| Background:Hepatocellular carcinoma(HCC),as the most common pathological pattern of liver cancer,is an aggressive malignancy with high morbidity and mortality.The pathogenic factors include hepatitis B virus infection,hepatitis C virus infection,exposure of aflatoxin,intaking alcohol and high-fat diet.China is a country with high incidence of hepatitis B virus infection,so the prevention and treatment of HCC is particularly important.Hepatic resection and liver transplantation are the main treatments for the early HCC,and the early stage HCC is asymptomatic,most patients are often in the moderate and late stages of the disease at the time of diagnosis.Therefore,only a small number of patients are suitable for radical treatment when they are diagnosed.In addition,recurrence and metastasis of HCC is another important factor affecting the prognosis of patients.Therefore,finding high-sensitivity and specific diagnostic biomarkers to improve the early diagnosis rate of HCC,so that more patients are suitable for radical treatment,and screening molecular targets and signaling pathways related to tumor recurrence and metastasis to undergo early and precise intervention in order to improve prognosis of HCC patients.Aims:1.Investigate and validate the potential application value of the new molecular biomarker-glutamine synthetase,in the diagnosis and prognosis of HCC.2.Study the effect of glutamine synthetase on the malignant biological behavior of HCC cells and to analyze its underlying molecular mechanism.Methods:1.We applied proteomics assay、enzyme-linked immunosorbent assay、immunohistochemistry to explore and validate the biomarker which is specifically high expression in tumor tissues and evaluate its clinical significance.Firstly,20 HCC samples(recognized by pathological diagnosis and underwent no other anti-tumor treatment before hepatic resection)were collected.The surgically resected tumor tissue and corresponding adjacent tissues were subjected to two-dimension difference gel electrophoresis(2D-DIGE)coupled with mass spectrometry.Differentially expressed proteins of HCC tissue and the matching adjacent tissue were screened for subsequent studies.Then,the concentration of candidate biomarker--glutamine synthetase(GS)and alpha-fetoprotein(AFP)in serum of patients with primary hepatocellular carcinoma(n=115)and healthy people(n=57)were detected by enzyme-linked immunosorbent assay(ELISA).The diagnostic value of GS and AFP was analyzed,especially for the AFP negative HCC patients(n=75).Subsequently,the immunohistochemical(IHC)staining was used to detect the expression level of GS in tumor tissues and the corresponding adjacent tissues of HCC patients(n=153).Clinicopathological features and prognosis information were collected.And the correlation between the expression of GS and the malignant degree or the prognosis of HCC were evaluated.The above findings were verified by means of an independent HCC cohort(n=364)from the TCGA database.2.We applied cellular function assay and mechanism analysis、metastatic animal model construction and tumor biomarker detection,to study the effect of glutamine synthetase on HCC cells and analyze the underlying molecular mechanism.In vitro,we used the lentivirus with GS-shRNA to reduce the expression of GS in Huh7 cells(with high expression level of GS in wild-type),and the lentivirus with plasmids of GS to infect HCCLM3 and BEL-7402 cells(with low expression level of glutamine synthetase in wild-type)to increase the expression of GS.The interference and overexpression efficiency was validated by q-PCR and western blot to construct the HCC cells with stably aberrant GS expression.The effects of glutamine synthetase on the proliferation,colony formation and migration of HCC cells were detected by MTT and trans well assays.The HCCLM3 cells with high metastatic ability was used to explore the effect of glutamine synthetase expression on the invasion ability of HCC in vivo.Western blot was used to detect the effect of GS on epithelial-mesenchymal transition(EMT)in HCC and biomarkers associated with mTOR/p-S6 signal pathway,and the underlying mechanism of its influence on HCC invasion and proliferation were explored preliminarily.At the same time,immunohistochemistry was used to detect GS,p-S6-S235/236 and p-S6-S240/244 in hepatocellular carcinoma tissues from HCC patients for correlation analysis,and to explore the mechanism of the influence of GS on malignant progression of hepatocellular carcinoma.Results:1.Proteomic analysis found that the expression level of 20 proteins in HCC tissues were significantly higher than that in the matching adjacent tissues(differential fold≥1.5 times),and the expression level of glutamine synthetase in tumor tissues was 20.12 times higher than the adjacent tissues(p=0.028).Therefore,it was selected as a candidate biomarker of this study for its clinical significance and cancer-promotion mechanism for subsequent exploration.The results of ELISA showed that the concentration of GS(853.2±785.4 ng/ml vs 228.5±113.8ng/ml,p<0.001)and AFP(235.2±584.4 ng/ml vs 18.3±6.6ng/ml,p<0.001)in serum of HCC patients was significantly higher than in that of healthy controls and the diagnostic value for HCC was favorable(The AUROC was 0.848 and 0.861,respectively).And the serological concentration of GS in AFP-negative HCC patients was also significantly higher than that in healthy controls(978.1±864.4 ng/ml vs 274.3±109.6ng/ml,p<0.001)with a satisfying diagnostic value(The AUROC was 0.913).IHC staining showed that the expression level of GS in HCC tissues was significantly higher than that in the matching adjacent tissues.Further analysis with the clinicopathological characteristics revealed that the level of GS in rumor tissues wasn’t associated with the clinicopathological features of HCC patients.Prognostic analysis found that in moderately differentiated HCC,the expression level of GS(p=0.045)and vascular invasion(p=0.003)in HCC were independent risk factors for the recurrence-free survival(RFS)of HCC patients,and Kaplan-Meier analysis demonstrated that the RFS rate of patients with high expression of GS or with vascular invasion was significantly lower than that of the control group(the post-operatively 3-year RFS:42%.vs 18%,p=0.001;36%.vs 8%,p<0.001,respectively).What’s more,combing analysis of the above two indicators could improve the predictive value for the prognosis of HCC(p<0.001).Analysis of the data of HCC cohort in TCGA database found that the expression level of GS-mRNA in tumor tissues was significantly higher than that in adjacent tissues(p<0.01).The prognosis analysis for hepatitis B related HCC patients found that the overall survival of patients with high level of GS-mRNA was worse than that with low level of GS-mRNA(post-operatively 5-year overall survival:61%.vs 43%,p=0.014).The same conclusion was made in alcoholic liver disease related HCC patients(post-operatively 5-year overall survival:53%.vs 28%,p<0.001)).2.In vitro,cell proliferation experiments showed that in the glutamine-free culture system,glutamine synthetase silencing significantly inhibited the proliferation(p<0.001)and clonal formation(p<0.01)of hepatocellular carcinoma cells,and overexpression of glutamine synthetase significantly increased the proliferation(p<0.001)and clonality(p<0.001)of hepatocellular carcinoma cells.Western blot analysis showed that silencing GS expression inhibited the phosphorylation of p-S6-S235/236 and p-S6-S240/244,while GS over expression enhanced the phosphorylation of p-S6-S235/236 and p-S6-S240/244.Glutamine synthetase has no significant effect on proliferation of hepatocellular carcinoma cells and the activation of mTOR/p-S6 signal pathway in glutamine-containing culture system.Trans well assay showed that overexpression of GS increases the migration ability of HCCLM3 and BEL-7402 cells(p<0.01 both),while silencing its expression inhibits the migration ability of Huh7 cells(p<0.01).Western blot analysis showed that silencing glutamine synthetase expression significantly decreased the expression of N-cadherin and increased the expression of E-cadherin.On the contrary,over-expressing glutamine synthetase increased N-cadherin expression and decreased the expression level of E-cadherin.The mouse lung and liver metastatic model of HCC showed that high expression of glutamine synthetase increased the ability of HCCLM3 cells to metastasize in mice(lung metastatic incidence:50%.vs 0%).Correlation analysis of immunohistochemistry for hepatocellular carcinoma tissues showed a significant positive correlation between GS and phosphorylated p-S6-S235/236 and between GS and phosphorylated p-S6-S240/244.Conclusions:1.GS may play a significant role in the development of hepatocellular carcinoma.2.GS is a useful biomarker for the diagnosis and prognosis of HCC.3.GS exerts influence on HCC cell proliferation and invasion through mediating mTOR/p-S6 signal pathway and epithelial-mesenchymal transition,respectively.4.GS may be a therapeutic target of inhibiting proliferation and invasion in HCC. |
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