The Mechanism Of Gli1 Upregulating YAP1 In Occurrence And Development Of Esophageal Squamous Cell Carcinoma

Esophageal cancer is the sixth most common malignant tumor in the world.Esophageal squamous cell carcinoma and esophageal adenocarcinoma are main histological types.Esophageal squamous cell carcinoma is the majority of patients in China.The familial aggregation of esophageal cancer has been found in the high incidence areas of esophageal cancer in northern China,suggesting that genetic susceptibility may play an important role in the occurrence of esophageal cancer.Gli1(glioma-associated oncogene transcription factor 1),as an important transcription factor in Hedgehog(Hh)signaling pathway,has been found to be highly expressed in human esophageal cancer samples and is associated with invasion,metastasis and poor prognosis,but the specific oncogenic role and mechanism of Gli1 in esophageal squamous cell carcinoma remain unclear.YAP1(yes associated protein1),a transcriptional coactivator in the Hippo signaling pathway,has been proven to be carcinogenic in esophageal squamous cell carcinoma,but the mechanism of activation in esophageal squamous cell carcinoma remains unclear.At present,existing studies have shown that Hh and Hippo signal interact with each other in digestive tract smooth muscle differentiation and liver regeneration,suggesting that Gli1 may be associated with YAP1 to some extent.Endoplasmic reticulum stress(ERS)is a protective stress response.Moderate ERS is a necessary step for cells returning to homeostasis and adapt to stress,thus playing a role in self-protection.However,excessive ERS can lead to apoptosis or necrosis.UPR is regulated by different ERS-related proteins,including GRP78,ATF6,IRE1?,and PERK.Previous studies have shown that activated ERS can promote tumorigenesis and development,but the mechanism of ERS in esophageal squamous cell carcinoma is still unclear.In addition,in hepatocellular carcinoma(HCC)cells,YAP1 can inhibit HCC cell death by regulating the nonfolding protein response by upregulating ATF6.Thus,weconcluded that Gli1 may promote the proliferation,invasion and migration of esophageal squamous cell carcinoma through synergism with YAP1,and participate in the regulation of ERS in esophageal squamous cell carcinoma.Objective:To investigate the role and mechanism of Gli1 and YAP1 in proliferation,invasion,migration and endoplasmic reticulum stress of esophageal squamous cell carcinoma.Methods:1.Real-time PCR assay was used to detect m RNA expression levels of Gli1 and YAP1 in TE1,EC109,EC9706,KYSE-450 cells of human ESCC cell line and NE-1 cells of esophageal epithelial immortalized cell line.Western blot assay was used to detect the protein expression levels of Gli1 and YAP1 in human ESCC cell lines and esophageal epithelial immortalized cell line.Immunohistochemical staining was used to detect the expression of Gli1 and YAP1 in human ESCC tissues.Kaplan-Meier survival analysis was used to study the correlation between the expression of Gli1 and YAP1 and the prognosis of patients with ESCC.Spearman nonparametric correlation analysis was used to investigate the correlation between Gli1 and YAP1 expression in human ESCC tissues.Kaplan-Meier survival analysis was conducted to investigate the correlation between simultaneous high expression of Gli1 and YAP1 in human ESCC and poor prognosis of patients.2.The efficiency of Gli1 or YAP1 inhibition was explored by western blot assays.The effect of Gli1 or YAP1 inhibition on the ESCC cell growth was explored by MTT assays and clonal formation assays.We assessed the the effect of Gli1 or YAP1 inhibition on cell migration and invasion using wound healing and transwell invasion assays.TE1 cells were treated with thapsigargin for 48 h to induce ERS,while the control group wastreated with dimethyl sulfoxide for 48 h.The effect of Gli1 knockdown on the viability of TE1 cells was detected by MTT assay.EC109 cells was treated with thapsigargin for48 h to induce ERS,while the control group was treated with dimethyl sulphoxide for48 h.MTT assays were performed to assess the effect of Gli1 overexpression on EC109 cell growth.Western blot assay was used to detect the effect of Gli1 knockdown on PARP splicing in TE1 and EC109 cells under ERS.3.The spatial correlation of Gli1 and YAP1 in TE1 and EC109 cells of ESCC lines and in human ESCC tissue samples was detected by double-label immunofluorescence assay.The binding of Gli1 and YAP1 in TE1 and EC109 cells was detected by immunoprecipitation assay.Western blot and real-time PCR experiments were used to explore the molecular mechanism of Gli1 and YAP1 mutual regulation.4.MTT and nude mouse tumorigenesis experiments were used to detect the effect of Gli1 regulation of YAP1 on the growth of ESCC cells in vitro and in vivo.5.Real-time PCR assay was used to detect the effect of Gli1 knockdown on XBP1 m RNA expression in TE1 cells under ERS.The effect of Gli1 inhibition on protein level of phosphorylated JNK1 and JNK1(a IRE1? signaling downstream target)was assessed by western blot assays.The effect of Gli1 knockdown on the expression of PERK signaling pathway target gene CHOP m RNA in TE1 cells under ERS was assessed by real-time PCR assays.Western blot and Real-time PCR were used to detect the effect of Gli1 knockdown on protein and m RNA expression of ATF6 in TE1 cells under ERS.Real-time PCR assay was used to detect the effect of Gli1 knockdown on the m RNA expression of Grp94,Ero1L? and Orp150 as ATF6 signaling pathway target genes in TE1 cells under ERS.Spearman nonparametric correlation analysis was used to investigate the correlation between Gli1 and ATF6 expression in human ESCC tissues.MTT,wound healing and transwell invasion assays were performed to assess the effect of ATF6 overexpression on the viability,migration and invasion in TE1 cells under ERS.The effect of ATF6 on the occurrence and development of Gli1-mediated ESCC was determined by rescue experiment combined with cell scratch,transwell invasion assay and lung metastasis model in nude mice.Whether YAP1 mediated the upregulation of ATF6 by Gli1 under ERS was explored by rescue experiments,western blot and real-time PCR assays.Results:1.Expression and correlation of Gli1 and YAP1 in ESCC1.1 Expression of Gli1 and YAP1 m RNA in ESCC linesThe expression of Gli1 and YAP1 m RNA in TE1,EC109,EC9706 and KYSE-450 cells of ESCC lines were significantly higher than those in NE-1 cells of esophageal epithelial immortalized cell line.1.2 Expression of Gli1 and YAP1 proteins in ESCC linesThe expression of Gli1 and YAP1 proteins in TE1,EC109,EC9706 and KYSE-450 cells of ESCC cell lines were higher than those in NE-1 cells of esophageal epithelial immortalized cell line.1.3 The expression of Gli1 and YAP1 in human ESCC tissues and the correlation with poor prognosis of patientsBoth Gli1 and YAP1 were highly expressed in human ESCC tissues,and the overall survival rate of patients with high Gli1 or YAP1 level was lower than that of patients with low Gli1 or YAP1 level.1.4 Correlation between the expression of Gli1 and YAP1 in human ESCC tissuesThe expression levels of Gli1 and YAP1 in human ESCC tissues were positively correlated.1.5 The correlation between the simultaneous high expression of Gli1 and YAP1 inhuman ESCC tissues and the poor prognosis of patientsPatients with simultaneous high expression of Gli1 and YAP1 had lower overall survival rate than those with simultaneous low expression of Gli1 and YAP1.2.Roles of Gli1 and YAP1 in proliferation,invasion,migration and ERS of ESCC in vitro2.1 Effects of Gli1 on proliferation,invasion and migration of ESCC in vitro Gli1 knockdown suppressed the survival,the number of clone formation,the2.1 Effects of Gli1 on proliferation,invasion and migration of ESCC in vitro Gli1 knockdown suppressed the survival,the number of clone formation,the healing range of cell scratches,and the number of invaded cells.2.2 The effect of YAP1 on proliferation,invasion and migration of ESCC in vitro YAP1 knockdown suppressed the survival,the number of clone formation,the healing range of cell scratches,and the number of invaded cells.2.3 The role of Gli1 in ESCC cells against ERS Under ERS,Gli1 knockdown decreased the survival rate of TE1 cells,and Gli1 overexpression increased the survival rate of EC109 cells.Gli1 knockdown up-regulated the splicing level of poly ADP ribose polymerase in TE1 and EC109 cells.3.Molecular mechanism of mutual regulation between Gli1 and YAP13.1 Spatial correlation of Gli1 and YAP1 in ESCCGli1 and YAP1 were co-localized in TE1 and EC109 cells and in human ESCC tissue samples.3.2 The binding of Gli1 and YAP1 in ESCCGli1 interacts with YAP1 in TE1 and EC109 cells.3.3 Molecular mechanism of mutual regulation between Gli1 and YAP13.3.1 Molecular mechanism of Gli1 up-regulation of YAP1 expressionIn TE1 and EC109 cells,Gli1 knockdown inhibited the expression of YAP1 protein.Overexpression of Gli1 can up-regulate the expression of YAP1 protein,andinhibit the expression of YAP1 phosphorylated at serine 127 and LATS1 phosphorylated at threonine 1079,but the total protein expression of LATS1 did not change significantly.Overexpression of Gli1 did not induce changes in YAP m RNA expression.3.3.2 Molecular mechanism of YAP1 up-regulation of Gli1 expressionIn TE1 and EC109 cells,YAP1 knockdown significantly inhibited Gli1 protein expression.The overexpression of YAP1 up-regulated Gli1 protein expression in a dose-dependent manner.Knocking down YAP1 did not affect Gli1 m RNA expression.In TE1 and EC109 cells,PI3K/Akt signaling inhibitor LY294002 can down-regulate Gli1 protein expression,while overexpression of YAP1 and inhibition of PI3K/Akt signaling can save the up-regulation of Gli1 protein expression caused by overexpression of YAP1.4.Gli1 regulates the effect of YAP1 on growth of ESCC cellsOverexpression of YAP1 or Gli1 can save the reduced proliferation ability of TE1 and EC109 cells induced by Gli1 or YAP1 knockdown.Overexpression of YAP1 or Gli1 saved the tumorigenesis reduction of TE1 cells induced by Gli1 or YAP1 knockdown in nude mice.5.Gli1 regulation YAP1 is involved in the molecular mechanism of ESCC cells' resistance to ERS5.1 Regulation of Gli1 on ERS-related signaling pathways5.1.1 Regulation of Gli1 on IRE1?-related signaling pathwayUnder ERS condition,Gli1 knockdown induced XBP1 m RNA clipped expression level in TE1 cells.Gli1 knockdown induced the expression of phosphorylated proteins of JNK1 as the downstream target molecule of the IRE1? signaling pathway,but the total JNK1 protein expression level did not change significantly.5.1.2 Regulation of Gli1 on PERK signalUnder ERS condition,Gli1 knockdown had no significant effect on the expression of CHOP m RNA as PERK signaling pathway target gene in TE1 cells.5.1.3 Regulation of Gli1 on ATF6 signalUnder ERS condition,Gli1 knockdown inhibited the expression levels of ATF6 protein and m RNA in TE1 cells.Gli1 knockdown inhibited the expression of Grp94 and Ero1L? m RNA as ATF6 downstream target genes in TE1 cells,but had no significant effect on Orp150 m RNA expression.The expression levels of Gli1 and ATF6 in human ESCC tissues were positively correlated.5.2 Gli1 regulation of ATF6 is involved in the occurrence and development of ESCC5.2.1 The effect of ATF6 on the occurrence and development of ESCC Under ERS condition,ATF6 knockdown inhibited proliferation of ESCC TE1 cells.ATF6 knockdown inhibits migration and invasion of ESCC TE1 cells in vitro.5.2.2 The role of ATF6 in the occurrence and development of Gli1-mediated ESCCATF6 knockdown inhibited the migration and invasion in vitro and tumor metastasis in vivo of Gli1-mediated ERCC TE1 cells.5.3 The mechanism of YAP1 mediates the up-regulation of ATF6 by Gli1 under ERSUnder normal condition and ERS condition,overexpression of Gli1 can up-regulate the expression of YAP1 protein in EC109 cells.Under ERS condition,YAP1 knockdown saved the upregulation of ATF6 protein caused by Gli1 overexpression in EC109 cells.Under ERS condition,YAP1 knockdown saved the up-regulation of Grp94 and Ero1L? m RNA expression as ATF6 downstream target genes induced by overexpression of Gli1 in EC109 cells.Conclusions:1.Both Gli1 and YAP1 are highly expressed in ESCC,and the high expression of both is positively correlated with poor prognosis of patients.The expression levels of Gli1 and YAP1 in human ESCC tissues were positively correlated.Patients of ESCC withsimultaneous high expression of Gli1 and YAP1 have a worse prognosis than patients with simultaneous low expression of Gli1 and YAP1.2.Both Gli1 and YAP1 promote the proliferation,migration and invasion of ESCC in vitro,and Gli1 can help ESCC cells to resist ERS-induced cell death.3.Gli1 and YAP1 interact with each other in ESCC,and they form positive feedback regulation.In mechanism,Gli1 upregulates the expression of YAP1 by regulating LATS1,and YAP1 upregulates the expression level of Gli1 by activating PI3K/ Akt signal.4.Gli1 regulates YAP1 to promote the growth of ESCC cells in vitro and in vivo.5.Gli1 induces ATF6 expression by upregulating YAP1,thereby promoting ESCC cells to resist ERS.