Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells. METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SW), were constructed by ligating into the vector, pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sail and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin-biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi. RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SW, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited. CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.

Quantitative analysis of hepatic hypoxia-inducible factor-1α and its abnormal gene expression during the formation of hepatocellular carcinoma

BACKGROUND:Hepatic hypoxia-inducible factor-1(HIF-1) is activated in the progression of hepatocellular carcinoma (HCC).This study aimed to investigate the dynamic alterations of HIF-1αand its gene expression so as to explore the relationship between HIF-1αexpression and hepatocarcinogenesis at the early stage of HCC. METHODS:A hepatoma model was made with 2-fluorenyl- acetamide(2-FAA)in male Sprague-Dawley rats.Morphological changes of rat hepatocytes were assessed pathologically (HE staining).The dynamic expression of hepatic and circulating HIF-1αwas quantitatively analyzed by ELISA. The gene fragments of hepatic HIF-1αmRNA were amplified by RT-PCR and confirmed by sequencing.The cellular distribution of hepatic HIF-1αexpression was confirmed by immunohistochemistry. RESULTS:Histological examination confirmed granulelike degeneration to atypical hyperplasia and HCC development in rat hepatocytes and progressive increases in the levels of hepatic and circulating HIF-1αand its gene expression during the course.The levels of HIF-1α expression in the liver and blood of rats with hepatoma were significantly higher than those in normal ratsand those with degeneration.Immunohistochemical analysis confirmed the positive expression and hepatocyte distribution of HIF-1αin the development of rat hepatoma. A positive relationship was found between HIF-1α expression in the liver and blood(P<0.01). CONCLUSIONS:The above observations support the hypothesis that the overexpression of HIF-1αand its gene are closely associated with the malignant transformation of hepatocytes and play an important role at the stage of hepatocarcinogenesis.

Alpha-fetoprotein-targeted reporter gene expression imaging in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers in Eastern Asia, and its incidence is increasing globally. Numerous experimental models have been developed to better our understanding of the pathogenic mechanism of HCC and to evaluate novel therapeutic approaches. Molecular imaging is a convenient and up-to-date biomedical tool that enables the visualization, characterization and quantification of biologic processes in a living subject. Molecular imaging based on reporter gene expression, in particular, can elucidate tumor-specific events or processes by acquiring images of a reporter gene&#x02019;s expression driven by tumor-specific enhancers/promoters. In this review, we discuss the advantages and disadvantages of various experimental HCC mouse models and we present in vivo images of tumor-specific reporter gene expression driven by an alpha-fetoprotein (AFP) enhancer/promoter system in a mouse model of HCC. The current mouse models of HCC development are established by xenograft, carcinogen induction and genetic engineering, representing the spectrum of tumor-inducing factors and tumor locations. The imaging analysis approach of reporter genes driven by AFP enhancer/promoter is presented for these different HCC mouse models. Such molecular imaging can provide longitudinal information about carcinogenesis and tumor progression. We expect that clinical application of AFP-targeted reporter gene expression imaging systems will be useful for the detection of AFP-expressing HCC tumors and screening of increased/decreased AFP levels due to disease or drug treatment.

Immunohistochemical analysis of p53,cyclinD1,RB1,c-fos and N-ras gene expression in hepatocellular carcinoma in Iran

AIM： TO study the effect of some genes especially those involved in cell cycle regulation on hepatocellular carcinoma. METHODS： Paraffin-embedded tissue samples of 25 patients （18 males and 7 females） with hepatocellular carcinoma were collected from 22 pathology centers in Tehran during 2000-2001, and stained using immunohistochemistry method （avidin-biotin-peroxidase） for detection of p53, cyclinD1, RB1, c-los and N-ras proteins. RESULTS： Six （24%）, 5 （20%）, 12 （48%） and 2 samples （8%） were positive for p53, cyclinDl, C-los and N-ras expression, respectively. Twenty-two （88%） samples had alterations in the （31 cell-cycle checkpoint protein expression （RBI or cyclinD1）. P53 positive samples showed a higher （9 times） risk of being positive for RBI protein than p53 negative samples. Loss of expression of RBI in association with p53 over-expression was observed in 4 （66.7%） of 6 samples. Loss of expression of RBI was seen in all cyclinD1 positive, 20 （90.9%） N-ras negative, and ii （50%） C-fos positive samples, respectively. CyclinD1 positive samples showed a higher （2.85 and 4.75 times） risk of being positive for c-los and N-ras expression than cyclinD1 negative samples. CONCLUSION： The expression of p53, RB1 and c-los genes appears to have a key role in the pathogenesis of hepatocellular carcinoma in Iran. Simultaneous overexpression of these genes is significantly associated with their loss of expression during development of hepatocellular carcinoma.

Monitoring microarray-based gene expression profile changes in hepatocellular carcinoma

AIM: To find out key genes responsible for hepatocarc inogenesis and to further understand the underlying molecular mechanism through investigating the differential gene expression between human normal liver tissue and hepatocellular carcinoma (HCC).METHODS: DNA microarray was prepared by spotting PCR products of 1 000 human genes including 445 novel genes, 540 known genes as well as 12 positive (housekeeping) and 3 negative controls (plant gene) onto treated glass slides. cDNA probes were prepared by labeling normal liver tissue mRNA and cancer liver tissue mRNA with Cy3-dUTP and Cy5-dUTP separately through reverse transcription. The arrays were hybridized against the cDNA probe and the fluorescent signals were scanned. The dataobtained from repeated experiments were analyzed. RESULTS: Among the 20 couple samples investigated (from cancerous liver tissue and normal liver tissue), 38 genes including 21 novel genes and 17 known genes exhibited different expressions. CONCLUSION: cDNA microarray technique is powerful to identify candidate target genes that may play important roles in human carcinogenesis. Further analysis of the obtained genes is helpful to understand the molecular changes in HCC progression and ultimately may lead to the identification of new targets for HCC diagnosis and intervention.

Detection of PRL-2 gene expression in hepatocellular carcinoma by real-time fluorescence quantitative PCR

Objective:To quantitatively detect the expression level of PRL-2 in primary hepatocellular carcinoma using real-time fluorescence quantitative PCR.Methods:Total RNA isolated from human HCC and liver tissue adjacent to the tumor was reversely transcribed into cDNA.Real-time fluorescence quantitative PCR(Q-PCR) method was used to analyze the expres-sion level of PRL-2 gene.Results:The Q-PCR method was performed successfully to precisely detect RNA level.PRL-2 was expressed in all portal vein tumor thrombosis(PVTT) and HCC,but only in some paratumor tissue.The highest expression level of PRL-2 gene was recorded in PVTT;meanwhile expression level of PRL-2 was higher than that in paratumor liver tis-sues and in HCC(P < 0.01),and it was higher in HCC than that in paratumor liver tissues.Conclusion:The Q-PCR may be the most precise method to quantitatively detect RNA level and can be used in gene expression changes.The PRL-2 gene has higher expression in PVTT than that in HCC and in paratumor liver tissue cells,indicating that it plays an important role in the development and metastasis of the HCC.

Analysis of in vivo patterns of caspase 3 gene expression in primary hepatocellular carcinoma and its relationship to p21^(WAF1) expression and hepatic apoptosis

AIM To detect the expression of caspase 3gene in primary human hepatocellular carcinoma（HCC）and investigate its relationship to p21WAF1gene expression and HCC apoptosis.METHODS In situ hybridization was employedto determine caspase 3 and p21WAF1expression inHCC.In situ end-labeling was used to detecthepatocytic apoptosis in HCC.RESULTS Twenty-one of 39（53.8%）cases ofHCC were found to express caspase 3transcripts,while 45.2% of HCC failed toexpress caspase 3.Non-cancerous adjacent livertissues showed more positive caspase 3（87.5%,7/8）as compared with HCC（P【0.05）.The expression of caspase 3 is correlated withHCC differentiation,72.2%（13/18）ofmoderately to highly differentiated HCC showedcaspase 3 transcripts positive,while only 38.1%of poorly differentiated HCC harbored caspase 3transcripts（P【0.05）.No relationship wasfound between caspase 3 expression and tumorsize or grade or metastasis,although 52.5%（5/8）of HCC with metastasis were caspase 3positive and a little higher than that with nometastasis（51.6%,P】0.05）.Expression of caspase 3 alone did not affect the apoptosisindex（AI）of HCC.The AI was 7.12%o in caspase3-positive tumors（n=21）,while in caspase 3-negative cases（n=18）6.59%0（P】0.05）.Expression of caspase 3 clearly segregated withp21WAF1positive tumors as compared withp21WAF1-negative cases（16 of 23,69.6% versus5 of 16,31.3%）with statistical significance（P=0.017）.In the cases with positive caspase 3and negative p21WAF1,the Al was found slightlyhigher,but with no statistical significance,thanthat with expression of p21WAF1and caspase 3（7.21‰ vs 6.98‰,P】0.05）.CONCLUSION Loss of caspase 3 expressionmay contribute to HCC carcinogenesis,althoughthe expression of caspase 3 does not correlatewell with cell apoptosis in HCC.p21WAF1may bemerely one of the inhibitors which can reducecaspase 3 mediated cell apoptosis in HCCs.

A review of the literature on the use of CRISPR/Cas9 gene therapy to treat hepatocellular carcinoma

Noncoding RNAs instruct the Cas9 nuclease to site speifillyl cleave DNA in the CRISPR/Cas9 system.Despite the high incidence of hepatocellular carcinoma(HCC),the patient's outcome is poor.As a result of the emergence of therapeutic resistance in HCC patients,dlinicians have faced difficulties in treating such tumor.In addition,CRISPR/Cas9 screens were used to identify genes that improve the dlinical response of HCC patients.It is the objective of this article to summarize the current understanding of the use of the CRISPR/Cas9 system for the treatment of cancer,with a particular emphasis on HCC as part of the current state of knowledge.Thus,in order to locate recent developments in oncology research,we examined both the Scopus database and the PubMed database.The ability to selectively interfere with gene expression in combinatorial CRISPR/Cas9 screening can lead to the discovery of new effective HCC treatment regimens by combining clinically approved drugs.Drug resistance can be overcome with the help of the CRISPR/Cas9 system.HCC signature genes and resistance to treatment have been uncovered by genome-scale CRISPR activation screening although this method is not without limitations.It has been extensively examined whether CRISPR can be used as a tool for disease research and gene therapy.CRISPR and its applications to tumor research,particularly in HCC,are examined in this study through a review of the literature.

Abnormal β-catenin gene expression with invasiveness of primary hepatocellular carcinoma in China

AIM To study the abnormal expression of β-catenin gene and its relationship with invasiveness of primary hepatocellular carcinoma among Chinese people.METHODS Thirty-four hepatocellular carcinoma (HCC) specimens and adjacent para-cancerous tissues, 4 normal liver tissues were immunohistochemically stained to study subcellular distribution of β-catenin. Semiquantitive analysis of expression of β-catenin gene exon 3 mRNA was examined by RT-PCR and in situ hybridization. The relationship between expressions of both β-catenin protein, mRNA and clinicopathological characteristics of HCC was also analyzed.RESULTS Immunohistochemistry showed that all normal liver tissues and para-cancerous tissues examined displayed membranous type staining for β-catenin protein,occasionally with weak expression in the cytoplasm.While 21 cases (61.8%) of HCC examined showed accumulated type in cytoplasms or nuclei. The accumuled type Labling Index (LI) of cancer tissue and paracancarous tissue was (59.9 ± 26.3) and (18.3 ± 9.7)respectively (P＜0.01). Higher accumulated type LI was closely related with invasiveness of HCC. Results of RTPCR showed the β-catenin gene exon 3 mRNA Expression Index (El) of 34 HCCs was higher than that of paracancerous tissue and normal liver tissue. Using in situ hybridization, the signal corresponding to β-catenin gene exon 3 mRNA was particularly strong in cytoplasm of HCC when compared with those of para-cancerous and normal liver tissues. Over expression of β-catenin exon 3 was also found to be correlated with high metastatic potential of HCC.CONCLUSION Abnormal expression of β-catenin gene may contribute importantly to the invasiveness of HCC among Chinese people.

Studied microRNA gene expression in human hepatocellular carcinoma by micro RNA microarray techniques

AIM: To achieve a better understanding of the molecular mechanisms of micro RNA expression changes involved in hepatocellular carcinoma.METHODS: In this research process, patients were not treated with antivirals, immunosuppressants or immunomodulators for at least 6 mo before collecting serum. The study population was composed of 35 outpatient hepatitis B virus(HBV) cases and 12 healthy control cases from the Affiliated Hospital of Inner Mongolia Medical University(Inner Mongolia, China) from July 2013 to April 2014. The 35 HBV cases were divided into two groups: a hepatocirrhosis group with 20 cases and a liver cancer group with 15 cases. All 35 cases carried HBs Ag. The diagnostic criteria followed the European Association for the Study of the Liver 2012(EASL2012) standards. Micro RNA(mi RNA) was extracted from a control group of patients, a group with hepatocirrhosis and a group with liver cancer and its quality was analyzed using the human V2 micro RNA expression beadchip. Cluster analysis and a radar chart were then applied to the mi RNA changes.RESULTS: The mi RNA-qualified rate of human serum samples was 93%. The concentration of a single sample was > 200 ng/μL and the volume was > 5 μL.All mi RNA serum samples were uncontaminated by the genome. The Mann-Whitney test showed significant differences in mi RNA between each group, with a detection P-value of < 0.05. Illumina software was set up with Diff Score set to ± 13, meaning that P = 0.001.There were significant changes in mi RNA expression between the three groups. mi RNA-183 was the most up-regulated, followed by mi RNA-373. mi RNA-129 and mi RNA-188 were both strongly down-regulated and mi RNA-378 was down-regulated a small amount. The liver cancer group had greater changes, which indicated that changes in mi RNA expression levels were caused by hepatocirrhosis. The liver cancer disease course then further increased these changes. In the pentagon created by these five mi RNAs, three groups showed significant deviation. The liver cancer group had a bigger deviation trend. The chart indicated that mi RNA expression changes occurred in the hepatocirrhosis group, which increased in the liver cancer disease course and were irreversible.CONCLUSION: There was a significant relationship between the irreversible up-regulation of mi RNA-183/373 and down-regulation of mi RNA-129/188/378 and incidences of hepatocirrhosis and liver cancer.

Difference in hTERT Gene Expressions between HbsAg-Positive and HbsAg-Negative Hepatocellular Carcinoma

To investigate the difference in expression of hTERT gene between HbsAg-positive human hepatocellular carcinoma (HCC) and HbsAg-negative HCC and to explore the relationship between HBV infection and hTERT gene expression in HCC. The expression of hTERT protein in 30 cases of HbsAg positive HCC and 17 cases of HbsAg negative HCC was detected by immunohistochemistry (SP method), and the expression of hTERT mRNA was analyzed by reverse transcription polymerase chain reaction (RT-PCR). t-test, Chi-squared test and cochran- armitage trend test were used to see whether there was an interrelation between HBsAg and hTERT gene in HCC. The expression of hTERT protein was mostly located in plasm and occasionally in the nucleus of liver cancer cells. The positive rate of hTERT protein and hTERT mRNA in HbsAg positive HCC- 93.33 % (28/30) and 83.33 % (25/30) respectively which were much higher than those in HbsAg negative HCC- 52.94 % (9/17), 47.06 % (8/17) (P<0.01) respectively. HbsAg is related to hTERT gene expression in human hepatocellular carcinoma. The hTERT gene activated by the efficacious ingredient of HBV may play an important role in hepatocellular transformation and carcinogenesis.

Hepatocellular carcinoma:An analysis of the expression status of stress granules and their prognostic value

BACKGROUND Hepatocellular carcinoma(HCC)is a global popular malignant tumor,which is difficult to cure,and the current treatment is limited.AIM To analyze the impacts of stress granule(SG)genes on overall survival(OS),survival time,and prognosis in HCC.METHODS The combined The Cancer Genome Atlas-Liver Hepatocellular Carcinoma(TCGA-LIHC),GSE25097,and GSE36376 datasets were utilized to obtain genetic and clinical information.Optimal hub gene numbers and corresponding coefficients were determined using the Least absolute shrinkage and selection operator model approach,and genes for constructing risk scores and corresponding correlation coefficients were calculated according to multivariate Cox regression,respectively.The prognostic model’s receiver operating characteristic(ROC)curve was produced and plotted utilizing the time ROC software package.Nomogram models were constructed to predict the outcomes at 1,3,and 5-year OS prognostications with good prediction accuracy.RESULTS We identified seven SG genes(DDX1,DKC1,BICC1,HNRNPUL1,CNOT6,DYRK3,CCDC124)having a prognostic significance and developed a risk score model.The findings of Kaplan-Meier analysis indicated that the group with a high risk exhibited significantly reduced OS in comparison with those of the low-risk group(P<0.001).The nomogram model’s findings indicate a significant enhancement in the accuracy of OS prediction for individuals with HCC in the TCGA-HCC cohort.Gene Ontology and Gene Set Enrichment Analysis suggested that these SGs might be involved in the cell cycle,RNA editing,and other biological processes.CONCLUSION Based on the impact of SG genes on HCC prognosis,in the future,it will be used as a biomarker as well as a unique therapeutic target for the identification and treatment of HCC.

Gene targets with therapeutic potential in hepatocellular carcinoma

Hepatocellular carcinoma(HCC)is the third leading cause of cancer-related deaths worldwide.Major treatments include liver transplantation,resection,and chemotherapy,but the 5-year recurrence rate remains high.Late diagnosis often prevents surgical intervention,contributing to poor patient survival rates.Carcinogenesis in HCC involves genetic alterations that drive the transformation of normal cells into malignant ones.Enhancer of zeste homolog 2(EZH2),a key regulator of cell cycle progression,is frequently upregulated in HCC and is associated with advanced stages and poor prognosis,making it a potential biomarker.Additionally,signal transducer and activator of transcription 3,which binds to EZH2,affects disease staging and outcomes.Targeting EZH2 presents a promising therapeutic strategy.On the other hand,abnormal lipid metabolism is a hallmark of HCC and impacts prognosis.Fatty acid binding protein 5 is highly expressed in HCC tissues and correlates with key oncogenes,suggesting its potential as a biomarker.Other genes such as guanine monophosphate synthase,cell division cycle associated 5,and epidermal growth factor receptor provide insights into the molecular mechanisms of HCC,offering potential as biomarkers and therapeutic targets.

New perspectives in prognostication of hepatocellular carcinoma:The role and clinical implications of transient receptor potential family genes

The study titled“Transient receptor potential-related risk model predicts prognosis of hepatocellular carcinoma patients”is a significant contribution to hepatocellular carcinoma(HCC)research,highlighting the role of transient receptor potential(TRP)family genes in the disease’s progression and prognosis.Utilizing data from The Cancer Genome Atlas database,it establishes a new risk assessment model,emphasizing the interaction of TRP genes with tumor proliferation pathways,key metabolic reactions like retinol metabolism,and the tumor immune microenvironment.Notably,the overexpression of the TRPC1 gene in HCC correlates with poorer patient survival outcomes,suggesting its potential as a prognostic biomarker and a target for personalized therapy,particularly in strategies combining immunotherapy and anti-TRP agents.

Tissue inhibitor of metalloproteinase-3 expression affects clinicopathological features and prognosis of aflatoxin B1-related hepatocellular carcinoma

BACKGROUND The dysregulation of tissue inhibitor of metalloproteinase-3(TIMP3)was positively correlated with the progression of hepatocellular carcinoma(HCC).However,it is not clear whether TIMP3 expression is associated with the clinico-pathological features and prognosis of aflatoxin B1(AFB1)-related HCC(AHCC).A retrospective study,including 182 patients with AHCC,was conducted to explore the link between TIMP3 expression in cancerous tissues and the clinico-pathological characteristics and prognosis of AHCC.TIMP3 expression was detected by immunohistochemistry and its effects on the clinicopathological features and prognosis of AHCC were evaluated by Kaplan-Meier survival analysis and Cox regression survival analysis.Odds ratio,hazard ratio(HR),median overall survival time(MST),median tumor recurrence-free survival time(MRT),and corresponding 95%confidential interval(CI)was calculated to RESULTS Kaplan-Meier survival analysis showed that compared with high TIMP3 expression,low TIMP3 expression in tumor tissues significantly decreased the MST(36.00 mo vs 18.00 mo)and MRT(32.00 mo vs 16 mo)of patients with AHCC.Multivariate Cox regression survival analysis further proved that decreased expression of TIMP3 increased the risk of death(HR=2.85,95%CI:2.04-4.00)and tumor recurrence(HR=2.26,95%CI:1.57-3.26).Furthermore,decreased expression of TIMP3 protein in tissues with AHCC was significantly correlated with tumor clinicopatho-logical features,such as tumor size,tumor grade and stage,tumor microvessel density,and tumor blood invasion.Additionally,TIMP3 protein expression was also negatively associated with amount of AFB1-DNA adducts in tumor tissues.CONCLUSION These findings indicate that the dysregulation of TIMP3 expression is related to AHCC biological behaviors and affects tumor outcome,suggesting that TIMP3 may act as a prognostic biomarker for AHCC.

Identification of immune cell-related prognostic genes characterized by a distinct microenvironment in hepatocellular carcinoma

BACKGROUND The development and progression of hepatocellular carcinoma(HCC)have been reported to be associated with immune-related genes and the tumor microenvir-onment.Nevertheless,there are not enough prognostic biomarkers and models available for clinical use.Based on seven prognostic genes,this study calculated overall survival in patients with HCC using a prognostic survival model and revealed the immune status of the tumor microenvironment(TME).AIM To develop a novel immune cell-related prognostic model of HCC and depict the basic profile of the immune response in HCC.METHODS We obtained clinical information and gene expression data of HCC from The Cancer Genome Atlas(TCGA)and International Cancer Genome Consortium(ICGC)datasets.TCGA and ICGC datasets were used for screening prognostic genes along with developing and validating a seven-gene prognostic survival model by weighted gene coexpression network analysis and least absolute shrinkage and selection operator regression with Cox regression.The relative analysis of tumor mutation burden(TMB),TME cell infiltration,immune check-points,immune therapy,and functional pathways was also performed based on prognostic genes.RESULTS Seven prognostic genes were identified for signature construction.Survival receiver operating characteristic curve analysis showed the good performance of survival prediction.TMB could be regarded as an independent factor in HCC survival prediction.There was a significant difference in stromal score,immune score,and estimate score between the high-risk and low-risk groups stratified based on the risk score derived from the seven-gene prognostic model.Several immune checkpoints,including VTCN1 and TNFSF9,were found to be associated with the seven prognostic genes and risk score.Different combinations of checkpoint blockade targeting inhibitory CTLA4 and PD1 receptors and potential chemotherapy drugs hold great promise for specific HCC therapies.Potential pathways,such as cell cycle regulation and metabolism of some amino acids,were also identified and analyzed.CONCLUSION The novel seven-gene(CYTH3,ENG,HTRA3,PDZD4,SAMD14,PGF,and PLN)prognostic model showed high predictive efficiency.The TMB analysis based on the seven genes could depict the basic profile of the immune response in HCC,which might be worthy of clinical application.

Identification of the key genes and mechanisms associated with transcatheter arterial chemoembolisation refractoriness in hepatocellular carcinoma

BACKGROUND Transcatheter arterial embolisation(TACE)is the primary treatment for intermediate-stage hepatocellular carcinoma(HCC)patients while some HCC cases have shown resistance to TACE.AIM To investigate the key genes and potential mechanisms correlated with TACE refractoriness in HCC.METHODS The microarray datasets of TACE-treated HCC tissues,HCC and non-HCC tissues were collected by searching multiple public databases.The respective differentially expressed genes(DEGs)were attained via limma R package.Weighted gene co-expression network analysis was employed for identifying the significant modules related to TACE non-response.TACE refractoriness-related genes were obtained by intersecting up-regulated TACE-associated and HCC-associated DEGs together with the genes in significant modules related to TACE nonresponse.The key genes expression in the above two pairs of samples was compared respectively via Wilcoxon tests and standard mean differences model.The prognostic value of the key genes was evaluated by Kaplan-Meier curve.Multivariate analysis was utilised to investigate the independent prognostic factor in key genes.Single-cell RNA(scRNA)sequencing analysis was conducted to explore the cell types in HCC.TACE refractoriness-related genes activity was calculated via AUCell packages.The CellChat R package was used for the investigation of the cell–cell communication between the identified cell types.RESULTS HCC tissues of TACE non-responders(n=66)and TACE responders(n=81),HCC(n=3941)and non-HCC(n=3443)tissues were obtained.The five key genes,DLG associated protein 5(DLGAP5),Kinesin family member 20A(KIF20A),Assembly factor for spindle microtubules(ASPM),Kinesin family member 11(KIF11)and TPX2 microtubule nucleation factor(TPX2)in TACE refractoriness-related genes,were identified.The five key genes were all up-regulated in the TACE non-responders group and the HCC group.High expression of the five key genes predicted poor prognosis in HCC.Among the key genes,TPX2 was an independent prognostic factor.Four cell types,hepatocytes,embryonic stem cells,T cells and B cells,were identified in the HCC tissues.The TACE refractoriness-related genes expressed primarily in hepatocytes and embryonic stem cells.Hepatocytes,as the providers of ligands,had the strongest interaction with embryonic stem cells that provided receptors.CONCLUSION Five key genes(DLGAP5,KIF20A,ASPM,KIF11 and TPX2)were identified as promoting refractory TACE.Hepatocytes and embryonic stem cells were likely to boost TACE refractoriness.

Identification of key genes underlying clinical features of hepatocellular carcinoma based on weighted gene co‑expression network analysis and bioinformatics analysis

Objective: To identify module genes that are closely related to clinical features of hepatocellular carcinoma (HCC) by weighted gene co‑expression network analysis, and to provide a reference for early clinical diagnosis and treatment. Methods: GSE84598 chip data were downloaded from the GEO database, and module genes closely related to the clinical features of HCC were extracted by comprehensive weighted gene co‑expression network analysis. Hub genes were identified through protein interaction network analysis by the maximum clique centrality (MCC) algorithm;Finally, the expression of hub genes was validated by TCGA database and the Kaplan Meier plotter online database was used to evaluate the prognostic relationship between hub genes and HCC patients. Results: By comparing the gene expression data between HCC tissue samples and normal liver tissue samples, a total of 6 262 differentially expressed genes were obtained, of which 2 207 were upregulated and 4 055 were downregulated. Weighted gene co‑expression network analysis was applied to identify 120 genes of key modules. By intersecting with the differentially expressed genes, 115 candidate hub genes were obtained. The results of enrichment analysis showed that the candidate hub genes were closely related to cell mitosis, p53 signaling pathway and so on. Further application of the MCC algorithm to the protein interaction network of 115 candidate hub genes identified five hub genes, namely NUF2, RRM2, UBE2C, CDC20 and MAD2L1. Validation of hub genes by TCGA database revealed that all five hub genes were significantly upregulated in HCC tissues compared to normal liver tissues;Moreover, survival analysis revealed that high expression of hub genes was closely associated with poor prognosis in HCC patients. Conclusions: This study identifies five hub genes by combining multiple databases, which may provide directions for the clinical diagnosis and treatment of HCC.

Expression of the Glypican-3 Gene in α-fetoprotein-negative Human Hepatocellular Carcinoma

Objective： To investigate the expression of Glypican-3 gene in （α-fetoprotein （AFP）-negative hepatocellular carcinoma （HCC） and clarify whether Glypican-3 expression is a useful parameter for the diagnosis of hepatocelhllar carcinoma （HCC）, especially for AFP-negative ones. Methods： Forty-one specimens of AFP-negative hepatocellular carcinoma and para-carcimoma tissue were studied for the expression of Glypican-3 by Northern blot. The expression of Glypican-3 protein was detected immunohistochemically with specific polyclonal antibody. Results： Northern blot analysis indicated that the expression of Glypican-3 mRNA was intensively detected in 30 of 41 AFP-negative HCC （73.17%）. In contrast, Glypican-3 mRNA was only weakly detected in 4 of the surrounding non-tumor liver tissues. There was significant difference in the Glypican-3 mRNA expression in large tumors （〉5 cm） （79.31%） and in small tumors （〈5 cm） （41.67%） （P〈0.01）. Gypican-3 mRNA was more frequently overexpressed in poorly differentiated HCC than in well differentiated ones （76.47% vs. 42.86%, P〈0.05）. The Glypican-3 expression was not correlated with age. sex. ttbsAg seropositivity, fibroeapsule, portal venous embolus and intrahepatic metastasis. The overexpression of Glypican-3 protein in HCC was targeted in tumor cells, not in bile duct cells and other interstitial cells. Conclusion： Glypican-3 was specially overexpressed in AFP-negative HCC, and its expression was closely correlated with the tumor size and tumor grade. Glypican-3 gene may play important roles in hepatocareinogenesis, and can be used as a new biochemical marker of HCC, especially for AFP-negative HCC.

Radiomics and molecular analysis:Bridging the gap for predicting hepatocellular carcinoma prognosis

This editorial examines a recent study that used radiomics based on computed tomography(CT)to predict the expression of the fibroblast-related gene enhancer of zeste homolog 2(EZH2)and its correlation with the survival of patients with hepatocellular carcinoma(HCC).By integrating radiomics with molecular analysis,the study presented a strategy for accurately predicting the expression of EZH2 from CT scans.The findings demonstrated a strong link between the radiomics model,EZH2 expression,and patient prognosis.This noninvasive approach provides valuable insights into the therapeutic management of HCC.