Anti HCV activity and expression inhibition of HCC markers by protein extract from Iberis gibraltarica / Atividade anti-HCV e inibição da expressão de marcadores HCC por extrato de proteína de Iberis gibraltarica

Hepatitis C virus infection (HCV) is the foremost reason of progressive hepatic fibrosis and cirrhosis, with an elevated risk of hepatocellular carcinoma (HCC) development. Medicinal plants have been used for human health benefits for several years, but their therapeutic potential needs to be explored. The main objective of this study was to figure out the in vitro antiviral and anticancer characteristics of total crude protein of Iberis gibraltarica against HCV and HCC. Total crude protein of Iberis gibraltarica was isolated and quantified. The level of cytotoxicity was measured against the HepG2 cell line and it shows no significant cytotoxicity at the concentration of 504µg/ml. The anti-HCV effect was determined by absolute quantification via real time RT-PCR method and viral titer was reduced up to 66% in a dose dependent manner against the total protein of Iberis gibraltarica. The anticancer potential of Iberis gibraltarica was also examined through mRNA expression studies of AFP and GPC3 genes against the total protein of Iberis gibraltarica-treated HepG2 cells. The results show up to 90% of the down-regulation expression of AFP and GPC3. The obtained results indicate the therapeutic potential of total protein of Iberis gibraltarica against HCV and hepatocellular carcinoma in vitro.

A infecção pelo vírus da hepatite C (HCV) é a principal causa de fibrose hepática progressiva e cirrose, com risco elevado de desenvolvimento de carcinoma hepatocelular (HCC). As plantas medicinais vêm sendo utilizadas para benefícios à saúde humana há vários anos, mas seu potencial terapêutico precisa ser explorado. O principal objetivo deste estudo foi descobrir as características antivirais e anticancerígenas in vitro da proteína bruta total de Iberis gibraltarica contra HCV e HCC. A proteína bruta total de Iberis gibraltarica foi isolada e quantificada. O nível de citotoxicidade foi medido contra a linha celular HepG2 e não apresenta citotoxicidade significativa na concentração de 504µg/ml. O efeito anti-HCV foi determinado por quantificação absoluta através do método RT-PCR em tempo real e o título viral foi reduzido em até 66% de forma dose-dependente contra a proteína total de Iberis gibraltarica. O potencial anticancerígeno de Iberis gibraltarica também foi examinado através de estudos de expressão de mRNA dos genes AFP e GPC3 contra a proteína total de células HepG2 tratadas com Iberis gibraltarica. Os resultados mostram até 90% da expressão de regulação negativa de AFP e GPC3. Os resultados obtidos indicam o potencial terapêutico da proteína total de Iberis gibraltarica contra HCV e carcinoma hepatocelular in vitro.

The synergism of hepatitis B and aflatoxicosis in hepatocellular carcinoma development: A mini-review

Hepatocellular carcinoma (HCC) is the twelfth most common cancer and the fifth leading cause of worldwide cancer related death. Chronic hepatitis B infection, caused by the hepatitis B virus (HBV) and exposure to aflatoxins is fundamental in the formation of HCC in developing countries. This review of scientific publications aims to establish the detrimental effects of aflatoxin-contaminated foods and highlights the correlation between aflatoxin and hepatitis B viral-associated hepatocellular carcinoma. Research has shown a significant increase in the occurrence of HCC in HBV-infected individuals exposed to fungal toxins. HBV demonstrates the ability to integrate and bind to p53 protein in the host DNA and propagate hepatocyte vulnerability through carcinogenic aflatoxin B1 (AFB1) damage. Although there has been clear evidence about the synergistic interaction of exposure to AFB1 and HBV infection in the induction of HCC, other literature has shown otherwise, mainly because incomplete and vague findings and hypotheses were made in regions where AFB1 and HBV pose a public health risk. Vaccination against hepatitis B and measures such as robust food safety systems to avoid hepatotoxicity and hepatocellular carcinogenesis induced by AFB1 is the most effective methods in the prevention of HCC induced by HBV and AFB1

Physical exercise suppresses hepatocellular carcinoma progression by alleviating hypoxia and attenuating cancer stemness through the Akt/GSK-3β/β-catenin pathway / 中西医结合学报

OBJECTIVE@#Physical exercise, a common non-drug intervention, is an important strategy in cancer treatment, including hepatocellular carcinoma (HCC). However, the mechanism remains largely unknown. Due to the importance of hypoxia and cancer stemness in the development of HCC, the present study investigated whether the anti-HCC effect of physical exercise is related to its suppression on hypoxia and cancer stemness.@*METHODS@#A physical exercise intervention of swimming (30 min/d, 5 d/week, for 4 weeks) was administered to BALB/c nude mice bearing subcutaneous human HCC tumor. The anti-HCC effect of swimming was assessed in vivo by tumor weight monitoring, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) detection of proliferating cell nuclear antigen (PCNA) and Ki67. The expression of stemness transcription factors, including Nanog homeobox (NANOG), octamer-binding transcription factor 4 (OCT-4), v-Myc avian myelocytomatosis viral oncogene homolog (C-MYC) and hypoxia-inducible factor-1α (HIF-1α), was detected using real-time reverse transcription polymerase chain reaction. A hypoxia probe was used to explore the intratumoral hypoxia status. Western blot was used to detect the expression of HIF-1α and proteins related to protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling pathway. The IHC analysis of platelet endothelial cell adhesion molecule-1 (CD31), and the immunofluorescence co-location of CD31 and desmin were used to analyze tumor blood perfusion. SMMC-7721 cells were treated with nude mice serum. The inhibition effect on cancer stemness in vitro was detected using suspension sphere experiments and the expression of stemness transcription factors. The hypoxia status was inferred by measuring the protein and mRNA levels of HIF-1α. Further, the expression of proteins related to Akt/GSK-3β/β-catenin signaling pathway was detected.@*RESULTS@#Swimming significantly reduced the body weight and tumor weight in nude mice bearing HCC tumor. HE staining and IHC results showed a lower necrotic area ratio as well as fewer PCNA or Ki67 positive cells in mice receiving the swimming intervention. Swimming potently alleviated the intratumoral hypoxia, attenuated the cancer stemness, and inhibited the Akt/GSK-3β/β-catenin signaling pathway. Additionally, the desmin+/CD31+ ratio, rather than the number of CD31+ vessels, was significantly increased in swimming-treated mice. In vitro experiments showed that treating cells with the serum from the swimming intervention mice significantly reduced the formation of SMMC-7721 cell suspension sphere, as well as the mRNA expression level of stemness transcription factors. Consistent with the in vivo results, HIF-1α and Akt/GSK-3β/β-catenin signaling pathway were also inhibited in cells treated with serum from swimming group.@*CONCLUSION@#Swimming alleviated hypoxia and attenuated cancer stemness in HCC, through suppression of the Akt/GSK-3β/β-catenin signaling pathway. The alleviation of intratumoral hypoxia was related to the increase in blood perfusion in the tumor. Please cite this article as: Xiao CL, Zhong ZP, Lü C, Guo BJ, Chen JJ, Zhao T, Yin ZF, Li B. Physical exercise suppresses hepatocellular carcinoma progression by alleviating hypoxia and attenuating cancer stemness through the Akt/GSK-3β/β-catenin pathway. J Integr Med. 2023; 21(2): 184-193.

Shikonin induces hepatocellular carcinoma cell apoptosis by suppressing PKM2/PHD3/HIF-1α signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the mechanism of shikonin-induced death of human hepatocellular carcinoma SMMC-7721 cells.@*METHODS@#Cultured SMMC-7721 cells and normal hepatocytes (L-02 cells) were treated with 4, 8, or 16 μmol/L shikonin, and the changes in cell viability was assessed using MTT assay. The levels of ATP and lactic acid in the cell cultures were detected using commercial kits. Co-immunoprecipitation and immunofluorescence staining were used to determine the relationship among pyruvate kinase M2 (PKM2), prolyl hydroxylase 3 (PHD3), and hypoxia-inducible factor-1α (HIF-1α). The expressions of PHD3, PKM2, HIF-1α, Bax, cleaved caspase-3, and Bcl-2 in SMMC-7721 cells were detected with Western blotting, and cell apoptosis was analyzed with annexin V-FITC/PI staining. The effects of RNA interference of PKM2 on PHD3 and HIF-1α expressions in SMMC-7721 cells were detected using Western blotting.@*RESULTS@#The IC50 of shikonin against SMMC-7721 and L-02 cells was 8.041 μmol/L and 31.75 μmol/L, respectively. Treatment with shikonin significantly inhibited the protein expressions of PKM2, HIF-1α and PHD3 and nuclear translocation of PKM2 and HIF-1α in SMMC-7721 cells. Coimmunoprecipitation and immunofluorescence staining confirmed that shikonin inhibited the formation of PKM2/PHD3/HIF-1α complex and significantly reduced the contents of lactic acid and ATP in SMMC-7721 cells (P < 0.05). The expressions of PHD3 and HIF-1α decreased significantly after PKM2 knockdown (P < 0.05). Shikonin treatment significantly increased the apoptosis rate, enhanced the expressions of Bax and cleaved caspase-3, and decreased Bcl-2 expression in SMMC-7721 cells (P < 0.05).@*CONCLUSIONS@#Shikonin induces apoptosis of SMMC-7721 cells possibly by inhibiting aerobic glycolysis through the PKM2/PHD3/HIF-1α signaling pathway to cause energy supply dysfunction in the cells.

Overexpression of CLEC5A inhibits cell proliferation and metastasis and reverses epithelial-mesenchymal transition in hepatocellular carcinoma / 南方医科大学学报

OBJECTIVE@#To evaluate the effects of CLEC5A expression level on cell proliferation, migration and invasion and epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) and explore the role of CLEC5A in the tumorigenesis and progression of HCC.@*METHODS@#The expression level of CLEC5A was detected in 50 pairs of HCC and adjacent tissues using immunohistochemical staining, and its association with clinicopathological parameters of HCC patients was analyzed. Cultured HCC cell line SK-HEP-1 was transfected with a lentiviral vector overexpressing CLEC5A, and the transfection efficiency was verified using real-time fluorescence quantitative PCR and Western blotting. The changes in proliferation, migration and invasion abilities of the transfected cells were analyzed using CCK-8, 5-ethynyl-29-deoxyuridine (EdU) and Transwell assays, and EMT of the cells was determined using Western blotting.@*RESULTS@#The protein expression level of CLEC5A was significantly lower in HCC tissues than in the adjacent tissues (P < 0.001). The expression level of CLEC5A was significantly correlated with tumor size (P=0.008), tumor number (P=0.010), histological differentiation (P=0.016), microvascular invasion (P=0.024) and BCLC stage (P=0.040). In SK-HEP-1 cells, overexpression of CLEC5A obviously inhibited the cell proliferation, migration and invasion and reversed EMT phenotype of the cells.@*CONCLUSION@#CLEC5A is a potential HCC suppressor gene and may serve as a promising therapeutic target for HCC.

Advances in post-operative prognostic models for hepatocellular carcinoma / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Hepatocellular carcinoma (HCC) is one of the most common malignancies and a leading cause of cancer-related death worldwide. Surgery remains the primary and most successful therapy option for the treatment of early- and mid-stage HCCs, but the high heterogeneity of HCC renders prognostic prediction challenging. The construction of relevant prognostic models helps to stratify the prognosis of surgically treated patients and guide personalized clinical decision-making, thereby improving patient survival rates. Currently, the prognostic assessment of HCC is based on several commonly used staging systems, such as Tumor-Node-Metastasis (TNM), Cancer of the Liver Italian Program (CLIP), and Barcelona Clinic Liver Cancer (BCLC). Given the insufficiency of these staging systems and the aim to improve the accuracy of prognostic prediction, researchers have incorporated further prognostic factors, such as microvascular infiltration, and proposed some new prognostic models for HCC. To provide insights into the prospects of clinical oncology research, this review describes the commonly used HCC staging systems and new models proposed in recent years.

Targeting TRMT5 suppresses hepatocellular carcinoma progression via inhibiting the HIF-1α pathways / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Accumulating evidence has confirmed the links between transfer RNA (tRNA) modifications and tumor progression. The present study is the first to explore the role of tRNA methyltransferase 5 (TRMT5), which catalyzes the m1G37 modification of mitochondrial tRNAs in hepatocellular carcinoma (HCC) progression. Here, based on bioinformatics and clinical analyses, we identified that TRMT5 expression was upregulated in HCC, which correlated with poor prognosis. Silencing TRMT5 attenuated HCC proliferation and metastasis both in vivo and in vitro, which may be partially explained by declined extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Mechanistically, we discovered that knockdown of TRMT5 inactivated the hypoxia-inducible factor-1 (HIF-1) signaling pathway by preventing HIF-1α stability through the enhancement of cellular oxygen content. Moreover, our data indicated that inhibition of TRMT5 sensitized HCC to doxorubicin by adjusting HIF-‍1α. In conclusion, our study revealed that targeting TRMT5 could inhibit HCC progression and increase the susceptibility of tumor cells to chemotherapy drugs. Thus, TRMT5 might be a carcinogenesis candidate gene that could serve as a potential target for HCC therapy.

Acteoside promotes autophagy and apoptosis of hepatoma cells by regulating JNK signaling pathway / 中国中药杂志

This study explored the molecular mechanism of acteoside against hepatoma 22(H22) tumor in mice through c-Jun N-terminal kinase(JNK) signaling pathway. H22 cells were subcutaneously inoculated in 50 male BALB/c mice, and then the model mice were classified into model group, low-dose, medium-dose, and high-dose acteoside groups, and cisplatin group. The administration lasted 2 weeks for each group(5 consecutive days/week). The general conditions of mice in each group, such as mental status, diet intake, water intake, activity, and fur were observed. The body weight, tumor volume, tumor weight, and tumor-inhibiting rate were compared before and after administration. Morphological changes of liver cancer tissues were observed based on hematoxylin and eosin(HE) staining, and the expression of phosphorylated(p)-JNK, JNK, B-cell lymphoma-2(Bcl-2), Beclin-1, and light chain 3(LC3) in each tissue was detected by immunohistochemistry and Western blot. qRT-PCR was performed to detect the mRNA expression of JNK, Bcl-2, Beclin-1, and LC3. The general conditions of mice in model and low-dose acteoside groups were poor, while the general conditions of mice in the remaining three groups were improved. The body weight of mice in medium-dose acteoside group, high-dose acteoside group, and cisplatin group was smaller than that in model group(P<0.01). The tumor volume in model group was insignificantly different from that in low-dose acteoside group, and the volume in cisplatin group showed no significant difference from that in high-dose acteoside group. Tumor volume and weight in medium-dose and high-dose acteoside groups and cisplatin group were lower than those in the model group(P<0.001). The tumor-inhibiting rates were 10.72%, 40.32%, 53.79%, and 56.44% in the low-dose, medium-dose, and high-dose acteoside groups and cisplatin group, respectively. HE staining showed gradual decrease in the count of hepatoma cells and increasing sign of cell necrosis in the acteoside and cisplatin groups, and the necrosis was particularly obvious in the high-dose acteoside group and cisplatin group. Immunohistochemical results suggested that the expression of Beclin-1, LC3, p-JNK, and JNK was up-regulated in acteoside and cisplatin groups(P<0.05). The results of immunohistochemistry, Western blot, and qRT-PCR indicated that the expression of Bcl-2 was down-regulated in the medium-dose and high-dose acteoside groups and cisplatin group(P<0.01). Western blot showed that the expression of Beclin-1, LC3, and p-JNK was up-regulated in acteoside and cisplatin groups(P<0.01), and there was no difference in the expression of JNK among groups. qRT-PCR results showed that the levels of Beclin-1 and LC3 mRNA were up-regulated in the acteoside and cisplatin groups(P<0.05), and the level of JNK mRNA was up-regulated in medium-dose and high-dose acteoside groups and cisplatin group(P<0.001). Acteoside promotes apoptosis and autophagy of H22 cells in mice hepatoma cells by up-regulating the JNK signaling pathway, thus inhibiting tumor growth.

Lamin B1 regulates the growth of hepatocellular carcinoma cells by influencing telomerase activity / 生物工程学报

Lamin B1 (LMNB1) is highly expressed in liver cancer tissues, and its influence and mechanism on the proliferation of hepatocellular carcinoma cells were explored by knocking down the expression of the protein. In liver cancer cells, siRNAs were used to knock down LMNB1. Knockdown effects were detected by Western blotting. Changes in telomerase activity were detected by telomeric repeat amplification protocol assay (TRAP) experiments. Telomere length changes were detected by quantitative real-time polymerase chain reaction (qPCR). CCK8, cloning formation, transwell and wound healing were performed to detect changes in its growth, invasion and migration capabilities. The lentiviral system was used to construct HepG2 cells that steadily knocked down LMNB1. Then the changes of telomere length and telomerase activity were detected, and the cell aging status was detected by SA-β-gal senescence staining. The effects of tumorigenesis were detected by nude mouse subcutaneous tumorigenesis experiments, subsequent histification staining of tumors, SA-β-gal senescence staining, fluorescence in situ hybridization (FISH) for telomere analysis and other experiments. Finally, the method of biogenesis analysis was used to find the expression of LMNB1 in clinical liver cancer tissues, and its relationship with clinical stages and patient survival. Knockdown of LMNB1 in HepG2 and Hep3B cells significantly reduced telomerase activity, cell proliferation, migration and invasion abilities. Experiments in cells and tumor formation in nude mice had demonstrated that stable knockdown of LMNB1 reduced telomerase activity, shortened telomere length, senesced cells, reduced cell tumorigenicity and KI-67 expression. Bioinformatics analysis showed that LMNB1 was highly expressed in liver cancer tissues and correlated with tumor stage and patient survival. In conclusion, LMNB1 is overexpressed in liver cancer cells, and it is expected to become an indicator for evaluating the clinical prognosis of liver cancer patients and a target for precise treatment.

Chinese expert consensus on intra-arterial drug and combined drug administration for primary hepatocellular carcinoma / 中华内科杂志

Transarterial interventional therapy is one of the most widely used treatment methods in patients with primary hepatocellular carcinoma. With the progress in interventional technology and the use of new drugs, transarterial interventional therapy has achieved favorable results in the treatment of primary hepatocellular carcinoma and has become the first choice non-surgical treatment for advanced liver cancer. However, at present, there are great differences in the drugs used in transarterial interventional treatment and the combined application of other drugs among centers, and there is no uniform consensus or guideline. Based on the latest research data and clinical practice experience, as well as the characteristics of Chinese patients, the Specialist Group of Interventional Drugs, Interventionalists Branch of the Chinese Medical Doctor Association was organized to formulate the Chinese expert consensus on intra-arterial drug and combined drug administration for primary hepatocellular carcinoma. The purpose of this consensus is to explore the efficacy and safety of drugs and drug combinations related to intra-arterial interventional therapy, the use of drugs in special populations, the management of adverse reactions, and adjuvant drugs to provide a reference for clinical practice.

Lenvatinib down-regulates IGF1R/Mek/Erk signaling pathway in the treatment of regorafenib-resistant hepatocellular carcinoma / 中华肿瘤杂志

Objective: To investigate the therapeutic effect and mechanism of lenvatinib on regorafenib-resistant hepatocellular carcinoma cells. Methods: CCK-8 and clone formation assay were used to observe the inhibitory effect of lenvatinib on the growth of hepatocellular carcinoma cells. Flow cytometry was used to detect the apoptosis of regorafenib-resistant hepatocellular carcinoma cells treated with lenvatinib. The expression levels of related proteins were detected by western blot and immunohistochemical staining. The inhibitory effect of lenvatinib on the tumor formation ability of regorafenib-resistant hepatocellular carcinoma cells in vivo was observed by subcutaneous tumor formation experiment in mice. Results: CCK-8 and clone formation assay showed that lenvatinib could inhibit the proliferation of regorafenib-resistant hepatocellular carcinoma cells. The number of clones of HepG2, SMMC7721 and regorafenib-resistant HepG2, SMMC7721 cells in lenvatinib group (120.67±11.06, 53.00±11.14, 55.00±9.54, 78.67±14.64) were all lower than those in control group (478.00±24.52, 566.00±27.87, 333.67±7.02, 210.00±12.77, all P<0.05). Flow cytometry showed that lenvatinib could promote apoptosis of regorafenib-resistant hepatocellular carcinoma cells, the apoptosis rates of HepG2, SMMC7721 and regorafenib-resistant HepG2, SMMC7721 cells in lenvatinib group [(12.30±0.70)%, (9.83±0.38)%, (15.90±1.32)%, (10.60±0.00)%] were all higher than those in control group [(7.50±0.87)%, (5.00±1.21)%, (8.10±1.61)%, (7.05±0.78)%, all P<0.05]. The apoptosis-related protein levels suggested that apoptosis was increased in the treatment of lenvatinib. The animal study showed that lenvatinib can inhibit the growth of regorafenib-resistant cells in vivo. Immunohistochemistry and western blot results showed that lenvatinib could down-regulate the abnormally activated IGF1R/Mek/Erk signaling pathway in regorafenib-resistant cells. Conclusion: Lenvatinib can reverse regorafenib resistance in hepatocellular carcinoma, possibly by down-regulating IGF1R/Mek/Erk signaling pathway.

Precision diagnosis of hepatocellular carcinoma / 中华医学杂志(英文版)

Hepatocellular carcinoma (HCC) is the most common type of primary hepatocellular carcinoma (PHC). Early diagnosis of HCC remains the key to improve the prognosis. In recent years, with the promotion of the concept of precision medicine and more in-depth analysis of the biological mechanism underlying HCC, new diagnostic methods, including emerging serum markers, liquid biopsies, molecular diagnosis, and advances in imaging (novel contrast agents and radiomics), have emerged one after another. Herein, we reviewed and analyzed scientific advances in the early diagnosis of HCC and discussed their application and shortcomings. This review aimed to provide a reference for scientific research and clinical practice of HCC.

Comparative effectiveness of different hepatocellular carcinoma screening intervals or modalities: a systematic review and meta-analysis / 中华医学杂志(英文版)

BACKGROUND@#Current guidelines recommend hepatocellular carcinoma (HCC) screening in high-risk populations. However, the ideal HCC screening interval and screening modality have not been determined. This study aimed to compare the screening efficacy among different modalities with various intervals.@*METHODS@#PubMed and other nine databases were searched through June 30, 2021. Binary outcomes were pooled using risk ratio (RR) with 95% confidence intervals (CIs). Survival rates were also pooled using RR with 95% CIs because most eligible studies only provided the number of survival patients instead of hazard ratio.@*RESULTS@#In all, 13 studies were included. Two random controlled trials (RCTs) and six cohort studies compared screening intervals for ultrasonography (US) screening and found no significant differences between shorter (3- or 4-month) and longer (6- or 12-month) screening intervals in terms of early HCC proportion, HCC significant mortality, 1-year survival rate; screening at 6-month interval significantly increased the proportion of early HCC (RR = 1.17, 95% confidence interval [CI]: 1.08-1.26) and prolonged the 5-year survival rate (RR = 1.39, 95% CI: 1.07-1.82) relative to the 12-month interval results. Three other RCTs and two cohort studies compared different screening modalities in cirrhosis or chronic hepatitis B, which indicated no statistical differences in the proportion of early HCC (RR = 0.89, 95% CI: 0.40-1.96) and HCC mortality (RR = 0.69, 95% CI: 0.23-2.09) between the biannual US and annual computed tomography (CT screening). Biannual US screening showed a lower proportion of early HCC than biannual magnetic resonance imaging (MRI) (RR = 0.60, 95% CI: 0.37-0.97) and biannual US combined with annual CT (RR = 1.31, 95% CI: 1.13-1.51) screening. The proportion of early HCC in the contrast-enhanced US group was slightly higher than that in the B-mode US (RR = 1.08, 95% CI: 1.00-1.23) group.@*CONCLUSIONS@#The evidence suggests that 6 months may be the best HCC screening interval for US screening. The effectiveness of CT and MRI is better than US during same screening intervals. However, MRI and CT are more expensive than US, and CT also can increase the risk of radiation exposure. The selection of CT or MRI instead of US should be carefully considered.@*REGISTRATION@#No. CRD42020148258 at PROSPERO website ( https://www.crd.york.ac.uk/PROSPERO/ ).

Procine recombinant NK-lysin inhibits hepatocellular carcinoma metastasis by downregulating FKBP3 and inhibiting oxidative phosphorylation and glycolysis: a proteomic analysis / 南方医科大学学报

OBJECTIVE@#To investigate the potential mechanisms that mediate the inhibitory effect of porcine recombinant NKlysin (prNK-lysin) against liver cancer cell metastasis.@*METHODS@#HPLC-tandem mass spectrometry was used to identify the differentially expressed proteins in prNK-lysin-treated hepatocellular carcinoma SMMOL/LC-7721 cells in comparison with the control and PBS-treated cells. GO functional annotation and KEGG pathway analysis of the differentially expressed proteins were performed using GO and KEGG databases. RT-qPCR was used to determine the mRNA expression levels of polypeptide-N-acetylgalactosaminotransferase 13 (GALNT13), transmembrane protein 51 (TMEM51) and FKBP prolyl isomerase 3 (FKBP3) in the cells, and the protein expression of FKBP3 was verified using Western blotting.@*RESULTS@#Proteomic analysis identified 1989 differentially expressed proteins in prNK-lysin-treated cells compared with the control cells, and 2753 compared with PBS-treated cells. Fifteen proteins were differentially expressed between PBS-treated and the control cells, and 1909 were differentially expressed in prNK- lysin group compared with both PBS and control groups. These differentially expressed proteins were involved mainly in the viral process, translational initiation and RNA binding and were enriched mainly in ribosome, protein process in endoplasmic reticulum, and RNA transport pathways. RT-qPCR showed that compared with the control group, prNK-lysin treatment significantly increased the mRNA expressions of GALNT13 (P < 0.05) and TMEM51 (P < 0.01) and lowered FKBP3 mRNA expression (P < 0.05). Western blotting also showed a significantly decreased expression of FKBP3 protein in prNK-lysin-treated cells (P < 0.001).@*CONCLUSION@#Treatment with prNK-lysin causes significant changes in protein expression profile of SMMOL/LC-7721 cells and inhibits hepatocellular carcinoma metastasis by downregulating FKBP3 protein and affecting the cellular oxidative phosphorylation and glycolysis pathways.

Prediction of microvascular invasion in hepatocellular carcinoma with magnetic resonance imaging using models combining deep attention mechanism with clinical features / 南方医科大学学报

OBJECTIVE@#To investigate the consistency and diagnostic performance of magnetic resonance imaging (MRI) for detecting microvascular invasion (MVI) of hepatocellular carcinoma (HCC) and the validity of deep learning attention mechanisms and clinical features for MVI grade prediction.@*METHODS@#This retrospective study was conducted among 158 patients with HCC treated in Shunde Hospital Affiliated to Southern Medical University between January, 2017 and February, 2020. The imaging data and clinical data of the patients were collected to establish single sequence deep learning models and fusion models based on the EfficientNetB0 and attention modules. The imaging data included conventional MRI sequences (T1WI, T2WI, and DWI), enhanced MRI sequences (AP, PP, EP, and HBP) and synthesized MRI sequences (T1mapping-pre and T1mapping-20 min), and the high-risk areas of MVI were visualized using deep learning visualization techniques.@*RESULTS@#The fusion model based on T1mapping-20min sequence and clinical features outperformed other fusion models with an accuracy of 0.8376, a sensitivity of 0.8378, a specificity of 0.8702, and an AUC of 0.8501 for detecting MVI. The deep fusion models were also capable of displaying the high-risk areas of MVI.@*CONCLUSION@#The fusion models based on multiple MRI sequences can effectively detect MVI in patients with HCC, demonstrating the validity of deep learning algorithm that combines attention mechanism and clinical features for MVI grade prediction.

EHHADH is a key gene in fatty acid metabolism pathways in hepatocellular carcinoma: a transcriptomic analysis / 南方医科大学学报

OBJECTIVE@#To explore the driving gene of hepatocellular carcinoma (HCC) occurrence and progression and its potential as new therapeutic target of HCC.@*METHODS@#The transcriptome and genomic data of 858 HCC tissues and 493 adjacent tissues were obtained from TCGA, GEO, and ICGC databases. Gene Set Enrichment Analysis (GSEA) identified EHHADH (encoding enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase) as the hub gene in the significantly enriched differential pathways in HCC. The downregulation of EHHADH expression at the transcriptome level was found to correlate with TP53 mutation based on analysis of the TCGA- HCC dataset, and the mechanism by which TP53 mutation caused EHHADH downregulation was explored through correlation analysis. Analysis of the data from the Metascape database suggested that EHHADH was strongly correlated with the ferroptosis signaling pathway in HCC progression, and to verify this result, immunohistochemical staining was used to examine EHHADH expression in 30 HCC tissues and paired adjacent tissues.@*RESULTS@#All the 3 HCC datasets showed signficnatly lowered EHHADH expression in HCC tissues as compared with the adjacent tissues (P < 0.05) with a close correlation with the degree of hepatocyte de-differentiation (P < 0.01). The somatic landscape of HCC cohort in TCGA dataset showed that HCC patients had the highest genomic TP53 mutation rate. The transcriptomic level of PPARGC1A, the upstream gene of EHHADH, was significantly downregulated in HCC patients with TP53 mutation as compared with those without the mutation (P < 0.05), and was significantly correlated with EHHADH expression level. GO and KEGG enrichment analyses showed that EHHADH expression was significantly correlated with abnormal fatty acid metabolism in HCC. The immunohistochemical results showd that the expression level of EHHADH in HCC tissues was down-regulated, and its expression level was related to the degree of hepatocytes de-differentiation and the process of ferroptosis.@*CONCLUSION@#TP53 mutations may induce abnormal expression of PPARGC1A to cause downregulation of EHHADH expression in HCC. The low expression of EHHADH is closely associated with aggravation of de-differentiation and ferroptosis escape in HCC tissues, suggesting the potential of EHHADH as a therapeutic target for HCC.

Effects of titanium dioxide nanoparticles on circRNA expression profiles in human hepatocellular carcinoma cells HepG2 / 北京大学学报(医学版)

OBJECTIVE@#To investigate the effect of titanium dioxide nanoparticles (TiO2 NPs) on the expression profile of circular ribonucleic acid (circRNA) in human hepatocytes through in vitro cell experiments, and to attempt to understand the potential mechanism of hepatotoxicity through bioinformatics analysis.@*METHODS@#TiO2 NPs were characterized from the aspects of particle size, shape and agglomeration state. The cell counting kit-8 (CCK8) was used to detect the cytotoxicity of TiO2 NPs against human hepatocellular carcinoma cells (HepG2) after exposure to 0, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 mg/L TiO2 NPs for 24 h or 48 h. The cells were treated at doses of 0 mg/L TiO2 NPs (control group) and 100 mg/L TiO2 NPs (treatment group), and collected after exposure for 48 h, and then RNA from the extracted cell samples was collected and sequenced. The differential circRNAs between the control and the TiO2 NPs treatment groups were screened, and then the enrichment pathway of the differential circRNA target gene was analyzed by multivariate statistics. According to the sequencing results, significantly altered genes and important genes in the significant enrichment pathways were screened, and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) was performed to verify the results.@*RESULTS@#TiO2 NPs were spherical anatase with a hydrated particle size of (323.50±85.44) nm and a Zeta potential of (-21.00±0.72) mV in a serum-free medium. The results of the CCK8 cytotoxicity assay showed that with the increase of TiO2 NPs concentration, cell viability gradually decreased. A total of 11 478 circRNAs were found by RNA sequencing. Compared with the control groups, TiO2 NPs treatment groups (100 mg/L) had a total of 89 differential circRNAs, of which 59 were up-regulated and 30 were down-regulated. Analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway showed that the targeted genes of differential circRNAs were mainly enriched in fatty acid degradation, Fanconi anemia pathway, and fatty acid metabolism. The expression levels of circRNA.6730, circRNA.3650 and circRNA.4321 were significantly different between the TiO2 NPs treatment group and the control group, which were consistent with the sequencing results.@*CONCLUSION@#TiO2 NPs can induce changes in circRNA expression profile, and epigenetics may play an important role in the mechanism of hepatotoxicity.

Study on F9 gene expression downregulation and its clinical value in hepatocellular carcinoma / 中华肝脏病杂志

Objective: To analyze the expression levels of the F9 gene and F9 protein in hepatocellular carcinoma by combining multiple gene chip data, real-time fluorescence quantitative PCR (RT qPCR), and immunohistochemistry. Additionally, explore their correlation with the occurrence and development of hepatocellular carcinoma, as well as with various clinical indicators and prognosis. Methods: The mRNA microarray dataset from the GEO database was analyzed to identify the F9 gene with significant expression differences associated with hepatocellular carcinoma. Liver cancer and adjacent tissues were collected from 18 cases of hepatocellular carcinoma. RT-qPCR method was used to detect the F9 gene expression level. Immunohistochemistry was used to detect the F9 protein level. Combined with the TCGA database information, the correlation between F9 gene expression level and prognostic and clinicopathological parameters was analyzed. The biological function of F9 co-expressed genes associated with hepatocellular carcinoma was analyzed by the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Statistical analysis was performed using Graphpad Prism software. Results: Meta-analysis results showed that the expression of the F9 gene was lower in HCC tissues than in non-cancerous tissues. Immunohistochemistry results were basically consistent with those of RT-qPCR. The data obtained from TCGA showed that the F9 gene had lower expression values in stages III-IV, T3-T4, and patients with vascular invasion. A total of 127 genes were selected for bioinformatics analysis as co-expressed genes of F9, which were highly enriched in redox processes and metabolic pathways. Conclusion: This study validates that the F9 gene and F9 protein are lower in HCC. The down-regulation of the F9 gene predicts adverse outcomes, which may provide a new therapeutic target for HCC.

Clinical study on early predictors of concurrent bile duct injury following TACE in patients with liver cancer / 中华肝脏病杂志

Objective: To explore the predictive factors of concurrent bile duct injury following transcatheter arterial chemoembolization (TACE) in patients with hepatocellular carcinoma (HCC). Methods: A retrospective study was conducted on 483 HCC patients in relation to TACE postoperative complications. A total of 21 cases of bile duct injury were observed following the TACE procedure. Laboratory data, imaging data, and clinically relevant medical histories were recorded before and after one week following the TACE procedure and follow-up. The χ (2) test, or Fisher's exact probability method, was used for categorical variables. The mean of the two samples was compared using a paired t-test or Wilcoxon rank sum test. The comparison of multiple mean values was conducted using an analysis of variance. Results: Twenty-one cases with bile duct injury had intrahepatic bile duct dilatation, bile tumors, hilar biliary duct stenoses, and other manifestations. 14.3% (3/21) of patients showed linear high-density shadows along the bile duct on a plain CT scan, while 76.2% (16/21) of patients had ALP > 200 U/L one week following TACE procedure, and bile duct injury occurred in later follow-up. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and γ-glutamyl transferase (GGT) were significantly increased in all patients following TACE procedure (t = -2.721, P = 0.014; t = -2.674, P = 0.015; t = -3.079, P = 0.006; t = -3.377, P = 0.003, respectively). Conclusion: The deposition of iodized oil around the bile duct on plain CT scan presentation or the continuous increase of ALP (> 200 U/L) one week following TACE procedure has a certain predictive value for the later bile duct injury.

Research progress of FAPI PET/CT in the diagnosis of malignant liver tumors / 中华肝脏病杂志

Malignant liver tumors have a high incidence and mortality rate. Therefore, it is of great significance to promptly learn about tumor advancement status through relevant examinations for patients' follow-up, diagnosis, and therapy as well as the improvement of the five-year survival rate. The primary lesions and intrahepatic metastases of malignant liver tumors have been better demonstrated in the clinical study with the use of various isotope-labeled fibroblast activating protein inhibitors because of their low uptake in liver tissues and high tumor/background ratio, which provides a new method for early diagnosis, precise staging, and radionuclide therapy. In light of this context, a review of the research progress of fibroblast-activating protein inhibitors for the diagnosis of liver malignant tumors is presented.