Regulatory effect of autophagy on the resistance of human liver cancer cell Huh7 to lenvatinib / 中国药房

OBJECTIVE To investigate the regulatory effect of autophagy on the resistance of human liver cancer cell Huh7 to lenvatinib. METHODS Using human liver cancer cell Huh7 as subject， the lenvatinib-resist cell model （Huh7-LR） was generated by the low-dose gradient method combined with long-term administration. The sensitivity of parental cell Huh7 and drug-resistant cell Huh7-LR to lenvatinib was detected by using CCK-8 assay and flow cytometry. Western blot assay and GFP-mCherry-LC3 plasmid transfection were performed to detect the expression levels of autophagic protein Beclin-1， autophagic adapter protein sequestosome 1 （p62）， microtubule-associated protein 1 light chain 3 （LC3） and autophagic level. Furthermore， an autophagy activation model was constructed by cell starvation， the protein expression of p62 and autophagy level were detected by using Western blot assay and GFP-mCherry-LC3 plasmid transfection， and the effect of autophagy activation on the sensitivity of Huh7-LR cells to lenvatinib was detected by flow cytometry. RESULTS Compared with parental cells， the drug resistance index of Huh7-LR cells was 6.2； protein expression of p62 was increased significantly， while apoptotic rate， protein expression of Beclin-1 and LC3Ⅱ/ LC3Ⅰ ratio were all reduced significantly （P＜0.05 or P＜0.01）； the level of autophagy was decreased to some extent. Autophagy activation could significantly increase the protein expression of p62 in Huh7-LR cells （P＜0.05） and autophagy level， and significantly increase its apoptotic rate （P＜0.05）. CONCLUSIONS Autophagy is involved in lenvatinib resistance， and activating autophagy can reverse the resistance of liver cancer cells to lenvatinib to some extent.

Renal injury caused by tenofovir disoproxil fumarate in a patient with liver cancer complicated with HBV infection：a case report and literature analysis / 中国药房

Tenofovir disoproxil fumarate （TDF） is a first-line treatment for chronic hepatitis B. With increasing use worldwide， the adverse events of renal injury caused by this drug have also attracted industry attention. This article reports a 61- year-old patient with liver cancer complicated with hepatitis B virus （HBV） infection. The patient started using TDF in mid-March 2022 and developed kidney injury after 2 months of treatment， during which he received 2 courses of donafenib combined with sintilimab chemotherapy and irregular administration of diclofenac for pain relief. In this paper， Naranjo’s assessment scale was used to evaluate the drugs that may be associated with renal injury， including TDF and sintilimab， and the drugs that are suspected to be associated with renal injury are donafenib and diclofenac. The renal injury caused by TDF can be judged according to the changes in the patient’s condition， the incidence of drug-induced renal injury， clinical manifestations， occurrence time， occurrence mechanism， drug combination， and high-risk factors. The changes of serum creatinine in patients with liver cancer complicated with HBV infection after TDF should be dynamically monitored in the clinic， and the dose of antiviral drugs should be adjusted if necessary and other antiviral drugs with less impact on renal function can be selected， to provide individualized medication recommendations for tumor patients， reduce the incidence of TDF-related renal injury.

Epidemiological trend analysis of liver cancer incidence in Luzhou City， Sichuan Province from 2016 to 2022 / 上海预防医学

ObjectiveTo analyze the epidemiological distribution and temporal trends of liver cancer incidence among Luzhou residents from 2016‒2022， and to provide a theoretical basis for improving liver cancer prevention and treatment strategies in Luzhou. MethodsData on liver cancer incidence among Luzhou residents from 2016 to 2022 were collected， and the incidence rate， age-specific incidence rate， and annual percentage change （APC） were calculated. A Joinpoint regression model was used to fit a time series segment to the monthly number of new cases in each district and county of Luzhou to explore the trend of liver cancer incidence rate. ResultsThe incidence rate of liver cancer in Luzhou increased from 22.96/105 in 2016 to 32.31/105 in 2022. The incidence rate of liver cancer in men was higher than that in women in both 2016 and 2022， and the incidence rate of liver cancer in men increased from 34.83/105 in 2016 to 47.95/105 in 2022， with an APC of 3.3%； the incidence rate of liver cancer in women increased from 10.50/105 in 2016 to 15.95/105 in 2022， with an APC of 3.0%， and the differences in the change trends were not statistically significant （P>0.05）.The incidence of liver cancer was low in the age group of 0‒<40 years from 2016 to 2022 and increased with age； the incidence of liver cancer in the age group of 55 years and above was increasing at an average annual rate of 16.4%. ConclusionThe overall incidence of liver cancer in Luzhou is on the rise， and the incidence of liver cancer in men is higher than that in women. Middle-aged and elderly men are the key population for liver cancer prevention and treatment， and liver cancer prevention and treatment should be carried out in a targeted manner， taking into account regional development differences.

A multidimensional platform of patient-derived tumors identifies drug susceptibilities for clinical lenvatinib resistance

Lenvatinib, a second-generation multi-receptor tyrosine kinase inhibitor approved by the FDA for first-line treatment of advanced liver cancer, facing limitations due to drug resistance. Here, we applied a multidimensional, high-throughput screening platform comprising patient-derived resistant liver tumor cells (PDCs), organoids (PDOs), and xenografts (PDXs) to identify drug susceptibilities for conquering lenvatinib resistance in clinically relevant settings. Expansion and passaging of PDCs and PDOs from resistant patient liver tumors retained functional fidelity to lenvatinib treatment, expediting drug repurposing screens. Pharmacological screening identified romidepsin, YM155, apitolisib, NVP-TAE684 and dasatinib as potential antitumor agents in lenvatinib-resistant PDC and PDO models. Notably, romidepsin treatment enhanced antitumor response in syngeneic mouse models by triggering immunogenic tumor cell death and blocking the EGFR signaling pathway. A combination of romidepsin and immunotherapy achieved robust and synergistic antitumor effects against lenvatinib resistance in humanized immunocompetent PDX models. Collectively, our findings suggest that patient-derived liver cancer models effectively recapitulate lenvatinib resistance observed in clinical settings and expedite drug discovery for advanced liver cancer, providing a feasible multidimensional platform for personalized medicine.

Intervention Mechanism of Biejiajian Wan on Primary Liver Cancer by Regulating lncRNA SNHG5/miRNA-26a-5p/GSK-3β Signal Axis / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Biejiajian Wan in the intervention of primary liver cancer based on long non-coding RNA SNHG5 （lncRNA SNHG5）/micro RNA-26a-5p （miRNA-26a-5p）/glycogen synthase kinase-3β （GSK-3β） signal axis. MethodDouble luciferase reporting assay was used to verify the targeted interaction between lncRNA SNHG5 and miRNA-26a-5p， miRNA-26a-5p， and GSK-3β in HepG2 cells. Nude-mouse transplanted tumor model of human HepG2 were established and randomly divided into model group， Biejiajian Wan low-dose group （0.5 g·kg-1）， medium-dose group （1.0 g·kg-1）， and high-dose group （2.0 g·kg-1）， and sorafenib group （100 mg·kg-1）， with 10 mice in each group. The mice were given intragastric administration of normal saline or drug for 28 days， and the tumor volume was measured at different time. Hematoxylin-eosin （HE） staining was used to observe the histological changes of tumors. The nucleic acid levels of lncRNA SNHG5， miRNA-26a-5p， GSK-3β， and β-catenin mPNA in tumor tissue were detected by real-time quantitative polymerase chain reaction （Real-time PCR）. The protein expression levels of GSK-3β and β-catenin in tumor tissue were detected by western blot. ResultCompared with the SNHG5-WT （wild type） + miRNA NC （negative control） group， the relative luciferase activities of the SNHG5-WT + miRNA-26a-5p mimic group were decreased （P<0.05）. Compared with the GSK-3β-WT + miRNA NC group， the relative luciferase activity of the GSK-3β-WT + miRNA-26a-5p mimic group was decreased （P<0.05）. Compared with the model group， the tumor volume of Biejiajian Wan low-dose， medium-dose， and high-dose groups was significantly decreased （P<0.05， P<0.01）. Compared with the model group， the cells in the tumor tissue of nude mice in each dose group of Biejiajian Wan were sparsely arranged with necrocytosis， which showed concentration-dependent changes. Compared with the model group， the expression levels of lncRNA SNHG5， GSK-3β， and β-catenin were decreased （P<0.05， P<0.01）， while the expression of miRNA-26a-5p was increased in each dose group of Biejiajian Wan （P<0.05， P<0.01）. Compared with the model group， the protein expression levels of GSK-3β and β-catenin were decreased in each dose group of Biejiajian Wan （P<0.05， P<0.01）. ConclusionBiejiajian Wan may affect the necrosis of liver cancer cells through lncRNA SNHG5/miRNA-26a-5p/GSK-3β signal axis and thus play an anti-tumor role. This research will provide more theoretical basis for the clinical application of Biejiajian Wan.

Regressão espontânea do carcinoma hepatocelular: foco nos fatores de risco associados / Spontaneous regression of hepatocellular carcinoma: focusing in the associated risk factors

ABSTRACT Background: Spontaneous regression (SR) is defined as the partial or complete disappearance of a tumor, in the absence of a specific treatment. Evidence of the SR in hepatocellular carcinoma (HCC) is rare. Objective: The authors aimed to review all the cases of SR of HCC in two reference centers of Southern Brazil, highlighting the main characteristics. Methods: Data of all patients with HCC were retrospectively reviewed looking for the occurrence of SR in patients from two tertiary centers in Southern Brazil, in the last five years. The diagnosis of cirrhosis was established according to clinical, laboratory and imaging data, as well as upper endoscopy or histopathological examination when necessary. The diagnosis of HCC was based on typical findings according to radiologic criteria (LIRADS) or histopathological examination. Spontaneous regression was defined as a partial or complete involution of a HCC in the absence of a specific therapy. Results: From all cases of HCC in the last 5 years (n=433), there were five cases of SR. Three (60%) were men, the mean age was 62.6 (50.0-76.0) years, and the etiology was HCV in 3 (60%). Complete regression was observed in three patients (60%), one patient (20%) presented partial regression, and one (20%) relapesed and died. The time of follow-up varied between 12 and 21 months. In this presentation, it was highlighted one case of SR observed after COVID-19 infection in a patient with cirrhosis. The possible mechanisms involved in this situation were reviewed, emphasizing the most common like hypoxia and immunological. There were also one patient submitted to a surgical procedure as a possible fator involved and three patients without obvious risk factors. Conclusion: This phenomenon will possibly contribute to a better understanding of the pathophysiological mechanisms of HCC.

RESUMO Contexto: A evidência da regressão espontânea (RE) no carcinoma hepatocelular (CHC) é rara. Objetivo: Revisar todos os casos de RE de CHC em dois centros de referência do Sul do Brasil, destacando as principais características. Métodos: Os dados de todos os pacientes com CHC foram revisados retrospectivamente buscando a ocorrência de RE em pacientes de dois centros terciários do Sul do Brasil, nos últimos 5 anos. O diagnóstico de cirrose foi estabelecido de acordo com dados clínicos, laboratoriais e de imagem, além de endoscopia digestiva alta ou exame histopatológico quando necessário. O diagnóstico de CHC foi baseado em achados típicos de acordo com critérios radiológicos (LIRADS) ou exame histopatológico. A RE foi definida como uma involução parcial ou completa de um CHC na ausência de terapia específica. Resultados: Do total de casos de CHC nos últimos 5 anos (n=433), houve cinco casos de RE. Três (60%) eram homens, a média de idade foi de 62,6 (50,0-76,0) anos, a etiologia foi virus da hepatite C em 3 (60%). A regressão completa foi observada em três pacientes (60%), um paciente (20%) apresentou regressão parcial e um (20%) apresentou recidiva e evoluiu a óbito. O tempo de seguimento variou entre 12 e 21 meses. Nesta apresentação foi destacado um caso (20%) de RE observado após infecção por COVID-19 em paciente com cirrose. Foram revisados os possíveis mecanismos envolvidos nesta situação, enfatizando os mais comuns como hipóxia e imunológicos. Houve também um paciente submetido a procedimento cirúrgico como possível fator envolvido e três pacientes sem fatores de risco evidentes. Conclusão: Este fenômeno possivelmente contribuirá para uma melhor compreensão dos mecanismos fisiopatológicos do CHC.

Ganoderma lucidum Polysaccharides Inhibit Malignant Phenotype of Hepatocellular Carcinoma Cells by Regulating PI3K/Akt Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Ganoderma lucidum polysaccharides （GLP） on the proliferation， migration， cycle， and apoptosis of hepatocellular carcinoma SK­HEP­1 and Huh­7 cells and to explore the underlying mechanism. MethodSK-HEP-1 and Huh-7 cells were classified into the blank group and low-， medium-， and high-dose GLP groups （3.5， 7， 14 g·L-1）. The proliferation of the cells was examined by cell counting kit-8 （CCK­8） assay， and the migration by scratch assay. Cell cycle was measured by flow cytometry and apoptosis was detected based on Hoechst33258 staining. In addition， the expression of phosphatidylinositol 3-kinase （PI3K）， protein kinase B （Akt）， phosphorylated PI3K （p­PI3K）， and phosphorylated Akt （p­Akt） in the cells was determined by Western blot. ResultCompared with the blank group， the three doses of GLP reduced the proliferation and migration of SK­HEP­1 and Huh­7 cells （P<0.05）， increased the percentage of cells in G1 phase （P<0.05）， and decreased percentage of cells in S and G2 phase （P<0.05）. In addition， the three doses can induce apoptosis of both SK-HEP-1 and Huh-7 cells， particularly the high dose. Moreover， the three doses of GLP lowered the levels of p­PI3K and p­Akt （P<0.05）. ConclusionGLP significantly inhibited the malignant phenotype of SK-HEP-1 and Huh-7 cells through the PI3K/Akt signaling pathway.

Efficacy Evaluation of Biejiajianwan in the Treatment of Primary Liver Cancer Based on Real-world Data of Traditional Chinese Medicine / 中国实验方剂学杂志

ObjectiveTo evaluate the efficacy and influencing factors of Biejiajianwan in the treatment of primary liver cancer based on real-world data of traditional Chinese medicine （TCM）. MethodClinical diagnosis and treatment data of patients with primary liver cancer admitted to five Grade-A tertiary hospitals in Henan Province from January 2015 to December 2020 were collected from the medical electronic database. The patients treated with Biejiajianwan for ≥30 days were assigned to the exposure group and those without treatment with Biejiajianwan or treated with Biejiajianwan for <30 days to the non-exposure group. The propensity score matching model was used to balance confounding factors between the two groups according to the 1∶1 genetic matching method. Kaplan-Meier method was used for survival analysis and survival curve plotting. Log-rank was used to test the difference in survival rate between the two groups. Univariate analysis of Biejiajianwan in the treatment of primary liver cancer was performed by Log-rank test combined with the Kaplan-Meier method. The factors with statistical significance （P<0.05） were combined with unbalanced factors by the propensity score matching model， and at the same time， clinical common sense and relevant prognostic factors by literature search were considered， which were subjected to multivariate analysis by Cox proportional hazards regression model. ResultA total of 2 207 electronic cases were collected，including 174 cases in the exposure group （Biejiajianwan group） and 2 033 cases in the non-exposure group. After propensity score matching， there were 174 cases in the exposure group and 174 cases in the non-exposure group. The Kaplan-Meier method was used for survival analysis on the matched data， and the Log-rank test results showed that the survival rate of patients with primary liver cancer in the Biejiajianwan group was higher than that in the control group （χ2=12.193， P<0.01）. Cox proportional hazards regression model analysis showed that the regression coefficient of Biejiajianwan was -0.916 4 with the hazard ratio （HR） ［95% confidence interval （CI）］=0.4 （0.239 5-0.668 0）， P<0.01， and the regression coefficient of radiofrequency ablation treatment was -0.976 5 with HR （95% CI）=0.376 6 （0.172 8-0.821 1， P<0.05）. Fibrinogen （FIB） abnormal regression coefficient was 0.481 4 with HR （95% CI）=1.618 4（1.022 0-2.562 9），P<0.05. ConclusionBiejiajianwan can prolong the survival period of patients with primary liver cancer. Radiofrequency ablation is an independent protective factor for Biejiajianwan in the treatment of primary liver cancer，while abnormal FIB are independent risk factors for Biejiajianwan in the treatment of primary liver cancer.

Anti-liver Cancer Mechanisms of Biejiajian Wan and Clinical Research： A Review / 中国实验方剂学杂志

Biejiajian Wan， a classical formula for liver diseases， originated from Synopsis of the Golden Chamber. It has been applied in clinical settings for more than 2 000 years. According to modern pharmacological studies， it has anti-tumor， anti-fibrosis， and immunity-enhancing effects and thus is widely used for the treatment of liver fibrosis， hepatitis， liver injury， and other diseases. In recent years， accumulating evidence has proven the efficacy of this formula in the treatment of malignant tumors， especially liver cancer. This paper summarizes relevant papers in the last 20 years and summed up the anti-liver cancer mechanisms of Biejiajian Wan as regulating biological behaviors of liver cancer cells， anti-precancerosis， inhibiting tumor angiogenesis， modulating signaling pathways， suppressing activity of relevant enzymes， and regulating immunity. Moreover， this prescription can inhibit the proliferation， invasion， and metastasis of liver cancer cells， promote apoptosis of cells， suppress tumor angiogenesis， and boost immunity. In addition， it regulates Wnt/β-catenin， interleukin-6/signal transducer and activator of transcription 3 （IL-6/STAT3）， NOD-like receptor protein 3 （NLRP3）， Janus kinase-signal transducers and activators of transcription （JAK-STAT）， Delta-like ligand 4-Notch （DLL4-Notch）， nuclear factor-κB （NF-κB）， Rho-associated kinase （Rho/ROCK）， transforming growth factor-β/Smad （TGF-β/Smad）， phosphatidylinositol 3-kinase/protein kinase B/glycogen synthase kinase-3β （PI3K/Akt/GSK-3β）， and other signaling pathways. Thus， Biejiajian Wan is confirmed to have anti-liver cancer effect based on the molecular mechanisms. According to the summary of Biejiajian Wan in anti-liver cancer treatment， Biejiajian Wan alone or in combination with other drugs can significantly alleviate the symptoms， reduce adverse reactions， prolong the survival time with definite efficacy. Thus， it is safe with no adverse reactions in long-term use， which should be further promoted in clinical application. This paper analyzed Biejiajian Wan in the treatment of liver cancer based on the molecular mechanisms and clinical studies， summarized the limitations in current research， and put forward suggestions， which can lay a basis for the future in-depth research on and clinical application of Biejiajian Wan and development of anti-tumor drugs.

The expression and function of PD-L1 in CD133(+) human liver cancer stem-like cells / 中华肿瘤杂志

Objective: To investigate the expression of programmed death protein-ligand 1 (PD-L1) in liver cancer stem-like cells (LCSLC) and its effect on the characteristics of tumor stem cells and tumor biological function, to explore the upstream signaling pathway regulating PD-L1 expression in LCSLC and the downstream molecular mechanism of PD-L1 regulating stem cell characteristics, also tumor biological functions. Methods: HepG2 was cultured by sphere-formating method to obtain LCSLC. The expressions of CD133 and other stemness markers were detected by flow cytometry, western blot and real-time quantitative polymerase chain reaction (RT-qPCR) were used to detect the expressions of stemness markers and PD-L1. The biological functions of the LCSLC were tested by cell function assays, to confirm that the LCSLC has the characteristics of tumor stem cells. LCSLC was treated with cell signaling pathway inhibitors to identify relevant upstream signaling pathways mediating PD-L1 expression changes. The expression of PD-L1 in LCSLC was down regulated by small interfering RNA (siRNA), the expression of stem cell markers, tumor biological functions of LCSLC, and the changes of cell signaling pathways were detected. Results: Compared with HepG2 cells, the expression rate of CD133 in LCSLC was upregulated [(92.78±6.91)% and (1.40±1.77)%, P<0.001], the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were also higher than those in HepG2 cells (P<0.05), the number of sphere-formating cells increased on day 7 [(395.30±54.05) and (124.70±19.30), P=0.001], cell migration rate increased [(35.41±6.78)% and (10.89±4.34)%, P=0.006], the number of transmembrane cells increased [(75.77±10.85) and (20.00±7.94), P=0.002], the number of cloned cells increased [(120.00±29.51) and (62.67±16.77), P=0.043]. Cell cycle experiments showed that LCSLC had significantly more cells in the G(0)/G(1) phase than those in HepG2 [(54.89±3.27) and (32.36±1.50), P<0.001]. The tumor formation experiment of mice showed that the weight of transplanted tumor in LCSLC group was (1.32±0.17)g, the volume is (1 779.0±200.2) mm(3), were higher than those of HepG2 cell [(0.31±0.06)g and (645.6±154.9)mm(3), P<0.001]. The expression level of PD-L1 protein in LCSLC was 1.88±0.52 and mRNA expression level was 2.53±0.62, both of which were higher than those of HepG2 cells (P<0.05). The expression levels of phosphorylation signal transduction and transcription activation factor 3 (p-STAT3) and p-Akt in LCSLC were higher than those in HepG2 cells (P<0.05). After the expression of p-STAT3 and p-Akt was down-regulated by inhibitor treatment, the expression of PD-L1 was also down-regulated (P<0.05). In contrast, the expression level of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) in LCSLC was lower than that in HepG2 cells (P<0.01), there was no significant change in PD-L1 expression after down-regulated by inhibitor treatment (P>0.05). After the expression of PD-L1 was knockdown by siRNA, the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were decreased compared with those of siRNA-negative control (NC) group (P<0.05). The number of sphere-formating cells decreased [(45.33±12.01) and (282.00±29.21), P<0.001], the cell migration rate was lower than that in siRNA-NC group [(20.86±2.74)% and (46.73±15.43)%, P=0.046], the number of transmembrane cells decreased [(39.67±1.53) and (102.70±11.59), P=0.001], the number of cloned cells decreased [(57.67±14.57) and (120.70±15.04), P=0.007], the number of cells in G(0)/G(1) phase decreased [(37.68±2.51) and (57.27±0.92), P<0.001], the number of cells in S phase was more than that in siRNA-NC group [(30.78±0.52) and (15.52±0.83), P<0.001]. Tumor formation in mice showed that the tumor weight of shRNA-PD-L1 group was (0.47±0.12)g, the volume is (761.3±221.4)mm(3), were lower than those of shRNA-NC group [(1.57±0.45)g and (1 829.0±218.3)mm(3), P<0.001]. Meanwhile, the expression levels of p-STAT3 and p-Akt in siRNA-PD-L1 group were decreased (P<0.05), while the expression levels of p-ERK1/2 and β-catenin did not change significantly (P>0.05). Conclusion: Elevated PD-L1 expression in CD133(+) LCSLC is crucial to maintain stemness and promotes the tumor biological function of LCSLC.

Basic and Clinical Research Progress of Immunotherapy for Hepatocellular Carcinoma / 肿瘤防治研究

Primary liver cancer, especially hepatocellular carcinoma, poses a serious threat to the life and health of the Chinese people. Given the insidious onset of liver cancer, less than 30% of hepatocellular carcinoma patients are considered for radical treatment at the initial diagnosis. Systemic anti-tumor therapy plays an important role in the treatment of advanced hepatocellular carcinoma. Immunotherapy of hepatocellular carcinoma has developed rapidly, and an increasing number of immunotherapy drugs, which can better control the progress of hepatocellular carcinoma and prolong the survival of patients, have become first- and second-line treatment options. This article reviews briefly the progress of immunotherapy for hepatocellular carcinoma in recent years.

Expression and Prognostic Value of Cuprotosis-related Genes in Liver Cancer / 肿瘤防治研究

Objective To explore the relationship of cuprotosis-related genes with survival rate and prognosis in patients with liver cancer. Methods By collecting clinical information and corresponding RNA-seq data of patients with liver cancer in the TCGA database, the differential expression levels of 10 cuprotosis-related genes in liver cancer and normal tissues was analyzed. Novel liver cancer subtypes were identified through consistent clustering, and differences in overall survival and clinicopathological factors were compared between the two subtypes. Univariate Cox regression analysis was used in screening cuprotosis genes associated with prognosis, and LASSO regression analysis was used in constructing a risk model. Results FDX1 was down-regulated, and the other nine genes were up-regulated in HCC tissues compared with normal tissues. Cluster analysis showed that the prognosis of Cluster1 was poor. Five prognostic genes (LIPT1, DLAT, MTF1, GLS, and CDKN2A) were screened out through univariate Cox regression analysis and LASSO regression analysis for risk model construction. The risk score of this prognostic model was identified as an independent prognostic factor compared with other clinical features. Conclusion Through bioinformatics analysis, a liver cancer prognosis model of five cuprotosis-related genes is constructed, which may be used as molecular markers for tumor diagnosis and are potential therapeutic targets.

Research Progress in Noninvasive Early Diagnosis of Hepatocellular Carcinoma / 肿瘤防治研究

Liver cancer is a common tumor that seriously threatens human life and health. Given that the early onset of liver cancer is insidious and lacks specific symptoms, hence it is difficult to screen through routine examination. Thus, clinical diagnosis of liver cancer is mostly in the advanced stage. However, advanced liver cancer has few treatment options, poor prognosis and high relapse rate, thereby causing a high mortality rate. Therefore, early diagnosis of liver cancer is particularly important. Currently, non-invasive screening of liver cancer widely used in clinical setups lacks sufficient sensitivity and specificity, hence, a more reliable diagnostic method needs to be found urgently. This article reviews the research progress of noninvasive early diagnosis of liver cancer to provide a reference for raising the early diagnosis rate of liver cancer.

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.

Long-term trend analysis of liver cancer survival rate in Qidong region, Jiangsu Province, 1972-2019 / 中华肝脏病杂志

Objective: To analyze the incidence and survival rate of liver cancer cases in the entire population in the Qidong region from 1972 to 2019, so as to provide a basis for prognosis evaluation, prevention, and treatment. Methods: The observed survival rate (OSR) and relative survival rate (RSR) of 34 805 cases of liver cancer in the entire Qidong region population from 1972 to 2019 were calculated using Hakulinen's method with SURV3.01 software. Hakulinen's likelihood ratio test was used for statistical analysis. Age-standardized relative survival (ARS) was calculated using the International Cancer Survival Standard. The Joinpoint regression analysis was performed with Joinpoint 4.7.0.0 software to calculate the average annual percentage change (AAPC) of the liver cancer survival rate. Results: 1-ASR increased from 13.80% in 1972-1977 to 50.20% in 2014-2019, while 5-ASR increased from 1.27% in 1972-1977 to 27.64% in 2014-2019. The upward trend of RSR over eight periods was statistically significant (χ (2) = 3045.29, P < 0.001). Among them, male 5-ASR was 0.90%, 1.80%, 2.33%, 4.92%, 5.43%, 7.05%, 10.78%, and 27.78%, and female 5-ASR was 2.33%, 1.51%, 3.35%, 3.92%, 3.84%, 7.18%, 11.45%, and 29.84%, respectively. There was a statistically significant difference in RSR between males and females (χ (2) = 45.68, P < 0.001). The 5-RSR for each age group of 25-34 years old, 35-44 years old, 45-54 years old, 55-64 years old, 65-74 years old, and 75 years old were 4.92%, 5.29%, 8.17%, 11.70%, 11.63%, and 9.60%, respectively. There were statistically significant differences in RSR among different age groups (χ (2) = 501.29, P < 0.001). The AAPC in Qidong region from 1972 to 2019 for 1-ARS, 3-ASR, and 5-ARS were 5.26% (t = 12.35, P < 0.001), 8.10% (t = 15.99, P < 0.001), and 8.96 % (t = 16.06, P < 0.001), respectively. The upward trend was statistically significant in all cases. The AAPC of 5-ARS was 9.82% in males (t = 14.14, P < 0.001), and 8.79% in females (t = 11.48, P < 0.001), and the upward trend was statistically significant in both. The AAPC of 25-34 years old, 35-44 years old, 45-54 years old, 55-64 years old, 65-74 years old, and 75 years old were 5.37% (t = 5.26, P = 0.002), 5.22% (t = 5.66, P = 0.001), 7.20% (t = 6.88, P < 0.001), 10.00% (t = 12.58, P < 0.001), 9.96% (t = 7.34, P < 0.001) and 8.83% (t = 3.51, P = 0.013), and the upward trend was statistically significant. Conclusion: The overall survival rate of registered cases of liver cancer in the Qidong region's entire population has greatly improved, but there is still much room for improvement. Hence, constant attention should be paid to the study on preventing and treating liver cancer.

Deubiquitinating enzyme MINDY1 is an independent risk factor for the maintenance of stemness and poor prognosis in liver cancer cells / 中华肝脏病杂志

Objective: To explore the key deubiquitinating enzymes that maintain the stemness of liver cancer stem cells and provide new ideas for targeted liver cancer therapy. Methods: The high-throughput CRISPR screening technology was used to screen the deubiquitinating enzymes that maintain the stemness of liver cancer stem cells. RT-qPCR and Western blot were used to analyze gene expression levels. Stemness of liver cancer cells was detected by spheroid-formation and soft agar colony formation assays. Tumor growth in nude mice was detected by subcutaneous tumor-bearing experiments. Bioinformatics and clinical samples were examined for the clinical significance of target genes. Results: MINDY1 was highly expressed in liver cancer stem cells. The expression of stem markers, the self-renewal ability of cells, and the growth of transplanted tumors were significantly reduced and inhibited after knocking out MINDY1, and its mechanism of action may be related to the regulation of the Wnt signaling pathway. The expression level of MINDY1 was higher in liver cancer tissues than that in adjacent tumors, which was closely related to tumor progression, and its high expression was an independent risk factor for a poor prognosis of liver cancer. Conclusion: The deubiquitinating enzyme MINDY1 promotes stemness in liver cancer cells and is one of the independent predictors of poor prognosis in liver cancer.

LncRNA SBF2-AS1 Regulates Invasion and Proliferation of Hepatocellular Carcinoma Cells Through miR-372-3p/CDK6 Axis / 肿瘤防治研究

Objective To investigate the effects of lncRNA SBF2-AS1 on the proliferation and invasion of hepatoma cells by regulating the miR-372-3p/CDK6 pathway. Methods Bel7402 and SK-hep1 cells were selected as research objects. The expression levels of SBF2-AS1, miR-372-3p, and CDK6 were up- or down-regulated according to different experimental stages, while the expression levels of miR-372-3p and CDK6 in cells were detected by real-time fluorescence quantitative PCR and Western blot. Dual luciferase reporter assay verified the targeting relationships between SBF2-AS1 and miR-372-3p as well as miR-372-3p and CDK6, respectively. CCK-8, colony formation assay, Transwell, cell cycle assay, and flow cytometry were used to analyze cell proliferation, colony formation, migration/invasion ability, cell cycle activity, and apoptosis. Results SBF2-AS1 was highly expressed in hepatocellular carcinoma cells (P<0.05). SBF2-AS1 knockdown resulted in decreased proliferation and invasion of Bel7402 and SK-hep1 cells (P<0.05). After miR-372-3p knockdown, the proliferation capacity and invasion number of Bel7402 cells were significantly increased. However, the above results were reversed after SBF2-AS1 knockdown (P<0.05). In addition, miR-372-3p targeted CDK6 and inhibited its expression, although over-expressing SFB2-AS1 could reverse the above results (P<0.05). Over-expressing CDK6 could reverse the inhibition of over-expressing miR-372-3p on the proliferation and invasion of Bel7402 cells. Conclusion LncRNA SBF2-AS1 can positively regulate the expression of CDK6 through miR-372-3p. It can also influence the distribution of cell cycle and affect the proliferation and invasion abilities of hepatocellular carcinoma cells.

Double-negative T cells: a promising avenue of adoptive cell therapy in transplant oncology / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Tumor recurrence is one of the major life-threatening complications after liver transplantation for liver cancer. In addition to the common mechanisms underlying tumor recurrence, another unavoidable problem is that the immunosuppressive therapeutic regimen after transplantation could promote tumor recurrence and metastasis. Transplant oncology is an emerging field that addresses oncological challenges in transplantation. In this context, a comprehensive therapeutic management approach is required to balance the anti-tumor treatment and immunosuppressive status of recipients. Double-negative T cells (DNTs) are a cluster of heterogeneous cells mainly consisting of two subsets stratified by T cell receptor (TCR) type. Among them, TCRαβ+ DNTs are considered to induce immune suppression in immune-mediated diseases, while TCRγδ+ DNTs are widely recognized as tumor killers. As a composite cell therapy, healthy donor-derived DNTs can be propagated to therapeutic numbers in vitro and applied for the treatment of several malignancies without impairing normal tissues or being rejected by the host. In this work, we summarized the biological characteristics and functions of DNTs in oncology, immunology, and transplantation. Based on the multiple roles of DNTs, we propose that a new balance could be achieved in liver transplant oncology using them as an off-the-shelf adoptive cell therapy (ACT).

Feasibility Study of Beam Angle Optimization Based on Scripts in Automated-planning for Liver Cancer / 中国医疗器械杂志

OBJECTIVE@#To study the feasibility and potential benefits of beam angle optimization (BAO) to automated planning in liver cancer.@*METHODS@#An approach of beam angle sampling is proposed to implement BAO along with the module Auto-planning in treatment planning system (TPS) Pinnacle. An in-house developed plan quality metric (PQM) is taken as the preferred evaluating method during the sampling. The process is driven automatically by in-house made Pinnacle scripts both in sampling and scoring. In addition, dosimetry analysis and physician's opinion are also performed as the supplementary and compared with the result of PQM.@*RESULTS@#It is revealed by the numerical analysis of PQM scores that only 15% patients whose superior trials evaluated by PQM are also the initial trials. Gantry optimization can bring benefit to plan quality along with auto-planning in liver cancer. Similar results are provided by both dose comparison and physician's opinion.@*CONCLUSIONS@#It is possible to introduce a full automated approach of beam angle optimization to automated planning process. The advantages of this procedure can be observed both in numerical analysis and physician's opinion.

Screening novel inhibitors of squalene epoxidase and their effects on hepatocellular carcinoma / 药学学报

Squalene epoxidase (SQLE) is a potential target for the treatment of liver cancer. Bioinformatics analysis indicated that the high expression of SQLE was closely related to the clinical stage and poor prognosis of patients with liver cancer. However, the existing inhibitors against SQLE 195 tyrosine residue (Y195) cannot be used clinically due to severe side effects. In this study, 35 small-molecule compounds targeting SQLE 335 tyrosine residue (Y335) were selected by computer virtual screening. Combined with MTT assay, 3 candidate compounds (19#, 31# and 35#) with significant inhibitory effects on the proliferation of Huh7 cell line were obtained. Further studies showed that these 3 compounds could inhibit the migration of Huh7 cells, reduce the contents of total and free cholesterol, up-regulate the expression of tumor suppressor gene PTEN, and down-regulate the expression of PI3K and AKT proteins. The results showed that the novel inhibitors 19#, 31# and 35# targeting SQLE Y335 could reduce cholesterol content, inhibit the proliferation and migration of Huh7, thus playing an anti-liver cancer role.