Gli Promote Epithelial-mesenchymal Transition In Esophageal Adenocarcinoma By PI3K/AKT Pathway

Objective: One of the most common malignant tumors of digestive tract in China is esophageal cancer whose morbidity and mortality rank the fifth and fourth place of malignant tumors respectively in China.Esophageal squamous carcinoma is the main pathological type of esophageal cancer in our country.Because of lack of obvious sympotoms,the patients are usually diagnosed at middle and late stage.Despite that we adopted integrated therapy including surgery and chemoradiation,the treatment effects were not improved obviously.In recent years for the molecular mechanism of tumor disease to take specific targeted therapy has become a hot research,Hedgehog(Hh)signaling pathway regulate differentiation and growth of various types of cells in embryo and is closely related to the maintenance of homeostasis of mature organs and development and transferation of mutiple malignant tumors of human.As the downstream regulation centre of Hh signal pathway,abnormal activation of Gli can activate the transcription of numerous tumorassociated genes downstream.Recently some studies indicate Gli in many human malignant tumors such as basal cell carcinoma,breast cancer and prostatic cancer can be abnormally activated and closely associated with generation,development,invasion and metastasis.Phosphatidvlinositol-3-kinases(PI3K)is involved in cell proliferation,apoptosis,invasion and metastasis through activating the downstream target protein AKT Epithelialmesenchymal transition(EMT)Epithelial-mesenchymal transition(EMT)refers to the epithelial cell in a specific physiological or pathological conditions to the mesenchymal cell phenotype transition process.It is an important mechanism of tumor invasion and metastasis.Studies have found that Hh pathway can affect the PI3 K pathway and thus affect the EMT process to promote tumor invasion and metastasis in the gastric cancer,but the current domestic and international researches on the relationship between Gli and human esophageal squamous carcinoma and its specific regulatory pathways of Gli are rare.This study aims to investigate the molecular mechanism through which Gli regulate esophageal squamous carcinoma KYSE-30 cells to epithelial-mesenchymal transition(EMT)and then find molecular targeted drugs which can effectively treat esophageal squamous cell carcinoma.Methods: The mRNA expression of Gli1,Gli2,Vimentin and N-cadherin in KYSE-30 cells were measured by real-time fluorescent quantitative PCR after GANT61 treatment for 24h;The expression of Gli1,Gli2,p-AKT,AKT,Vimentin and N-cadherin protein in KYSE-30 cells was observed by Western blot after 24 h treatment;Effects of GANT61 on migration and invasion of esophageal squamous cell carcinoma were observed by Transwell invasion experiment;At the same time,after N-Shh was used to stimulate the Hedgehog pathway of KYSE-30 cells for 24 hours,the corresponding mRNA and protein expression of Gli1,Gli2,Vimentin and N-cadherin were were measured,and the invasion ability was assessed by the Transwell invasion experiment.Correspondingly we observed the mRNA and protein expression of Gli1,Gli2,Vimentin and N-cadherin and the invasion ability via RT-PCR,Western blot and invasion experiment respectively after GANT61&N-Shh was used for 24 h.Results:1 The mRNA expression of Gli1,Gli2,Vimentin and N-cadherin in esophageal squamous cell carcinoma KYSE-30 cells were significantly decreased in Gli inhibitor GANT61 group compared with DMSO group(all P<0.01);Western blot showed that GANT61 could downregulate the expression of Gli1,Gli2,p-AKT,Vimentin and N-cadherin in KYSE-30 cells(all P<0.01),but the expression of AKT protein was not statistically significant(P> 0.05).Invasion assay showed that GANT61 could significantly depress the invasion ability of KYSE-30 cells(P< 0.01).2 The mRNA level of Gli1,Gli2,Vimentin and N-cadherin in esophageal squamous cell carcinoma KYSE-30 cells were significantly upregulated by Gli agonist N-Shh administration(all P< 0.01);Western blot showed that N-Shh could upregulate the expression of Gli1,Gli2,p-AKT,Vimentin and N-cadherinin the KYSE-30 cells(all P<0.01),but the expression of AKT protein was not statistically significant(P>0.05),That inhibited the invasion ability of tumor cells in the invasion experiment(P< 0.01).3 GANT61&N-Shh group can upregulate the mRNA expression of Gli1,Gli2,Vimentin and N-cadherin in KYSE-30 cells(all P<0.01)compared with GANT61 group;Western blot displayed GANT61&N-Shh can upregulate the expression of Gli1,Gli2,p-AKT,Vimentin and N-cadherin proteins(all P<0.01),but the expression of AKT protein was not statistically significant(P>0.05).Transwell invasion assay showed that GANT61&N-Shh group promoted the invasion of KYSE-30 cells compared with GANT61(P< 0.01).Conclusions: The decreased expression of Gli1 and Gli2 inhibited the epithelial mesenchymal transition process and the invasion and metastasis of esophageal squamous cell carcinoma and the expression of p-AKT protein.However,after Gli1 and Gli2 were increased by N-Shh,the epithelial mesenchymal transition process was obviously promoted and p-AKT was highly expressed,and the cell invasion ability was also increased.When GANT61 and N-Shh were administered at the same time,the epithelial mesenchymal transition and cell invasion ability was partially restored.Therefore,the Gli expression in esophageal squamous cell carcinoma is closely related to the epithelial mesenchymal transformation process,and Gli may promote epithelial mesenchymal transformation through PI3K/AKT pathway.Gli is expected to be an effective target for inhibiting esophageal cancer metastasis.