| BackgroundHepatocellular carcinoma(HCC)is the most common primary liver cancer and the major leading cause of cancer mortality worldwide.Tumor micro-environment(TME)actively participates in hepatic fibrosis,HCC,invasive and metastasis.Tumor pathogenesis is a high generalization of TME heterogeneity,and the remarkable characteristics of the treatment of HCC with TCM is to regulate and improve TME through multi-target effects.Autophagy plays a dual role in HCC,and the three main types of autophagy terminate in lysosomes,referred to as autophagy-lysosomal pathway(ALPs).Artesunate(ART)has been indicated as a candidate drug for HCC with multiple targets and multiple pathways.However,whether ART intervenes the malignant progression of HCC via targeting ALPs remains to be defined.Herein,HCC markers that reveal the features of TME were screened and their clinical significance was explored sequentially based on the paired samples of HCC and non-cancerous liver tissues from clinical cohorts.Multi-dimensional experiments from molecular,cellular to animal levels were carried out.Given that therapeutic advantages of ART in improving the TME of HCC,investigation was undertaken on whether ART could affect the expression of the identified HCC marker to interfere the malignant transformation from hepatic fibrosis to HCC.In addition,the underlying mechanisms of autophagy-lysosomal pathway effected by ART were investigated to provide a new interpretation of the molecular events of the malignant transformation from Hepatic fibrosis to HCC.This study may provide a new therapeutic target and method for the clinical treatment of HCC.Methods&Results1.Identification of the candidate marker genes related to the malignant progression from liver fibrosis to HCC and verification of the clinical relevance.This study was approved by the Research Ethics Committee of the Institute of Chinese Materia Medica and the Fifth Medical Centre of Chinese PL A General Hospital(License No:2016003D).1.1.Clinical microarray detection was carried out on clinical cohort samples to identify HCC related differential gene expression profiling.Using the clinical cohort collected(a total of 99 cancerous and 18 adjacent noncancerous liver tissue samples from 100 HCC patients)from the Fifth Medical Centre of Chinese PLA General Hospital,mRNA expression profiling was carried out on 3 pairs of HCC tissues and adjacent non-cancerous liver tissues.The microarray data showed that compared with non-cancerous liver tissues,a total of 1766 differential genes were found in HCC tissues,including 1248 up-regulated genes and 518 downregulated genes.1.2.Clinical qPCR experiments and biomolecular network mining were integrated to screen HCC candidate marker genes.The interaction network of HCC related differential genes was constructed.Following the calculation of network topological characteristics and functional modular analysis,glucocerebrosidase(GCase,encoded by GBA gene)was indicated to play a key role in the malignant progression from liver fibrosis to HCC.In addition,clinical microarray results showed that GBA was one of the significantly overexpressed genes(cancer and non-cancer,7.54±1.10 vs 5.73±1.02,P=0.03).Consistently,clinical qPCR experiments showed that GBA gene was highly expressed in HCC(cancer and noncancerous,0.07±0.07 vs 0.01±0.01,P<0.001).1.3.Clinical relevance analysis based on our clinical cohort and TCGA database revealed the associations of GBA with clinicopatholoical features and patients’prognosis.In order to evaluate the clinical significance of GBA in human HCC,taking the median expression of GBA gene(0.506)in all 99 HCC tissues as the cutpoint value,the patients were divided into GBA high expression group and low expression group(n=50 and 49 cases respectively).The results showed that HCC patients in the GBA high expression group had higher serum AFP level(0.01<p<0.05)and tumor grade(P<0.01)than those in the GBA low expression group.The statistical analysis of TCGA data set showed that the overexpression of GBA was more frequently occurred in HCC patients with the old age(P=0.02),the presence of metastasis(0.01<p<0.05)and vascular invasion(P=0.04),and the short overall survival(P=0.01).The Kaplan Meier survival analysis based on our clinical cohort and TCGA data set showed that the overall survival(P=0.0274 and P=0.0151,respectively)and the disease-free survival(P=0.0377 and P=0.0405,respectively)of GBA high expression group were markedly worse than that of low expression group.2.Oncogenic roles of GBA in malignant characteristics of HCC.2.1.Cellular functional experiments was carried out to investigate the oncogenic roles of GBA in HCC.Western blot was carried out to detect the expression of GBA protein in two human HCC cell lines HepG2 cells and MHCC-97H cells,with WRL68 human normal hepatocytes as the control.Using lentivirus overexpression GBA vector and cell transfection,the GBA overexpressed cell line was constructed,and Western blot experiment was carried out to verify the transfection efficacy.GBA specific small interfering RNA(siRNA)was used to construct transient transfection vectors(si-GBA313,si-GBA-1255 and si-GBA-1620).GBA knockdown expression cell lines were constructed through cell transfection.Western blot results showed that the above specific siRNA transfection could significantly inhibit the expression of GBA protein in HepG2 and MHCC-97H cells.Considering that GBA gene interferes with lysosomal and autophagy functions by regulating the expression and activity of GCase hydrolase,this study used GBA enzyme activity agonist LTI-291 as the control of GBA function enhancement experiment in following research.The results of cellular functional experiment showed that the proliferation and cloning ability of HepG2 and MHCC97H cells decreased significantly after knockdown of GBA expression(P<0.05,P<0.01).Knockdown of GBA effectively induced apoptosis by increasing the percentage of early and late apoptotic cells(P<0.01),and showed G1 phase arrest of cell cycle(P<0.05).Accordingly,GBA activator LTI-291 treatment significantly reversed the inhibitory effects of GBA down-regulation on the proliferation,cell cycle and cell colony of two HCC cells and its inhibitory effect on apoptosis(all P<0.05).2.2.Immunohistochemistry and immunofluorescence were carried out to detect the expression of GBA and autophagy markers in clinical HCC samples.The HCC samples from the same patient were used for immunohistochemical and immunofluorescence analysis to detect the expression of GBA protein and autophagy flux[microtubule associated protein 1 light chain-3b(LC3B),sequentosome 1(SQSTM1,p62)]protein.The results showed that HCC tissues showed strong positive staining for GBA protein and SQSTM1/p62 accumulation,and the increase in the number of LC3B fluorescent spot(P<0.001).2.3.Western blot and transmission electron microscope were unfolded to study the effect of GBA on autophagy flux of HCC cells.Western blot showed that the expression levels of LC3B and SQSTM1/p62 proteins were significantly up-regulated in GBA knockdown HepG2 cells compared with GBA knockdown and BAF(late autophagy inhibitor)treatment group(P<0.05).Transmission electron microscopy showed the ultrastructure of HepG2 cells in different treatment groups.A large number of multimembrane autophagy vesicles(autophagy bodies)were found in GBA knockdown cells.GBA knockdown and BAF combined treatment increased autophagy bodies(P<0.01).The treatment group with GBA activator LTI-291 decreased the number of autophagosomes(P<0.01),indicating that the knockdown of GBA may lead to the impairment of autophagic degradation of HCC cells,and the activation of GBA partially reversed this result.3.Identification and validation of GBA as a direct target of ART.Using the biological probe with artemisinin activity,taken HepG2 cell line as the carrier and human normal hepatocyte line WRL68 as the control,affinity proteomics was down to identify the direct target spectrum of ART on HCC cells.The direct targeting relationship between ART and GB A was found and verified by probe labeled liquid chromatography and tandem mass spectrometry(LC-MS/MS),pull-down/WB and surface plasmon resonance technology(SPR).The binding activity between ART and GBA was expressed by KD value,which was 40 μM.4.At the cellular level,the mechanism of ART’s anti HCC effect by targeting and regulating ALPs mediated by GBA was studied.4.1.GBA is involved in the inhibitory effect of ART on HCC cells.The results showed that ART inhibited the proliferation and clone formation of HepG2 and MHCC-97H cells in a dose-dependent manner,blocked the G1 phase of cell cycle and induced HCC cells apoptosis(P<0.05).The combined treatment of LTI291 reversed the inhibitory effect of ART on the proliferation,cell cycle and cell clone of HCC cells and its induction of apoptosis(P<0.05).4.2.ART inhibits autophagic degradation of HCC cells by targeting GBA.Different doses of ART showed that ART promoted the transformation from LC3B-I to LC3B-II,increased the expression of SQSTM1/p62,but decreased the expression of GBA protein in HepG2 cells(P<0.05).Transmission electron microscopy showed that ART treatment significantly increased the number of autophagosomes in HepG2 cells,which was enhanced in the combined si-GBA transfection group,but reversed in the combined LTI-291 group(P<0.05).5.At the animal model level,the mechanism of ART playing an anti HCC role by targeting and regulating Alps mediated by GBA was studied.Male Sprague-Dawley(SD)rats(n=80,240-260g in weight)were purchased from Guangdong Medical Laboratory Animal Center(production license no.SCXK 2013-0002,Guangzhou,China).BALB/c nude mice(weight:15±2 g;age:7-8 weeks old)were purchased from Guangdong Medical Laboratory Animal Center,Guangzhou,China(License No:SCXK-2018-0186).Animal experiments were carried out according to the guidelines for the care and use of laboratory animals of the Center for Laboratory Animal Care,China Academy of Chinese Medical Sciences,Beijing,China(License No:IACUC-AEWC-F2005003).5.1.ART may inhibit the overexpression of GBA and reduce the occurrence of inflammation induced HCC.The results of gross anatomical observation and pathological tissue section showed by hematoxylin eosin(HE staining)indicated that ART effectively reversed the pathological changes of the liver by reducing the level of inflammation and malignancy at the specified time.The body weight and liver index of rats in DEN injury model and DEN+ART treatment group were significantly lower than those in normal control group(CON group)and ART group(P<0.05).There was no significant difference in body weight and liver index between CON group and ART group,DEN group and DEN+ART group.ART effectively reduced the increase of serum biochemical indexes[alanine aminotransferase(ALT),glutamic oxaloacetic transaminase(AST),alkaline phosphatase(ALP),hyaluronic acid(HA)and alpha fetoprotein(AFP)]induced by den(P<0.05).The results of immunohistochemistry and immunofluorescence showed that the anti-HCC effect of ART was related to GBA induced autophagy degradation.After treatment,the expression level of GBA protein was significantly decreased,while the expression level of SQSTM1/p62 protein was significantly increased(all P<0.05).Immunofluorescence detection showed that LC3B fluorescent spots after DEN administration increased after ART treatment(P<0.05).5.2.ART may block GBA-mediated autophagic degradation and reduce tumor burden in tumor-bearing in situ nude mice.The results of gross anatomy showed that ART treatment effectively reduced the size of tumor compared with the model group.Transmission electron microscopy showed that ART significantly enhanced the accumulation of autophagy bodies in tumor tissues(P<0.05).Compared with ART and LTI-291 alone,the number of autophagosomes decreased and increased when ART and LTI-291 were administered together.Immunofluorescence analysis revealed the co-localization of GBA with LC3B and SQSTM1/p62 proteins in different groups of tumor tissues.The levels of LC3B and SQSTM1/p62 protein increased and GBA decreased in the ART group,which was reversed by LTI-291.Conclusion1.GBA is significantly overexpressed in clinical HCC tissues and is significantly associated with HCC aggressive progression and poor prognosis.2.Both in vivo and in vitro experiments show that GBA may play an oncogenic role in promoting HCC progression,and the inhibition of the abnormally high expression of GBA may effectively reduce the malignant behavior of HCC cells.3.In clinical HCC samples,liver tumor tissues of DEN-induced HCC rat model and orthotopic mouse model,and human HCC cell lines,the abnormally high expression of GBA is closely related to the excessive increase of autophagy activity of HCC cells.4.GBA is one of the direct targets of ART.Both in vivo and in vitro experiments showed that ART treatment may inhibit the expression of GBA,leading to the block of late stage of autophagy(ALPs impaired),and this effect may be partially reversed by the GBA agonist LTI-291.5.ART exerts its anti-HCC effect by targeting the GBA-mediated ALPs. |
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