| BackgroundIn China,most of liver cancer is chronic hepatitis B(CHB)related hepatocellular carcinoma(HCC)(CHB-HCC),which is highly malignant.Patients with CHB-HCC are prone to have portal vein tumor thrombus(PVTT)and extrahepatic metastasis(EHM).Occurrences of PVTT and EHM are both bottlenecks in the curative efficacy and prognosis of CHB-HCC patients,but there are problems of delayed diagnosis or over examinations in clinic.At present,there is no risk prediction tool for the occurrence of PVTT or EHM in CHB-HCC patients.Traditional Chinese medicine(TCM)shows unique advantages in the treatment of liver cancer,TCM syndrome types and syndromes have potential predictive ability for the metastasis of CHB-HCC patients,but there is no research on this topic.Earlier,our team extracted the core prescription named "Shugan Jianpi Yangxue Decoction" treating liver cancer from abundant prescriptions of a famous old TCM doctor through data mining.Meanwhile,we used network pharmacology method to predict this decoction and its primary bioactive component quercetin(QUR)could be used to control the growth and metastasis of liver cancer by regulating CHB and other signal pathways related to cancer survival and invasion,but their actual effects and mechanisms on HCC were not clear.Competitive endogenous RNA(ceRNA)is an important mechanism of posttranscriptional gene expression regulation,but effects of QUR on the ceRNA network in HCC are unknown.Objective1.To learn the clinical characteristics of patients with firstly-diagnosed CHB-HCC,and to explore correlations between the occurrences of PVTT and EHM and clinical factors(including TCM syndrome types and syndromes),thus exploring the predictive efficacy of TCM syndrome types and syndromes on CHB-HCC metastasis,and deepen the understanding of mechanisms of CHB-HCC metastasis from perspectives of both traditional C hinese and Western medicine.Risk prediction nomograms of the occurrence of PVTT and EHM in patients with firstly-diagnosed CHB-HCC were established respectively,to assist individualized medical decision-making.2.To preliminarily determine the effects of Shugan Jianpi Yangxue Decoction and its primary component QUR on HCC cells.Due to the limitation of conditions,only the QUR monomer was further explored.The overall mechanisms,key targets,and ceRNA mechanisms of QUR in the treatment of HCC were explored based on transcriptome sequencing,to help explain the action mechanisms of Shugan Jianpi Yangxue Decoction and QUR in the prevention and treatment of HCC,and provide references for their clinical applications and follow-up scientific researches.Methods1.Clinical study(1)A risk prediction nomogram of the occurrence of PVTT in patients with firstlydiagnosed CHB-HCC:①The clinical data of 200 patients with firstly-diagnosed CHB-HCC in Shenzhen traditional Chinese medicine Hospital from January 2014 to May 2020 were retrospectively collected.The patients were divided into two groups according to whether PVTT occurred or not,then the clinical characteristics of the two groups were compared.②Univariate and multivariate logistic regression analyses were applied to explore correlations between the occurrence of PVTT and clinical factors(including TCM syndrome types and syndromes)in CHB-HCC patients.③A nomogram for PVTT risk prediction was constructed using the independent risk factors,applying the package of R software.The receiver operating characteristic(ROC)curve,calibration curve,and decision curve analysis(DCA)were used to evaluate the nomogram.(2)A risk prediction nomogram of the occurrence of EHM in patients with firstlydiagnosed CHB-HCC:The patients’ data and methods were the same as those of the above.The patients were divided into two groups according to whether EHM occurred or not,and the existence of PVTT was investigated as one of the correlated factors.2.Experimental study(1)Effects of Shugan Jianpi Yangxue Decoction and its primary bioactive component QUR on HepG2 cells:①Human hepatoma HepG2 cells were intervened with different concentrations of Shugan Jianpi Yangxue Decoction(0,2,4,6,8,and 10 mg/mL)and QUR(0,20,40,80,160,and 320 μM)for 24,48 and 72 hours respectively in vitro,the effects of drugs on cell viability were detected using CCK-8 method.②Different concentrations of Shugan Jianpi Yangxue Decoction(0,4,and 6 mg/mL)and QUR(0,160,and 320 μM)were used to intervene HepG2 cells for different lengths of time respectively;the effects of drugs on cell cloning ability were detected using clone formation experiment,and the effects of drugs on cell migration were evaluated by wound-healing experiment.③Due to limited conditions,only different concentrations of QUR(0,160,320 μM)were used to intervene HepG2 cells for 48 hours.After cells were stained with annexin V-FITC/PI reagents,the cell apoptosis was detected by flow cytometry.(2)A study of the mechanisms of Chinese medicine monomer QUR regulating the ceRNA network in HCC:①HepG2 cells were treated with zero and 320 μM QUR as control and QUR groups respectively,each group had three samples.The total RNA of all samples was extracted and the expression of transcriptome was detected through high-throughput RNA sequencing.②The DESEq2 method was applied to screen significantly differentiallyexpressed mRNAs(DEmRNAs),DElncRNAs,and DEmiRNAs between the control and QUR groups.③Gene ontology(GO)and Kyoto encyclopedia of Genes and Genomes(KEGG)function enrichment analyses were performed for DEmRNAs.④Protein-protein interaction(PPI)analysis of DEmRNAs was performed,then the PPI network was constructed,the core genes and the main functional modules were screened.⑤Survival analysis of DEmRNA genes was conducted,the genes significantly associated with the survival of HCC patients were regarded as key DEmRNAs.⑥Miranda,TargetScan,and RNAhybrid databases were used to predict DEmiRNA-key DEmRNA and DEmiRNADElncRNA relationship pairs.Combining with co-expression correlations,the core ceRNA network in HCC regulated by QUR was established.⑦Openly available databases were used to discover the expression of core ceRNA network components in HCC patients,also their associations with clinical features and prognosis.⑧HepG2 cells were treated with zero and 320 μM QUR for 48 hours respectively,then the expression of various RNAs in the two groups was detected using real-time quantitative polymerase chain reaction(RT-qPCR).Results1.Clinical study(1)A risk prediction nomogram of the occurrence of PVTT in patients with firstlydiagnosed CHB-HCC:①Among the 200 patients with newly-diagnosed CHB-HCC,77(38.5%)of them had PVTT,the incidence rate of type Ⅲ PVTT was 50.65%.②The gender,diabetes history,hypertension history,TCM syndrome types(liver depression and spleen deficiency,Qi stagnation and blood stasis,damp-heat accumulation,damp-heat and blood stasis toxin,and liver-kidney Yin deficiency),TCM syndromes(Qi stagnation,spleen deficiency,blood stasis,damp heat,and Yin deficiency),serum albumin(ALB),glutamic pyruvic transaminase(ALT),aspartate aminotransferase(AST),y-glutaminyl transferase(GGT),alpha-fetoprotein(AFP),platelet count(PLT),prothrombin time(PT),fibrinogen(FIB)and hepatitis B e antigen(HBeAg)of firstly diagnosed CHB-HCC patients had no significant correlation with the occurrence of PVTT(P>0.10).However,the age,tumor gross pathological classification,tumor number,tumor location,portal hypertension,esophageal and gastric varices,ascites,liver cirrhosis,serum total bilirubin(TBIL),neutrophil count(NEUT),lymphocyte count(LYM),neutrophil to lymphocyte ratio(NLR),platelet to lymphocyte ratio(PLR),and HBV DNA load were the correlated factors of the occurrence of PVTT(P<0.05).③Tumor gross pathological type of massive type(OR=8.13,P=4.32E-06)and diffuse type(OR=11.11,P=6.17E-04),multiple tumors(OR=3.29,P=4.85E-03),TBIL≥ 17.1 μmol/L(OR=3.92,P=7.56E-03),PLR ≥ 128.8(OR=3.82,P=9.89E-04),and aging(OR=0.94,P=3.16E-04)were the independent associated factors of PVTT occurrence in patients with firstly-diagnosed CHB-HCC.④The PVTT risk nomogram was composed of five indexes:tumor gross classification,tumor number,TBIL,PLR,and age;the diagnostic threshold of which was 140 of total scores,namely 25.08%of the prediction probability.The area under the ROC curve of the PVTT nomogram was 0.883,the sensitivity was 93.5%,and the specificity was 68.3%.The calibration was good,and the nomogram could provide over 75%of clinical net benefits.(2)A risk prediction nomogram of the occurrence of EHM in patients with firstlydiagnosed CHB-HCC:①Among the 200 patients with newly diagnosed CHB-HCC,31(15.5%)of them had EHM,and lungs were the most invaded(48.39%).②The gender,age,diabetes history,hypertension history,TCM syndrome types(liver depression and spleen deficiency,Qi stagnation and blood stasis,damp-heat accumulation,damp-heat and blood stasis toxin,and liver-kidney Yin deficiency),TCM syndromes(Qi stagnation,spleen deficiency,blood stasis,damp heat,and Yin deficiency),TBIL,PT,FIB,LYM,ALT,AST,GGT,AFP,HBeAg,and HBV DNA load of firstly diagnosed CHB-HCC patients showed no significant correlation with the occurrence of EHM(P>0.10).The tumor gross pathological classification,tumor number,tumor location,PVTT existence,liver cirrhosis,ascites,ALB,NEUT,NLR,and PLR were the correlated factors of EHM(P<0.05),portal hypertension and PLT were the potential correlated factors of EHM(P<0.10).③In the first model of multivariate analysis,tumor gross pathological type of massive type(OR=7.98,P=0.009)and diffuse type(OR=7.80,P=0.023),multiple tumors(OR=4.15,P=0.013),and NEUT≥6.3×109/L(OR=7.84,P=1.64E-05)were the independent risk factors of EHM occurrence in firstly-diagnosed CHB-HCC patients.In the second model,PVTT existence(OR=2.92,P=0.018)and NEUT≥ 6.3×109/L(OR=8.04,P=2.74E-06)were independent risk factors of EHM occurrence.④The EHM risk nomogram was composed of three indicators:tumor general classification,tumor number,and NEUT;the diagnostic threshold of which was 140 of total scores,namely 12.3%of the prediction probability.The area under the ROC curve of the EHM nomogram was 0.862,the sensitivity was 90.3%,and the specificity was 66.3%,which had good calibration and clinical benefits of over 60%.2.Experimental study(1)Effects of Shugan Jianpi Yangxue Decoction and its primary bioactive component QUR on HepG2 cells:①2～10 mg/mL Shugan Jianpi Yangxue Decoction and 20～320 μM QUR significantly inhibited the proliferation of HepG2 cells(P<0.05),the effect increased significantly with the increase of drug concentrations and intervention times(P<0.05).②Four and six mg/mL Shugan Jianpi Yangxue Decoction and 160 and 320 μM QUR significantly inhibited the clonal formation(P<0.001)and migration of HepG2 cells(P<0.05).③QUR significantly induced cell apoptosis(P<0.05),mainly late apoptosis(P<0.01).(2)A study of the mechanisms of Chinese medicine monomer QUR regulating the ceRNA network in HCC:①There were a total of 647 DEmRNAs,304 DElncRNAs,and 17 DEmiRNAs between the control and QUR groups.②The down-regulated DEmRNAs were responsible for the metabolism of nuclear corpuscles,chromatin,and telomere;while the upregulated DEmRNAs were involved in cytokine activity and immune cell differentiation.Signaling pathways of metabolism,cancer,interactions between cytokines and their receptors,PI3K/AKT,viral carcinogenesis,transcriptional disorders,and CHB were involved.③There were five dominant function modules in the DEmRNA PPI network,which included 10 core genes and 12 key DEmRNAs significantly associated with the survival of HCC patients(P<0.05).④The constructed core ceRNA network included three DEmiRNAs(hsa-miR-1260b,hsa-miR-423-5p,and hsa-miR-449a),five DEmRNAs(CSF2,TNF,IFIT1,KIF20A,and CCNB1)and 33 DElncRNAs.The ceRNA network might participate in the processes of foot assembly,viral genome replication,exogenous apoptosis,antigen-stimulated chronic inflammatory responses,and cell biosynthesis.⑤Compared with normal liver tissues,the expression of CSF2,KIF20A,and CCNB 1 in HCC tissues were significantly elevated;and the high expression of KIF20A and CCNB1 was correlated with the deteriorations of pathological stages,histological grades,and the prognosis of HCC(P<0.05),However,the increased expression of CSF2,TNF,and IFIT1 was correlated with the better prognosis(P<0.05).⑥Compared with normal liver tissues,the expression of hsa-miR-423-5p and hsamiR-449a was significantly increased in HCC(P<0.001),and the high-expression of hsamiR-423-5p was positively correlated with the progression of HCC stages and grades,but not with the prognosis of patients.⑦Compared with normal liver tissues,the expression of lncRNAs LOC730668,NEAT,NSUN5P1,NSUN5P2,BCRP2,and SP2-AS1 was significantly increased in HCC(P<0.001),and the high-expression of BCRP2 was associated with the poorer survival of patients(P<0.05).⑧The results of RT-qPCR showed that 320 μM QUR significantly increased the expression of CSF2,TNF,hsa-miR-449a,BCRP2,and SP2-AS1 in HepG2 cells(P<0.001);while decreasing the expression of IFIT1,KIF20A,CCNB1,hsa-miR-1260b,hsa-miR-423-5p,LOC730668,NEAT1,NSUN5P1 and NSUN5P2(P<0.01),compared with the control group,which were consistent with the results of RNA sequencing.Conclusion1.Clinical studyThis study had not found a significant correlation between TCM syndrome types or syndromes and the occurrence of PVTT or EHM in patients with CHB-HCC.The established PVTT and EHM risk nomogram could accurately predict the occurrence risk of PVTT and EHM in patients with firstly-diagnosed CHB-HCC,which might help the early diagnosis and treatment of high-risk patients,reduce the unwanted panic and examinations of low-risk patients,and improve clinical benefits.2.Experimental studyShugan Jianpi Yangxue Decoction and QUR effectively inhibited the proliferation and migration of HCC cells,and QUR monomer induced cell apoptosis.QUR might up-regulate BCRP2/hsa-miR-423-5p/TNF and SP2-AS1/hsa-miR-1260b/CSF2 axes,and down-regulate(NEAT1,LOC730668,NSUN5P1,NSUN5P2,and LINC00473)/hsa-miR-449a/(IFIT1,KIF20A,and CCNB1)axes to regulate the expression of multiple tumor suppressor/promoter factors benignly,thus inhibiting metabolism,mitosis,and transcription of HCC cells,and enhance cytokine-mediated anti-cancer immune responses and exogenous apoptosis to treat HCC through multiple mechanisms,with advantages on CHB-HCC.This study preliminarily confirmed the inhibitory effects of Shugan Jianpi Yangxue Decoction and QUR on HCC and reported the ceRNA mechanism of QUR against HCC for the first time.This study might provide references for the clinical application and follow-up research of the decoction and QUR. |
