Abnormal Expression Of ESE3 And Its Clinical Significance In Hepatocellular Carcinoma

[Background]Hepatocellular carcinoma（HCC）is a kind of malignant tumor derived from liver cells.HCC is the sixth most common cancer and the fourth most common cause of cancer-related deaths.The occurrence of HCC is a complex pathological process,main reasons are chronic virus infection,alcoholic,nitrosamine and aflatoxin etc.In recent years,patients with nonalcoholic fatty liver disease and autoimmune liver disease are increasing.HCC is the fourth most common cancer and the fourth most common cause of cancer-related deaths in China.According to the data of the global cancer burden in 2020 from WHO,45.27%of the global incidence of HCC and 47.12%of the global mortality are from China.HCC brings a huge burden to the safety of people’s lives and property.Surgical resection is the first choice of the treatments.But a lot of patients are diagnosis in advanced stage of HCC which leads them losing operation opportunities.In recent years,the treatments of HCC increasingly mature,such as,transarterial chemoembolization,radiofrequency ablation,microwave ablation,radiation therapy,liver transplantation and biological target therapy,immune therapy etc.Combined with liver cancer staging system,personal willingness and therapeutic response,patients can receive individualized treatment plan.However,due to the high recurrence rate of HCC,these conventional treatment methods have certain limitations.Different patients with the same tumor stage may have completely different prognosis.The follow-up management plan should be made individually.Therefore,it is urgent to find new potential therapeutic targets for HCC to improve the therapeutic effect,and to fine new and effective prognostic indicators for HCC,especially for early-stage patients.ETS（E26 transformation-specific）superfamily is one of the largest families of transcription regulators.The superfamily is structurally divided into 11 subfamilies.All ETS transcription factors have a highly conserved ETS DNA binding domain composed of 85 amino acids.The ETS domain regulates downstream gene transcription by specifically binding to the promoter’s core sequence[5’-GGA（A/T）-3’].ETS transcription factors play an important role in cell proliferation,differentiation,migration,apoptosis,vascular development and organ formation.Abnormal protein expression of family members is related to the occurrence and progression of tumors,and has become an important indicator for predicting tumor prognosis.Epithelium-specific ETS（ESE）transcription factors are a subfamily of the ETS superfamily.It is divided into two subgroups:（1）ELF3,ELF5,ESE3,（2）SPDEF.In the occurrence and development of many cancers,the specific ESE factor can play different roles.Epithelium-specific ETS factor family member 3（ESE3）,also known as E26 transformation specific homologous factor（EHF）,is a member of the ESE subfamily and can directly involve in epithelial-mesenchymal transformation（EMT）,stem cell-like characteristics and tumor progression.ESE3 expression mainly locates in the nucleus of the epithelial cells.ESE3 plays different roles in different tumors.ESE3 plays a promoting role in gastric cancer,thyroid papillary cancer,ovarian cancer,and breast cancer,while it plays an anti-cancer role in prostate cancer,pancreatic cancer,colon cancer and esophageal cancer.Intracellular location change was found in the study of esophageal squamous cell carcinoma（ESCC）,ESE3 expression is mainly in the nucleus of normal esophageal epithelial cells,and in the cytoplasm of ESCC.ESE3 negatively regulates the proliferation of ESCC cells.The overexpression of ESE3 can restore the nuclear expression of ESCC cells and play an anti-tumor role.There is no relevant research on the expression location and the role of ESE3 in HCC.In this research,we investigated the expression pattern of ESE3 in HCC and its role in the progression of HCC,the relationship between its expression level and the clinicopathological characteristics and prognosis of patients,and the possibility of being a clinical prognostic indicator.We verified the effects of overexpression or knockdown expression of ESE3 on the cell proliferation,apoptosis and cell cycles in vitro.Conclusions were verified in vivo.Part 1.Abnormal expression of ESE3 in HCC and its relationship with clinicopathological characteristics and prognosis.[Objective]1.To study the location and expression level of ESE3 in HCC tissue and adjacent liver tissue;2.To analysis the correlation between the expression level of ESE3 in HCC and the clinicopathological features of patients including clinical stage,tumor size,number of tumor nodule,vascular invasion,and so on.3.To analysis the correlation between the expression level of ESE3 in HCC and the clinicopathological features and prognosis of recurrence and survival.To analysis the predictive value of the expression level of ESE3 in HCC for postoperative recurrence and survival with different clinical stages and pathological features.[Methods]1.The tissue microarrays were prepared with specimens of patients underwent curative resection.Study the location and expression level of ESE3 in HCC tissue and peritumoral tissues with Western Blot and immunohistochemistry.2.Using Image Pro software to analysis the Integrated Optical Density（IOD）of immunohistochemical stain.Analysis the correlation between expression level of ESE3 in HCC tissue and clinicopathological features of patients with the LogIOD value.3.Survival analysis and responding curve were carried out with the Kaplan-Meier method and Cox risk model.These statistical methods were used to analysis the predictive value of the expression level of ESE3 in HCC tissue for postoperative recurrence and survival with different clinical stages and pathological features.[Results]1.Subcellular localization and expression level of ESE3 in HCC tissues and corresponding peritumoral tissues.（1）ESE3 mainly located in the cytoplasm of HCC and peritumoral tissues,while rarely expressed in nucleus.（2）The LogIOD of ESE3 expression in HCC tissues was significantly lower than that in corresponding peritumoral tissues.（3）Using 4.758 as the cut-off value,we found that the proportion of low ESE3 expression in the HCC tissues was significantly higher than that in corresponding peritumoral tissues.（4）The LogIOD of ESE3 expression in Edmondson grade Ⅰ HCC tissues was significantly higher than that in Edmondson grade Ⅱ or Ⅲ.（5）Using Western Blot,expression of ESE3 protein was further demonstrated in five paired fresh samples tested.The expression of ESE3 protein was significantly lower in HCC tissue than that in corresponding peritumoral tissue.2.The relationship between ESE3 expression and clinicopathologic features in HCC TMA.Patients with low ESE3 expression in HCC was positively correlated with the aggressive clinical feature,such as presence of vascular invasion,capsular invasion,multiple tumor nodules,BCLC stage（B+C）and worse Edmondson grade.No statistical relationship was found between ESE3 expression and patient’s age,gender,liver cirrhosis or tumor size.3.The predictive value of the expression level of ESE3 in hepatocellular carcinoma for postoperative recurrence and survival with different clinical stages and pathological features.（1）Kaplan-Meier analysis showed that HCC patients in the low ESE3 expression group has worse RFS and OS than those in the high ESE3 expression group.（2）Univariate survival analysis showed that ESE3 expression level,tumor size,BCLC stage,vascular invasion were significantly associated with RFS and OS.Worse Edmondson grade has prognostic significance for poor RFS.（3）A multivariable analysis using Cox regression model was performed.The tumor size,Edmondson gradeand ESE3 expression level were identified as independent risk factors for RFS,while ESE3 expression leveland tumor size were recognized as independent prognostic factors for OS.The hazard ratios for low ESE3 expression level for RFS and OS were 0.595（95%CI:0.354 to 1.000）and 0.528（95%CI 0.296 to 0.942）,respectively.4.The significance of ESE3 loss and gain in HCC tissues compared to peritumoral normal tissues.According to the intensity of ESE3 staining in HCC tissues and corresponding peritumoral normal tissues,the patients were classified two groups:the ESE3 loss group（ESE3T0.05,except for RFS in the tumor size≥5cm,multiple tumor nodules and Edmondson grade Ⅲ group respectively,p<0.05）.（2）In HCC "Early-stage" subgroup,patients with low ESE3 expression level exhibited a significantly poorer OS and RFS than those with high ESE3 expression level regardless of clinical features.[Conclusion]1.ESE3 mainly located in cytoplasm of HCC tissue and corresponding peritumoral tissues.2.Low ESE3 expression in HCC tissues related to HCC progression and poor prognostic.expression level of ESE3 may be a clinical prognostic indicator for postoperative recurrence and survival,especially in HCC "Early-stage" patients.Part 2.Expression of ESE3 in HCC cells and its effects on biological functions of hepatocellular carcinoma cells.[Objective]1.To study the location and expression level of ESE3 in HCC cell lines;2.To study the biological functions of ESE3 expression in HCC cell lines.Overexpression or knockdown ESE3 in HCC cell lines.Analysis the influences on cell proliferation,apoptosis and cell cycles.3.To evaluate the effect of ESE3 on HCC progression in vivo of xenograft mice tumor model.[Methods]1.ESE3 mRNA and protein expression were analyzed by q-PCR and Western Blot in HCC cell lines,respectively.Immunofluorescence analysis was performed to characterize the subcellular localization of ESE3 in HCC cell lines.2.Construct recombinant ESE3 overexpressed lentiviruses.Western Blot was used to verify the effect of overexpression in HepG2 cell.Construct three ESE3 interference fragments.Western Blot was used to verify the efficiency of knockdown expression of ESE3 in Hep3B.3.MTT assay was used to evaluate the effects on cell proliferation.Flow Cytometer was used to evaluate the effects on cell apoptosis.4.Flow Cytometer was used to evaluate the effects on cell cycle.5.HepG2 cells stably transfected with the recombinant ESE3 overexpressed lentivirus or mock control were subcutaneously injected into the flanks of nude mice.The tumor volumes were measured every 3 days for 30 days.Tumor volumes and weights were detected after execute.Evaluate the roles of ESE3 on HCC progression in vivo.[Results]1.The location and expression level of ESE3 in HCC cell lines and LO2 cell.（1）Relative densities（RD）by normalizing with β-tubulin showed that the ESE3 expression level in HCC cell lines was remarkably lower than that in LO2 cell.（2）Consistent with the ESE3 protein expression pattern,the q-PCR results also confirmed the low expression of ESE3 mRNA in HCC cell lines than that of LO2 cell.（3）ESE3 was mainly located in cytoplasm of Hep3B and nucleus of pancreatic carcinoma cell line BxPC-3 respectively,whereas the negative control showed the absence of specific fluorescence signal.2.Overexpression in HepG2 cell and knockdown ESE3 in Hep3B cell.（1）ESE3-SiRNA（ESE3-SiRNA-1/2/3）was transfected to Hep3B cell.Compared with ESE3-SiNC control group,expressions of ESE3 in ESE3-SiRNA-1 group and ESE3-SiRNA-3 group were significantly lower.ESE3-SiRNA-3 group was chosen in the following experiments.（2）Significantly elevated expression of ESE3 in infected HepG2 cells was confirmed by Western Blot.3.The impacts of overexpression or knockdown ESE3 on the ability of cell proliferation and apoptosis of HCC cell lines.（1）MTT assay was used to evaluate the effects on cell proliferation.Overexpression ESE3 in HepG2 cells significantly inhibited cell proliferation at the 72h,96h,120h observation points.Overexpression ESE3 in Hep3B cells significantly inhibited cell proliferation at the 24h and 72h observation points.Knockdown ESE3 in Hep3B cells significantly promoted cell proliferation at the 72h observation points.（2）Flow Cytometer was used to evaluate the effects on cell apoptosis.Overexpression ESE3 in Hep3B cells significantly promoted cell apoptosis.Overexpression ESE3 in HepG2 cells significantly promoted cell apoptosis.Knockdown ESE3 in Hep3B cells significantly inhibited cell apoptosis.Knockdown ESE3 in HepG2 cells inhibited cell apoptosis,but the result has no significant difference.4.Flow Cytometer was used to evaluate the effects on cell cycle.Overexpression ESE3 in Hep3B cells and HepG2 cells,the percentage of G0/G1 cells is significantly higher than that in the control group,the percentage of S cells is significantly lowerthan that in the control group.Knockdown ESE3 in Hep3B cells,the percentage of G0/G1 cells is significantly lower than that in the control group,the percentage of S cells is significantly higher than that in the control group.Knockdown ESE3 in HepG2 cells,the percentage of G0/G1 cells is lowerthan that in the control group,the percentage of S cells is higher than that in the control group.But the results have no significant difference.5.Effects of ESE3 on HCC progression in vivo of xenograft mice tumor model.Growth curve of the formed tumor showed that overexpression ESE3 group has lower growth rate than the mock control group.Analysis of the excised tumors showed that the weight and volume of overexpression ESE3 group were lower than those in the mock control group.[Conclusion]1.Expression level of ESE3 in HCC cell lines is lower than that in normal liver cell.2.ESE3 can inhibit HCC cell proliferation,promote HCC cell apoptosis and stop HCC cells in G0/G1.ESE3 may be used as a new therapeutic target of HCC.