioinformatics. TRUST4 was used to analyze the differences in the immune repertoire between the two groups. Another 11 blood samples from patients with HCC who had received LT were collected for RT-qPCR verification of key genes. Results: A total of 131 upregulated and 157 downregulated genes were identified using RNA-seq, and GO enrichment analysis revealed that the top 15 pathways were immune-related. The PPI network identified 10 key genes. Immune infiltration analysis revealed a significant difference in the five immune cell types between the two groups. A total of 83 intersecting genes were obtained by intersecting DEGs and immune genes. 6 key genes, including MX1, ISG15, OAS1, PRF1, SPP1, and THBS1 were obtained according to the intersection of DEGs, PPI network top 10 genes and immune intersecting genes. Immune repertoire analysis showed that the usage frequency of variable (V) and joining (J) genes in the normal group was higher than that in the relapse group. RT-qPCR validation showed that the expression levels of key genes were consistent with the RNA-seq results. Conclusion: Our study identified key pathways and genes that could help determine whether transplant recipients are more prone to HCC relapse. Immune repertoire analysis revealed a difference in the usage frequency of VJ genes between the normal and relapse groups, providing a research direction for immunotherapy in patients with HCC relapse after liver transplantation.

Identification of Recurrence-Related mRNAs and Noncoding RNAs in Hepatocellular Carcinoma Following Liver Transplantation.

BACKGROUND: This study aimed to determine whether altered mRNA, long non-coding RNA, and circular RNA expression is related to hepatocellular carcinoma recurrence after liver transplantation. METHODS: Five recurrent and 5 non-recurrent primary hepatocellular carcinoma samples were used to perform RNA sequencing. Then, differentially expressed mRNAs, differentially expressed long non-coding RNAs, and differentially expressed circular RNAs between recurrent group and non-recurrent group were identified. In addition, differentially expressed long non-coding RNA/differentially expressed circular RNA-differentially expressed mRNA co-expression network, and competing endogenous RNA (differentially expressed circular RNA/differentially expressed long non-coding RNA-miRNA-differentially expressed mRNA) regulatory network were constructed. Finally, real-time quantitative polymerase chain reaction was performed for verification. RESULTS: Five hundred forty-one differentially expressed mRNAs, 239 differentially expressed long non-coding RNAs, and 16 differentially expressed circular RNAs in the recurrent group were obtained. Gene set enrichment analysis indicated that these differentially expressed mRNAs may affect hepatocellular carcinoma recurrence through multiple pathways, such as E2F, epithelial-mesenchymal transition, G2M, and oxidative phosphorylation. Then, 993 differentially expressed long non-coding RNA-differentially expressed mRNA co-expression pairs and 28 differentially expressed circular RNA/differentially expressed mRNA co-expression pairs were obtained. The competing endogenous RNA network contained 10 circular RNA-miRNA pairs, 12 long non-coding RNA-miRNA pairs, and 36 miRNA- mRNA pairs. Real-time quantitative polymerase chain reaction results showed the same expression trend as RNA-seq results. CONCLUSION: Our results reveal key mRNAs, long non-coding RNAs, and circular RNAs associated with recurrence in hepatocellular carcinoma patients after liver transplantation.

Endocrine disrupting chemicals: A promoter of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disorder. With the improvement in human living standards, the prevalence of NAFLD has been increasing in recent years. Endocrine-disrupting chemicals (EDCs) are a class of exogenous chemicals that simulate the effects of hormones in the body. There has been growing evidence regarding the potential effects of EDCs on liver health, especially in NAFLD. This paper aims to summarize the major EDCs that contribute to the growing burden of NAFLD and to raise public awareness regarding the hazards posed by EDCs with the objective of reducing the incidence of NAFLD.

Exploring the underlying molecular mechanism of liver cancer cells under hypoxia based on RNA sequencing.

BACKGROUND: The aim of our study was to use the differentially expressed mRNAs (DEmRNAs) and differentially expressed miRNAs (DEmiRNAs) to illustrate the underlying mechanism of hypoxia in liver cancer. METHODS: In this study, a cell model of hypoxia was established, and autophagy activity was measured with western blotting and transmission electron microscopy. The effect of hypoxia conditions on the invasion of liver cancer cell was evaluated. RNA sequencing was used to identify DEmRNAs and DEmiRNAs to explore the mechanism of hypoxia in liver cancer cells. RESULTS: We found that autophagy activation was triggered by hypoxia stress and hypoxia might promote liver cancer cell invasion. In addition, a total of 407 shared DEmRNAs and 57 shared DEmiRNAs were identified in both HCCLM3 hypoxia group and SMMC-7721 hypoxia group compared with control group. Furthermore, 278 DEmRNAs and 24 DEmiRNAs were identified as cancer hypoxia-specific DEmRNAs and DEmiRNAs. Finally, we obtained 19 DEmiRNAs with high degree based on the DEmiRNA-DEmRNA interaction network. Among them, hsa-miR-483-5p, hsa-miR-4739, hsa-miR-214-3p and hsa-miR-296-5p may be potential gene signatures related to liver cancer hypoxia. CONCLUSIONS: Our study may help to understand the potential molecular mechanism of hypoxia in liver cancer.

Mycophenolate mofetil enhances the effects of tacrolimus on the inhibitory function of regulatory T cells in patients after liver transplantation via PD-1 and TIGIT receptors.

OBJECTIVE: Regulatory T cells (Tregs) induce immune tolerance in patients after organ transplantation. Various immunosuppressors can affect Tregs function through different mechanisms. PD-1 and TIGIT are important receptors on Tregs surface. Here, we investigated the effects of Tacrolimus and mycophenolate mofetil (MMF) on the inhibitory function of Tregs and explored the regulatory mechanism in patients after liver transplantation. METHODS: Thirty patients that underwent a liver transplant and 15 healthy people were enrolled. Fifteen patients received Tacrolimus only, and 15 received a combination of Tacrolimus and MMF. Tregs and effector T cells (Teffs) were isolated using magnetic beads and were mixed at different ratios of 0:1, 1:4, 1:2 and 1:1. An inhibition assay was performed by adding anti-PD-1 and anti-TIGIT when the mixture ratio was 1:1. The Tregs inhibition rate was determined and the levels of IFN-γ and TNF-α were measured. RESULTS: As the ratios of Tregs to Teffs in the mixture increased, the Tregs inhibition rate increased and the levels of IFN-γ and TNF-α decreased. At each mixture ratio, Tacrolimus + MMF group had the highest Tregs inhibition rate compared to Tacrolimus and control group. At the specific mixture ratio of 1:1, the addition of both anti- PD-1 and anti-TIGIT led to lower Tregs inhibition rate and higher IFN-γ and TNF-α levels in all three groups as opposed to the addition of each antibody separately. Additionally, both the decrease in the Tregs inhibition rate and the increase in the IFN-γ and TNF-α levels were the most for Tacrolimus + MMF group among all cases, either adding antibodies alone or mixed. CONCLUSION: Tacrolimus and MMF enhanced the function of Tregs by synergistically affecting PD-1 and TIGIT in liver transplant patients.

Uncovering the potential differentially expressed miRNAs as diagnostic biomarkers for hepatocellular carcinoma based on machine learning in The Cancer Genome Atlas database.

The present study aimed to identify novel diagnostic differentially expressed microRNAs (miRNAs/miRs) in order to understand the molecular mechanisms underlying hepatocellular carcinoma. The expression data of miRNA and mRNA were downloaded for differential expression analysis. Optimal diagnostic differentially expressed miRNA biomarkers were identified via a random forest algorithm. Classification models were established to distinguish patients with hepatocellular carcinoma and normal individuals. A regulatory network between optimal diagnostic differentially expressed miRNA and differentially expressed mRNAs was then constructed. The GSE63046 dataset and in vitro experiments were used to validate the expression of the optimal diagnostic differentially expressed miRNAs identified. In addition, diagnostic and prognostic analyses of optimal diagnostic differentially expressed miRNAs were performed. In total, 14 differentially expressed miRNAs (all upregulated) and 2,982 differentially expressed mRNAs (1,989 upregulated and 993 downregulated) were identified. hsa­miR­10b­5p, hsa­miR­10b­3p, hsa­miR­224­5p, hsa­miR­183­5p and hsa­miR­182­5p were considered as the optimal diagnostic biomarkers for hepatocellular carcinoma. The mRNAs targeted by these five miRNAs included secreted frizzled related protein 1 (SFRP1), endothelin receptor type B (EDNRB), nuclear receptor subfamily 4 group A member 3 (NR4A3), four and a half LIM domains 2 (FHL2), NK3 homeobox 1 (NKX3­1), interleukin 6 signal transducer (IL6ST) and forkhead box O1 (FOXO1). 'Bile acid biosynthesis and cholesterol' was the most enriched signaling pathways of these target mRNAs. The expression validation of the five miRNAs was consistent with the present bioinformatics analysis. Notably, hsa­miR­10b­5p and hsa­miR­10b­3p had a significant prognosis value for patients with hepatocellular carcinoma. In conclusion, the five differentially expressed miRNAs may be considered as diagnostic biomarkers for patients with hepatocellular carcinoma. In addition, the differential expression levels of the targets of these five mRNAs, including SFRP1, EDNRB, NR4A3, FHL2, NKX3­1, IL6ST and FOXO1, may be involved in hepatocellular carcinoma tumorigenesis.

Bioinformatics analysis to reveal key biomarkers for early and late hepatocellular carcinoma.

BACKGROUND: The aim of this study was to find the long non-coding RNAs (lncRNAs) and mRNAs acting as biomarker of early and late hepatocellular carcinoma (HCC). METHODS: The Cancer Genome Atlas (TCGA) dataset was used to identify shared differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) between early and late HCC and normal tissue. Functional annotation and protein-protein interaction network of shared DEmRNAs were performed. Furthermore, DElncRNAs-DEmRNAs co-expression network of early and late HCC were also performed. The expression of selected candidate genes were validated by the quantitative real time polymerase chain reaction (qRT-PCR). RESULTS: A total of 1,201 shared DEmRNAs and 162 shared DElncRNAs were identified in both early and late HCC compared with normal controls. Cell cycle, p53 signaling pathway, retinol metabolism and metabolism of xenobiotics by cytochrome P450 were four significantly enriched pathways. Base on the protein-protein interaction network, CDK1, AURKA, CDC20, PLK1, AURKB, HIST1H2BG, BUB1B, CCNA2, CCNB1 and CDT1 were key protein. CTD-2510F5.4 and HAND2-AS1 were hub lncRNAs in both early and late HCC. Overall, the confirmation results of qRT-PCR were generally consistent with our integrated analysis. CONCLUSIONS: A total of 4 DEmRNAs (CDK1, KIFC1, CENPF and ECM1) and 2 DElncRNAs (CTD-2510F5.4 and HAND2-AS1) were identified as key biomarkers for HCC. This study may contribute to reveal the pathogenesis of early and late HCC and provide new and accurate therapeutic targets for HCC.

Effects of tacrolimus (FK506) and mycophenolate mofetil (MMF) on regulatory T cells and co-inhibitory receptors in the peripheral blood of human liver allograft patients.

Context: Recent studies have shown that a combination treatment of mycophenolate mofetil (MMF) and tacrolimus (FK506) may be an option for organ transplantation patients. Objective: In this study, we detected the effects of FK506 and MMF on the expressions of regulatory T cells (Tregs) and co-inhibitory receptors on Tregs in peripheral blood mononuclear cells (PBMC) of patients with stable phase after liver transplantation. Materials and methods: A total of 35 patients with stable stage after 6 months of liver transplantation were divided into two groups including 20 patients were treated with FK506 monotherapy (FK506 group), and 15 patients with FK506 and MMF combination (FK506 + MMF group). 15 healthy subjects were served as the control. Results: It is found that percentages of CD3+, CD3+CD4+ and CD3+CD8+ T cells in FK506 group are lowered compared to the control group but they are elevated in FK506 + MMF group. Amount of CD4+CD25+CD127low/-Treg cells in CD3+ CD4+T cells in FK506 + MMF group was higher than that in FK506 group and control group. The expressions of co-inhibitory receptors (CTLA-4, PD-1, Tim-3, LAG-3 and TIGIT) on Tregs in FK506 + MMF group were significant higher than those in the FK506 group and control group. The levels of the relative cytokines (TGF-ß and IL-10) in FK506 group are down-regulated compared to the control group. Conclusion: The application of FK506 combined with MMF may be superior to FK506 monotherapy for the patients to further induce the immune tolerance after liver transplantation.

Pancreaticoduodenectomy versus duodenum-preserving pancreatic head resection for the treatment of chronic pancreatitis.

OBJECTIVE: The objective of this study was to assess the efficacy and safety of pancreaticoduodenectomy (PD) and duodenum-preserving pancreatic head resection (DPPHR) for the treatment of chronic pancreatitis (CP). METHODS: The 123 patients with CP who underwent pancreatic head resection between January 2004 and June 2009 were retrospectively analyzed. The preoperative variables, operative data, postoperative complications, and follow-up information were examined. RESULTS: There were no significant differences in clinical and morphological characteristics, pain relief, and jaundice status between the PD and DPPHR groups. The duration of operation was shorter (251.8 [SD, 43.1] vs 324.5 [SD, 41.4] minutes, P < 0.001), blood loss was less (464.4 [SD, 203.6] vs 646.5 [SD, 242.9] mL, P < 0.001), and overall postoperative morbidity was lower (3% vs 19%, P = 0.006) in DPPHR group. The duration of hospital stay was also significantly different (9.9 [SD, 1.8] vs 13.7 [SD, 2.8] days, P < 0.001). Most functional and symptom scales revealed a better quality of life in DPPHR group. The proportion of patients with exocrine and endocrine insufficiency was higher in PD group as compared with DPPHR group. CONCLUSIONS: Both procedures are equally effective in pain relief, but DPPHR is superior to PD in operative data, postoperative morbidity, improving quality of life, and preservation of exocrine and endocrine function.

[Diagnosis and surgical interventions for the chronic obstructive pancreatitis due to the inflammatory lesions at the opening of the pancreatic duct].

OBJECTIVE: To explore the diagnostic methods and reasonable surgical interventions for the chronic obstructive pancreatitis due to the inflammatory lesions at the opening of the pancreatic duct. METHODS: From January 2002 to November 2010 the data of 28 patients who were diagnosed as the chronic obstructive pancreatitis (COP) was retrospectively reviewed. Out of the 28 patients, it was analyzed that the clinical manifestations, diagnostic methods, surgical finding and surgical interventions of the 13 patients who were diagnosed as COP due to the inflammatory lesions at the opening of the pancreatic duct in the exploratory operation accompanying recurrent acute abdominal pain with increased serum amylase and lipase, dilation of entire pancreatic duct on imaging before surgery. The conditions included pain recrudescence, quality of life, pancreatic changes on imaging and the serum amylase and lipase after surgery were recorded. RESULTS: All the 13 patients had clinical manifestations of COP. However, 12 patients had different manifestations on imaging from those chronic pancreatitis imaging due to tumors at the duodenal papilla, ampulla or inner pancreatic duct. Via exploratory operation and magnetic resonance cholangiopancreatography (MRCP), there were short pancreaticobiliary common channel or pancreas divisum existing in most patients. There was no acute abdominal pain with the increased serum amylase and lipase in the 12 patients who receiving the transduodenal mastoid, ampulla and pancreatic ductal opening incision and plasty, the paramastoideus incision and plasty in the visit. CONCLUSIONS: The imaging character of COP due to the inflammatory lesions at the opening of the pancreatic duct is the dilation of the pancreatic duct without the chronic obstruction in the bile duct. The patients with short pancreaticobiliary common channel or pancreas divisum easily suffer COP due to the stenosis of the pancreatic ductal opening caused by the duodenal mastoiditis or paramastoiditis. The local plasty surgery to correct the stenosis at the pancreatic ductal opening and improve the drainage of the pancreatic duct is an easy and effective management.