Upregulation of lncRNA PTOV1-AS1 in hepatocellular carcinoma contributes to disease progression and sorafenib resistance through regulating miR-505.

Increasing evidence has displayed the vital influence of lncRNA in tumorigenesis and chemoresistance of cancer treatment. This study investigated the function of lncRNA PTOV1-AS1 in hepatocellular carcinoma (HCC) and its role in sorafenib resistance. The relative expression of lncRNA and miRNA was measured by RT-qPCR. The cellular activities including cell proliferation and invasion were explored by CCK-8 and Transwell assays. Bioinformatics analysis and dual-luciferase reporter assay were used to predict the targeting miRNA of PTOV1-AS1. The expression levels of PTOV1-AS1 were higher in HCC tissues than that in the normal tissues and associated with patients' overall survival. Knockdown of PTOV1-AS1 decreased cell proliferation rate and invasion number. After treatment with different concentrations of sorafenib, the sorafenib-resistant hepatoma cells were conducted. PTOV1-AS1 expression levels were increased in HepG2-SR and Huh7-SR cells. PTOV1-AS1 knockdown repressed the proliferation, invasion, and drug resistance of sorafenib-resistant HCC cells by targeting the expression of miR-505. In conclusion, the expression of PTOV1-AS1 is increased in HCC and sorafenib-resistance HCC cells, as well as is associated with patients' prognosis. Inhibition of PTOV1-AS1 expression can reduce the resistance of sorafenib-resistant HCC cells, which may play a role by targeting the negative regulation of miR-505 expression.

Toripalimab in combination with Anlotinib for unresectable hepatocellular carcinoma after SBRT: A prospective, single-arm, single-center clinical study.

Objective: Exposing tumor antigens to the immune system is the key to ensuring the efficacy of immunotherapy. SBRT is the main way to reveal the specifical antigens of tumors which can enhance the immune response. We aimed to explore the clinical efficacy and safety of Toripalimab combined with Anlotinib for uHCC after SBRT. Methods: This is a prospective, single-arm, explorative clinical study. uHCC patients with an ECOG PS score of 0-1, Child-Pugh class A or B, and BCLC stage B or C were included and treated with SBRT(8Gy*3) followed by 6-cycle combinational therapy with Toripalimab and Anlotinib. The primary endpoint was progression-free survival (PFS) and the secondary endpoints were objective response rate (ORR), disease control rate (DCR), overall survival (OS), and incidence of treatment-related adverse events (TRAEs). Continuous variables were presented as medians and ranges. Survivals were studied with the Kaplan-Meier method. Categorical data were expressed as n (percentage). Results: Between June 2020 and October 2022, a total of 20 patients with intermediate-advanced uHCC were enrolled. All cases had multiple intrahepatic metastases, or macrovascular invasion, or both, among whom 5 cases with lymph node or distant metastases. Until September 2022, the median follow-up time was 7.2 months (range, 1.1-27.7 months). Median survival time could not be assessed at the moment, based on iRecist, median PFS was 7.4 months (range, 1.1-27.7 months), ORR 15.0%, and DCR 50.0%. 14 patients experienced treatment-related adverse events with an incidence of 70%. The overall survival rates at 18 months and 24 months were 61.1% and 50.9%, respectively. And the progression-free survival rates were 39.3% and 19.7%. Conclusion: Exposure of specific antigens of HCC via SBRT may improve the efficacy of combinational therapy with Toripalimab and Anlotinib for uHCC with manageable adverse effects, which deserves further exploration. Clinical trial registration: www.clinicaltrials.gov, identifier ChiCTR2000032533.

SSTDP: Supervised Spike Timing Dependent Plasticity for Efficient Spiking Neural Network Training.

Spiking Neural Networks (SNNs) are a pathway that could potentially empower low-power event-driven neuromorphic hardware due to their spatio-temporal information processing capability and high biological plausibility. Although SNNs are currently more efficient than artificial neural networks (ANNs), they are not as accurate as ANNs. Error backpropagation is the most common method for directly training neural networks, promoting the prosperity of ANNs in various deep learning fields. However, since the signals transmitted in the SNN are non-differentiable discrete binary spike events, the activation function in the form of spikes presents difficulties for the gradient-based optimization algorithms to be directly applied in SNNs, leading to a performance gap (i.e., accuracy and latency) between SNNs and ANNs. This paper introduces a new learning algorithm, called SSTDP, which bridges the gap between backpropagation (BP)-based learning and spike-time-dependent plasticity (STDP)-based learning to train SNNs efficiently. The scheme incorporates the global optimization process from BP and the efficient weight update derived from STDP. It not only avoids the non-differentiable derivation in the BP process but also utilizes the local feature extraction property of STDP. Consequently, our method can lower the possibility of vanishing spikes in BP training and reduce the number of time steps to reduce network latency. In SSTDP, we employ temporal-based coding and use Integrate-and-Fire (IF) neuron as the neuron model to provide considerable computational benefits. Our experiments show the effectiveness of the proposed SSTDP learning algorithm on the SNN by achieving the best classification accuracy 99.3% on the Caltech 101 dataset, 98.1% on the MNIST dataset, and 91.3% on the CIFAR-10 dataset compared to other SNNs trained with other learning methods. It also surpasses the best inference accuracy of the directly trained SNN with 25~32× less inference latency. Moreover, we analyze event-based computations to demonstrate the efficacy of the SNN for inference operation in the spiking domain, and SSTDP methods can achieve 1.3~37.7× fewer addition operations per inference. The code is available at: https://github.com/MXHX7199/SNN-SSTDP.

Upregulation of microRNA miR-652-3p is a prognostic risk factor for hepatocellular carcinoma and regulates cell proliferation, migration, and invasion.

As powerful regulatory factors, microRNAs (miRNAs) are involved in tumor progression. The current research aimed to excavate the prognostic significance and potential regulatory mechanisms of miR-652-3p in hepatocellular carcinoma (HCC). Expression of miR-652-3p in HCC tissues and cells was exposed by Quantitative real-time polymerase chain reaction (RT-qPCR) assay, and we found that miR-652-3p was elevated in HCC tissues and cells than in the control group (P < 0.05). Then, the relationship between miR-652-3p levels and clinical characteristics was obtained from the Chi-square test. Kaplan-Meier survival analysis and Cox regression model to explore the outcome of miR-652-3p on the prognosis of HCC. The results investigated that overexpression of miR-652-3p was related to clinical tumor-node-metastasis (TNM) stage (P = 0.020) and differentiation (P = 0.031). HCC patients with elevated miR-652-3p levels were correlated with poor overall survival (log-rank, P = 0.007), and maybe a possible prognostic marker for HCC. Finally, CCK-8, colony formation, wound healing and Transwell assay was detected after transfection of HCC cells with miR-652-3p mimic or inhibitor. And the results confirmed that elevation miR-652-3p promoted the proliferation, migration, and invasion of tumor cells (P < 0.05). All data indicated that elevated miR-652-3p is a prognostic marker and would be able to participate in tumor progression of HCC by regulating cell proliferation, migration, and invasion.

Protective Effect of Retrograde Reperfusion Against Hepatic Autophagy Impairment in Rat Liver Transplantation.

The retrograde reperfusion (RTR) technique was introduced in orthotopic liver transplantation (OLT) to improve initial postoperative liver function, but the related mechanisms remain unexplained. We investigated the influences of different reperfusion sequences, including initial portal reperfusion (IPR) and RTR, on hepatic ischemia/reperfusion (I/R) injury and autophagic activity in a simplified rat orthotopic liver transplantation (ROLT) model. METHODS: First, we established an ROLT model of male Sprague-Dawley rats to simulate either the IPR or RTR technique. The operative times and survival rates until postoperative day (POD) 7 were recorded. Liver enzyme levels, histologic damage, and in situ apoptosis were assessed. Second, we evaluated differences in the autophagic flux of liver grafts at 1, 2, and 6 hours after reperfusion between the IPR and RTR techniques. All experimental procedures involving animals were approved by the Institutional Animal Ethics Committee of the 900th Hospital of PLA. RESULTS: In the first experiment, all animals survived to POD 7. In contrast to the IPR sequence, the RTR technique decreased the extent of graft I/R injury. In the second experiment, reperfusion markedly impaired the autophagic flux of ischemic liver grafts, but the RTR technique could alleviate and postpone the reduction in autophagy after I/R. CONCLUSIONS: A feasible modified ROLT model with the cuff method was described and could flexibly simulate 2 reperfusion techniques: IPR and RTR. The use of the RTR sequence exhibited a protective effect against I/R injury and impairment of autophagy in liver grafts.

microR-505/heterogeneous nuclear ribonucleoprotein M inhibits hepatocellular carcinoma cell proliferation and induces cell apoptosis through the Wnt/ß-catenin signaling pathway.

Aim: This study aimed to investigate the expression of microRNA-505 (miR-505) and explore its clinical significance, biological function and mechanisms in hepatocellular carcinoma (HCC). Methods: Expression of miR-505 was measured in 128 paired HCC tissues and five cell lines by quantitative real-time polymerase chain reaction (qRT-PCR). MTT assay, Transwell migration, invasion assays and apoptosis assay were performed to explore the functional role of miR-505. The target gene of miR-505 was assessed using the bioinformatics assay and the related signaling pathway was confirmed using western blot. Results: Expression of miR-505 in HCC serum and tissues were downregulated. The overexpression of miR-505 in HCC cells inhibited cell proliferation and metastasis, as well as enhanced cell apoptosis by directly downregulating heterogeneous nuclear ribonucleoprotein M (HNRNPM). The activity of the Wnt/ß-catenin signaling pathway was suppressed by the overexpression of miR-505 but was promoted by the upregulation of HNRNPM. Conclusion: The results suggest that the regulation of miR-505/HNRNPM may be a novel strategy to improve the targeted therapy of HCC.

Glycochenodeoxycholic acid impairs transcription factor E3 -dependent autophagy-lysosome machinery by disrupting reactive oxygen species homeostasis in L02 cells.

Cholestasis represents pathophysiologic syndromes defined as impaired bile flow from the liver. As an outcome, bile acids accumulate and promote hepatocyte injury, followed by liver cirrhosis and liver failure. Glycochenodeoxycholic acid (GCDCA) is relatively toxic and highly concentrated in bile and serum after cholestasis. However, the mechanism underlying GCDCA-induced hepatotoxicity remains unclear. In this study, we found that GCDCA inhibits autophagosome formation and impairs lysosomal function by inhibiting lysosomal proteolysis and increasing lysosomal pH, contributing to defects in autophagic clearance and subsequently leading to the death of L02 human hepatocyte cells. Notably, through tandem mass tag (TMT)-based quantitative proteomic analysis and database searches, 313 differentially expressed proteins were identified, of which 71 were increased and 242 were decreased in the GCDCA group compared with those in the control group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that GCDCA suppressed the signaling pathway of transcription factor E3 (TFE3), which was the most closely associated with autophagic flux impairment. In contrast, GCDCA-inhibited lysosomal function and autophagic flux were efficiently attenuated by TFE3 overexpression. Specifically, the decreased expression of TFE3 was closely related to the disruption of reactive oxygen species (ROS) homeostasis, which could be prevented by inhibiting intracellular ROS with N-acetyl cysteine (NAC). In summary, our study is the first to demonstrate that manipulation of ROS/TFE3 signaling may be a therapeutic approach for antagonizing GCDCA-induced hepatotoxicity.

miRNA-26a blocks interleukin-2-mediated migration and proliferation of non-small cell lung cancer cells via vascular cell adhesion molecule-1.

BACKGROUND: Literatures have confirmed role of inflammatory mediators like interleukin-2 (IL-2) in non-small cell lung cancer (NSCLC). microRNA-26a (miR-26a) is involved in variety of signaling pathways and functions as tumor suppressor and regulating the responsible genes at posttranscriptional level. The aim of study was to study the correlation of IL-2 and miR-26a in lung cancer (LC) cells. METHODS: For the study we selected 32 subjects reported for NSCLC and 20 with chronic obstructive pulmonary disease (COPD) as control in the present study. For in vitro analysis, H-460 and H-226 cell lines were selected and received treatment of varying dose of IL-2 to study the effect of IL-2 on expression of miR-26a and vascular cell adhesion molecule-1 (VCAM-1). miR-26a mimic and miR-26a inhibitor were transfected to study the effect on progression and migration of tumor cell as well as on levels of VCAM-1. RESULTS: We evidenced that, expression of miR-26a was suppressed, whereas levels of IL-2 and VCAM-1 were increased in NSCLC tissues. IL-2 down-regulated the levels of miR-26a but upregulated the levels of VCAM-1 in H-460 and H-226 cells. miR-26a modulated the expression level of VCAM-1 via binding the 3'-UTR region. We found that miR-26a attenuated the migration and proliferation of H-460 and H-226 cells. IL-2 modulated the levels of miR-26a via activating p65 but not STAT-3. CONCLUSIONS: All together, the finding of our study show that miR-26a blocks IL-2 mediated proliferation and migration of NSCLC via targeting VCAM-1.

Suppression of microRNA­27a protects against liver ischemia/reperfusion injury by targeting PPARγ and inhibiting endoplasmic reticulum stress.

Liver ischemia­reperfusion (I/R) injury is an important clinical issue related to liver transplantation. Recent studies suggest that microRNAs are implicated in various biological and pathological processes, including liver I/R injury. This study aimed to investigate the role and potential mechanism of miR­27a during liver I/R injury. A liver I/R model was induced via 60 min of ischemia and reperfusion for 6 h in rats. Cells were transfected with miR­27a mimics or the miR­27a inhibitor to examine the effect of miR­27a on liver I/R. Apoptotic cells were detected by flow cytometry and TUNEL staining. The expression of miR­27a was measured by real­time PCR. The expression of peroxisome proliferator­activated receptor γ (PPARγ); gastrin­releasing peptide 78 (GRP78) and C/EBP homologous protein (CHOP) were detected by western blot analysis. The results showed that miR­27a was significantly upregulated during I/R injury in vivo and in vitro. In addition, miR­27a inhibitors attenuated hypoxia/reoxygenation (H/R)­induced oxidative stress, endoplasmic reticulum stress (ERS) and apoptosis in AML12 cells. By contrast, miR­27a mimics promoted hypoxia/reoxygenation­induced ERS, and apoptosis. Furthermore, PPARγ was identified as a target gene of miR­27a using bioinformatic analysis and a dual­luciferase reporter assay. Knockdown of PPARγ significantly abrogated the inhibitory effect of miR­27a inhibitors on the ERS pathway. Moreover, the miR­27a antagomir attenuated liver I/R injury in rats, a finding manifested by reduced ALT/AST, hepatocyte apoptosis, oxidative stress and inhibition of the ERS pathway. Taken together, these findings demonstrate that suppression of miR­27a protects against liver I/R injury by targeting PPARγ and by inhibiting the ERS pathway.

Dihydromyricetin protects against liver ischemia/reperfusion induced apoptosis via activation of FOXO3a-mediated autophagy.

Liver ischemia and reperfusion (I/R) injury is characterized by defective liver autophagy accompanied by alterations to the endogenous defense system. Dihydromyricetin (DHM) is a natural flavonoid that demonstrates a wide range of physiological functions, and has been implicated as a regulator of autophagy. This study investigates the protective effects of DHM pretreatment on liver injury caused by ischemia/reperfusion (I/R) and elucidates the potential mechanism of DHM-mediated protection. Mice were subjected to 60 minutes of ischemia followed by 5 hours of reperfusion. DHM (100 mg/kg bw/day) or the vehicle was administered daily by gavage 7 days before ischemia and immediately before reperfusion. In this study, DHM markedly decreased serum aminotransferase activity and inhibited liver I/R -stimulated apoptosis. Moreover, DHM exerted hepatoprotective effects by upregulating mRNA levels of various essential autophagy-related genes including ATG5, ATG12, BECN1, and LC3. Autophagy inhibitor chloroquine or Atg5 knockdown blocked DHM -mediated elevation in liver function. Specifically, DHM significantly increased FOXO3a expression, and enhanced FOXO3a nuclear translocation and Ser588 phosphorylation modification. Importantly, the inhibition of FOXO3a with FOXO3a-siRNA in mice decreased DHM-induced autophagy-related genes and diminished the protective effects of DHM against liver I/R injury. In summary, these findings identify DHM as a novel hepatoprotective small molecule by elevating FOXO3a expression and nuclear translocation, stimulating autophagy-related genes and suppressing liver I/R-induced apoptosis, suggesting FOXO3a may have therapeutic value in liver cell protection in liver I/R injury.

Mitofusin 2 protects hepatocyte mitochondrial function from damage induced by GCDCA.

Mitochondrial impairment is hypothesized to contribute to the pathogenesis of chronic cholestatic liver diseases. Mitofusin 2 (Mfn2) regulates mitochondrial morphology and signaling and is involved in the development of numerous mitochondrial-related diseases; however, a functional role for Mfn2 in chronic liver cholestasis which is characterized by increased levels of toxic bile acids remain unknown. Therefore, the aims of this study were to evaluate the expression levels of Mfn2 in liver samples from patients with extrahepatic cholestasis and to investigate the role Mfn2 during bile acid induced injury in vitro. Endogenous Mfn2 expression decreased in patients with extrahepatic cholestasis. Glycochenodeoxycholic acid (GCDCA) is the main toxic component of bile acid in patients with extrahepatic cholestasis. In human normal hepatocyte cells (L02), Mfn2 plays an important role in GCDCA-induced mitochondrial damage and changes in mitochondrial morphology. In line with the mitochondrial dysfunction, the expression of Mfn2 decreased significantly under GCDCA treatment conditions. Moreover, the overexpression of Mfn2 effectively attenuated mitochondrial fragmentation and reversed the mitochondrial damage observed in GCDCA-treated L02 cells. Notably, a truncated Mfn2 mutant that lacked the normal C-terminal domain lost the capacity to induce mitochondrial fusion. Increasing the expression of truncated Mfn2 also had a protective effect against the hepatotoxicity of GCDCA. Taken together, these findings indicate that the loss of Mfn2 may play a crucial role the pathogenesis of the liver damage that is observed in patients with extrahepatic cholestasis. The findings also indicate that Mfn2 may directly regulate mitochondrial metabolism independently of its primary fusion function. Therapeutic approaches that target Mfn2 may have protective effects against hepatotoxic of bile acids during cholestasis.

Damage to mtDNA in liver injury of patients with extrahepatic cholestasis: the protective effects of mitochondrial transcription factor A.

Oxidative stress and mitochondrial dysfunction are involved in the pathogenesis of chronic liver cholestasis. Mitochondrial DNA (mtDNA) is highly susceptible to oxidative stress and mtDNA damage leads to mitochondrial dysfunction. This study aimed to investigate the mtDNA alterations that occurred during liver injury in patients with extrahepatic cholestasis. Along with an increase in malondialdehyde (MDA) levels and a decrease in ATP levels, extrahepatic cholestatic patients presented a significant increase in mitochondrial 8-hydroxydeoxyguanosine (8-OHdG) levels and decreases in mtDNA copy number, mtDNA transcript levels, and mtDNA nucleoid structure. In L02 cells, glycochenodeoxycholic acid (GCDCA) induced similar damage to the mtDNA and mitochondria. In line with the mtDNA alterations, the mRNA and protein levels of mitochondrial transcription factor A (TFAM) were significantly decreased both in cholestatic patients and in GCDCA-treated L02 cells. Moreover, overexpression of TFAM could efficiently attenuate the mtDNA damage induced by GCDCA in L02 cells. However, without its C-tail, ΔC-TFAM appeared less effective against the hepatotoxicity of GCDCA than the wild-type TFAM. Overall, our study demonstrates that mtDNA damage is involved in liver damage in extrahepatic cholestatic patients. The mtDNA damage is attributable to the loss of TFAM. TFAM has mtDNA-protective effects against the hepatotoxicity of bile acid during cholestasis.

Operative timing of liver transplantation for patients with severe hepatitis.

BACKGROUND: Fulminant hepatic failure manifests a rapid onset, serious complications, and a high mortality, but still there is a possibility of recovery. Once the patient is able to pass a crisis, the liver is able to regenerate completely and regain its normal function. Therefore it is of vital importance to determine the eligible timing for transplantation. Premature surgery might result in a loss of the chance of internal medical treatment and misuse of liver resources, whereas delayed surgery might increase the difficulty of treatment in the preoperative period and the possibility of complications and medical expense, which eventually result in decreased rate of success and survival. This problem remains worldwide how to choose the optional timing of operation. METHODS: Thirty-six patients with severe hepatitis were treated by orthotopic liver transplantation. The distribution of MELD scores in these patients was: 10-19 in 8 patients, 20-29 in 10, 30-39 in 11, and 40 in 7. They were divided into two groups: MELD score <30 and MELD score >or=30. Parameters (1-year survival rate, complications, preoperative use of artificial liver, operative time, volume of bleeding and blood transfusion, and average hospital costs) were examined as prognostic factors after liver transplantation. RESULTS: The 1-year survival rate of the MELD score <30 group was higher than that of the >or=30 group (77.8% and 33.3%, P=0.007), and the rate of complications in the <30 group was lower (P=0.012). There were no differences in the timing of artificial liver treatment, operative time, operative hemorrhage, and transfusion between the two groups (P=0.742). But the average daily hospital cost in the MELD score >or=30 group was higher (P=0.008). CONCLUSION: This study shows that when the MELD score is <30 it may be the optimal time to perform liver transplantation for patients with severe hepatitis.

[Hand-assisted laparoscopic hepatectomy for large liver cancer in 56 cases].

OBJECTIVE: To investigate the practical value of hand-assisted laparoscopic hepatectomy for large liver cancer in peripheral segments. METHODS: From March 2004 to December 2007, 56 patients with large liver cancer underwent hand-assisted laparoscopic hepatectomy including 53 cases of hepatocellular carcinoma, 2 cases of cholangiocellular carcinoma, 1 case of hepatic metastatic squamous carcinoma. RESULTS: The operation procedures were completed safely in all patients including 27 left lateral segment hepatectomy, 6 left hemi-hepatectomy and 23 atypical right hepatectomy. Thirty-one cases with hepatic hilum blocking in the procedure and the mean time was 16.7 minutes. Mean surgical time was 105.3 minutes. Mean blood loss was 97 ml. Mean gross tumor size was 8.6 cm. Mean excisional hepatic tissue volume was 10.5 cm. No serious postoperative complications occurred. Mean eating time was 2.1 days. The mean postoperative hospital stay was 7.3 days. CONCLUSION: Hand-assisted laparoscopic hepatectomy for large liver carcinoma is feasible and safe for selected patients.

Liver transplant for 70 patients with end-stage liver diseases.

BACKGROUND: Liver transplantation has evolved as a successful treatment for patients with end-stage liver cirrhosis and acute liver failure. Postoperative survival rates have increased to 90% in 1 year and 80% in 5 years as a result of improvements in immunosuppression, perioperative management and surgical techniques. However, a wide range of postoperative complications are of technical or medical origin. This study was undertaken to determine the relationship between the technical improvements and optimal timing of surgery and its outcome. METHODS: From April 1999 to October 2005, typical orthotopic or piggyback liver transplantation was performed in 70 patients (58 men and 12 women, aged 19-74 years). Twenty-four patients had liver carcinoma and cirrhosis, and 46 had benign liver disease. RESULTS: All patients survived the operation and 14 died in the first month after surgery because of respiratory failure (6), respiratory failure accompanied by acute renal failure (4), intra-abdominal hemorrhage and infection (2), and cerebral edema (2). A total of 76 complications occurred in the 70 patients after operation: pneumonia (34), right pleural effusion (11), bile leakage (7), postoperative intra-abdominal hemorrhage and infection (4), acute renal failure (4), acute rejection (3), wound infection (2), biliary tract stenosis (2), severe cholangitis derived from cholelith (2), morphological alteration of biliary tree (2), cerebral edema (2), empyema (1), chronic rejection (1), and wound hematoma (1). Finally, 33 patients survived more than 6 months, 16 more than 1 year, 4 more than 2 years, and 2 more than 6 years after operation. The perioperative survival rate was 80% in this series. CONCLUSIONS: Liver transplantation is an effective treatment for patients with end-stage liver disease. To obtain good results, improvements of surgical technique, optimal timing and better postoperative care are needed.