| Esophageal cancer(EC)is the 7th most common cancer and the 6th leading cause of death among all cancers worldwide,which becomes one of the major diseases threatening human health.Esophageal squamous cell carcinoma(ESCC)is the predominate EC subtype in China,accounting for more than 90% of cases.Surgery combined with adjuvant treatments remains the main therapy for early stage of EC.However,the 5-year survival rate is only approximately 30-50%.Local recurrence and distant metastasis are the main factors affecting the prognosis of patients.In recent years,targeted therapy has shown effective results in some malignant tumors.However,the progress of targeted therapy is relatively slow for ESCC.It is one of the current strategies for delaying ESCC progress by exploring several effective targets at the molecular level to reduce the recurrence and metastasis of ESCC,which has important clinical value and significance.Epithelial mesenchymal transition(EMT)refers to the process of epithelial cells transforming into mesenchymal cells under certain conditions.EMT is involved in the formation of gastrula,histomorphology and wound healing under physiological conditions.However,EMT could lead to the loss of polarity,decreased adhesion and enhanced migration ability of epithelial cells under pathological conditions,which endows the cells with the ability of invasion and metastasis.Therefore,EMT is widely recognized as one of the mechanisms of tumor recurrence and metastasis.LEF1 is a key nuclear transcription factor in Wnt/β-cantenin signaling pathway.Under physiological conditions,LEF1 plays an essential role in stem cell maintenance and organ development.However,abnormal expression of LEF1 may disrupt the regulatory mechanism of cells,causing abnormal cell proliferation and tumor formation.It has been confirmed that LEF1 plays a vital role in the cell proliferation,invasion,metastasis,and the maintenance of tumor stem cells.Thus,it could be a potential prognostic marker and therapeutic target of tumors.The study of LEF1 in ESCC is limited.Our previous study had showed that LEF1 was highly expressed in ESCC compared with normal esophageal epithelial tissue.In addition,the prognosis of patients with high LEF1 was worse than those with low LEF1,which suggested that LEF1 could be used as a prognostic marker of ESCC.However,the biological function of LEF1 and its regulatory mechanism in ESCC have not been reported until now.We speculate that LEF1 may play an important role in the occurrence and progression of ESCC,and now we continue to explore the function and mechanism of LEF1 in ESCC.Part 1 The role and mechanism of LEF1 in ESCC Objective: To explore the biological function and downstream molecular mechanism of LEF1 in ESCC.Methods: 1.Oncomine database was used to analyze the expression level of LEF1 in ESCC tissues and normal tissues.2.Stable LEF1-knockdown and LEF1-overexpression ESCC cell lines were established by lentivirus.3.m RNA level of LEF1 was detected by q RT-PCR and protein level of LEF1 was detected by western blot.4.CCK-8 assay was employed to examine the cell proliferation ability,wound-healing assay and transwell assay were employed to detect cell migration and invasion ability.5.Xenograft mice was employed to compare the tumorigenicity in vivo.6.Transcriptome sequencing was employed to find the downstream target genes.7.JASPAR database was used to predict the binding sites between transcription factors and downstream target genes.8.Luciferase reporter assay and chromatin immunoprecipitation(Ch IP)assay were employed to verify the binding of transcription factors and downstream target genes and determine the binding sites.9.Rescue experiment was used to verify the phenotypic rescue ability.10.Consecutive section of immunohistochemistry(IHC)were used to determine the association between two proteins.Result:1.LEF1 is highly expressed in ESCC: Compared with normal esophageal tissue,LEF1 was highly expressed ESCC tissues.2.LEF1 enhances the proliferation and tumorigenicity of ESCC cells:(1)Compared with the control group,overexpression of LEF1 enhanced the proliferation of Eca109 and TE1 cells,while knockdown of LEF1 inhibited the proliferation of Eca109 and TE1 cells.(2)Compared with the control group,the tumor volume of nude mice inoculated with LEF1-overexpression cells was larger(P < 0.05),while those inoculated with LEF1-knockdown cells was smaller(P < 0.01).3.LEF1 enhances the migration,invasion and EMT of ESCC cells:(1)Overexpression of LEF1 significantly decreased the expression of epithelial marker E-cadherin while increased the expression of mesenchymal marker N-cadherin.On the other hand,knockdown of LEF1 significantly increased the expression of epithelial marker E-cadherin while decreased the expression of mesenchymal marker N-cadherin.(2)Overexpression of LEF1 not only significantly promoted the migratory ability of Eca109 and TE1 cells(P < 0.01,P < 0.01),but also promoted their invasive ability(P < 0.01,P < 0.01);While knockdown of LEF1 inhibited the migratory ability of Eca109 and te1 cells(P < 0.01,P < 0.01),and also inhibited their invasive ability(P < 0.01,P < 0.01).4.LEF1 exerts its biological function by directly binding to ID3:(1)According to the results of transcriptome sequencing,LEF1 could activate TGF beta signaling pathway.According to the results of enrichment analysis,q RT-PCR and Jaspar database were used to confirm that ID1 and ID3 are likely to be downstream target genes of LEF1.(2)Luciferase reporter assay showed that LEF1 could enhance the promoter activity of both ID1 and ID3.Chip PCR showed that LEF1-binding site was located at-1631 ～-1616 of the ID1 promoter and-1100 ～-1114 of the ID3 promoter.(3)By detecting the expression of LEF1,ID1 and ID3 in 14 pairs of fresh ESCC tissues,LEF1 was more closely related to ID3 than ID1.The rescue experiment showed that knockdown with ID3 had a greater impact on the proliferation and EMT mediated by LEF1 than knockdown with ID1,suggesting that LEF1 played a carcinogenic role by regulating the expression of ID3.(4)Consecutive section of IHC showed that there was a closely association between LEF1 and ID3.Conclusion: LEF1 is highly expressed in ESCC.Overexpression of LEF1 could promote the growth,migration,invasion and EMT of ESCC cells.Mechanistically,LEF1 regulates the expression of ID3 to produce its biological functions by directly binding to ID3.Part 2 The role and mechanism of ID3 in ESCC Objective: To explore the biological function and downstream molecular mechanism of ID3 in ESCC.Methods: 1.Oncomine and UALCAN database were used to analyze the expression level of ID3 in ESCC tissues and normal tissues,and IHC was used to verify it in tissue specimens.2.The association between the expression level of ID3 and clinical data of ESCC patients,as well as the prognosis was analyzed.3.Stable ID3-knockdown and ID3-overexpression ESCC cell lines were established by lentivirus.4.m RNA level was detected by q RT-PCR and protein level was detected by western blot.5.CCK-8 assay and colony formation assay were employed to examine the cell proliferation ability.Wound-healing assay and transwell assay were employed to detect cell migration and invasion ability.6.Xenograft mice were employed to compare the tumorigenicity in vivo.And lung metastasis model was established by injecting tail vein with tumor cells.7.Transcriptome sequencing was used to explore the downstream target of gene.8.Rescue experiment was used to verify the phenotypic rescue ability.Result:1.The high expression of ID3 in ESCC is correlated with poor clinical characteristics and prognosis:(1)IHC of 92 paraffin embedded specimens showed that high expression of ID3 account for 64.13% in ESCC tissues while only 27.17% in normal esophageal tissues(P<0.01).Compared with normal esophageal tissues,ID3 was highly expressed in ESCC tissues.(2)Analysis of the association between ID3 level and clinical data of ESCC patients showed that patients with high ID3 expression had worse histological differentiation(P=0.011),higher pathological T stage(P<0.01)and TNM grade(P<0.01).(3)The overall survival(OS)of patients with high expression of ID3 was significantly worse than that with low expression of ID3(P<0.001).Cox univariate analysis showed that histological differentiation(P=0.035),pathological N stage(P=0.025)and ID3 expression level(P=0.002)were risk factors affecting OS.Multivariate analysis showed that ID3 expression level was an independent prognostic factor for OS(P=0.007).ID3 could be used as an independent prognostic factor to predict the OS of patients.In addition,LEF1 and ID3 were combined for comparing the OS.Patients with high expression of LEF1 and high expression of ID3 had the worst prognosis,while the patients with low expression of LEF1 and low expression of ID3 had the best prognosis,indicating that combination of LEF1 and ID3 had more accurate outcome for predicting the prognosis of ESCC patients.2.ID3 promotes the proliferation,migration,invasion and EMT of ESCC cells:(1)CCK-8assay showed that overexpression of ID3 could enhance the proliferation activity of Eca109 and TE1 cells compared with the control group(P < 0.01,P < 0.01).Colony formation assay showed that overexpression of ID3 could significantly increase the clone number of Eca109 and TE1 cells(P<0.01,P<0.05).However,the proliferation activity of Eca109 and TE1 cells was significantly decreased by CCK-8 after ID3 knockdown,and the clone number of Eca109 and TE1 cells was significantly reduced by interfering with ID3 in colony formation assay.(2)Wound-healing assay showed that the distances of Eca109 and TE1 cells decreased significantly after overexpression of ID3(P<0.05,P < 0.01),while the cell distances increased after ID3 knockdown.Transwell assay showed that overexpression of ID3 not only significantly promoted the migratory ability of Eca109 and TE1 cells(P<0.01,P<0.05),but also promoted their invasive ability(P<0.01,P<0.05),while knockdown of ID3 significantly inhibited the migratory and invasive ability of Eca109 and TE1 cells.(3)Overexpression of ID3 significantly decreased the epithelial markers E-cadherin while increased the mesenchymal markers N-cadherin,Snail,Slug,Vimentin,MMP2 and MMP9.On the other hand,knockdown of ID3 significantly increased the epithelial markers E-cadherin while decreased the mesenchymal markers N-cadherin,Snail,Slug,Vimentin,MMP2 and MMP9.These results suggest that ID3 could promote EMT in ESCC.(4)Overexpression of ID3 significantly increased the tumor volume of nude mice(P<0.01),while knockdown of ID3 significantly reduced the tumor volume(P<0.01),suggesting that ID3 promoted the growth of ESCC cells in vivo.(5)The number of lung metastases in nude mice injected with ov-ID3 cells were more than those in ov-NC group.The average number of nodules in ov-NC group was 6 ± 0.82 while that in ov-ID3 group was 16.75 ± 1.11,with a significant difference(P<0.01).On the other hand,the number of lung metastases in nude mice injected with shID3 group were lower than those in sh-NC group.The average number of nodules in sh-NC group was 12.25 ± 1.03 while that in sh-ID3 group was 2.25 ± 0.63,with a significant difference(P<0.01).These findings indicated that overexpression of ID3 could promote the lung metastasis of ESCC cells,while knockdown of ID3 could inhibit the lung metastasis of ESCC cells.3.ID3 activates ERK/MAPK signaling pathway,and ERK/MAPK pathway could also induce the expression of ID3 in turn.A local positive feedback loop exists between ID3 and ERK/MAPK pathway:(1)According to the results of RNA-seq and further validation,ID3 could activate ERK/MAPK pathway,but had no effect on JNK/SAPK and p38 MAPK pathways.(2)U0126(an inhibitor of ERK/MAPK pathway)was applied for rescue experiment.After added into ov-ID3 Eca109 and TE1 cells,the proliferation,invasion,migration and EMT of cells were significantly inhibited.This result revealed that the biological function of ID3 was mediated by ERK/MAPK pathway in ESCC cells.(3)TPA(an activator of ERK/MAPK pathway)significantly increased the expression of ID3 in Eca109 and TE1 cells.The expression of ID3 decreased slightly with the increase of the concentration of U0126.These results indicated that ERK/MAPK pathway could regulate the expression of ID3.Activation of ERK/MAPK pathway could significantly increase the expression of ID3,while inhibition of ERK/MAPK pathway could significantly reduce the expression of ID3.4.ID3 activates ERK/MAPK signaling pathway by increasing the expression of HRAS:(1)q RT-PCR and WB showed that HRAS was significantly increased in terms of the m RNA levels and protein levels.(2)After transferring sh-HARS plasmid into ov-ID3 Eca109 and TE1 cells,the proliferation,invasion,migration and EMT ability of cells were significantly inhibited,indicating that ID3 produced its biological function via activating HRAS and ERK/MAPK pathway.Conclusion: ID3 could activate ERK/MAPK signaling pathway through promoting the expression of HRAS,and then produce biological function.The activated ERK/MAPK signaling pathway could promote the expression of ID3 in turn,and amplify the local promotion through positive feedback regulation. |
PDF Full Text Request