| Objective:By revealing the relationship between the expression level of ACSS2 and the prognosis and survival of patients who had been diagnosed as ESCC,we further analyzed the effect of ACSS2 on EMT of ESCC cells under nutrient stress,and preliminarily explored the key roles that ACSS2 played in histone H3acetylation/TGF-βpathway which was involved in the process of tumor metastasis.Methods:1.Immunohistochemistry was used to detect the expression and distribution characteristics of ACSS2 in ESCC and adjacent tissues;The correlation between the expression and distribution of ACSS2 and the number of vascular endothelial cells(CD31 labeled),MVD was analyzed by immunofluorescence and immunohistochemi-stry;the expression of ACSS2 protein in ESCC cell lines KYSE-150 and ECA-109under nutrient stress and/or hypoxia conditions was detected by Western blotting;Image J Pro Plus software was used to evaluate the expression of ACSS2,and then analyse the relationship between ACSS2 and disease-free survival(DFS)and overall survival(OS)of patients with ESCC.2.The effects of nutrient stress on invasion and migration of ESCC cell lines were detected by Transwell,combined with si RNA treatment,the key role that ACSS2plays was identified;the effect of up-regulating and down-regulating ACSS2 on EMT related molecular markers on m RNA level in ESCC cells was detected by real time q PCR(RT-PCR);the effect of ACSS2 inhibitor on EMT related molecular markers and TGF-βexpression in KYSE-150 and ECA-109 was detected by Western blotting;under normal and nutrient stress conditions,the changes of VEGF-A,VEGF-C,MMP-2 and MMP-9 in ECA-109 cells before and after the knockdown of ACSS2gene were detected by ELISA.3.Combined to target ACSS3,RT-PCR was used to detect the expression differences of various cytokines that involved in EMT under normal and nutrient stress conditions;Western blotting was used to detect the changes of EMT related molecular markers,SNAI1 and Slug after downregulating TGF-βexpression in KYSE-150,which had been overexpressed ACSS2;ELISA was used to detect the changes of TGF-βsecreted by KYSE-150 and ECA-109 before and after the addition of 0.6μM ACSS2 inhibitor for 48 h under the nutrient stress;The migration ability of ECA-109 was detected by Transwell after adding 0.6 mg/m L TGF-βneutralizing antibody or 10μM TGF-βreceptor inhibitor SB-431542 both for 48 h under the nutrient stress;the expression of ACSS2 and TGF-βin ESCC and adjacent tissues was detected by RT-PCR,and the correlation between ACSS2 and TGF-βwas then analyzed.4.Combined to si RNA-ACSS2,Western blotting was used to detect the effect of nutrient stress and ACSS2 on histone acetylation in ESCC cells;under normal and nutrient stress conditions,RT-PCR was used to detect the effect of 5 n M A-485 for 48h on the expression of TGF-βat m RNA level in ESCC cells;Western blotting and ELISA were used to detect the effect of A-485 on TGF-βin at protein level and exocrine level KYSE-150 that had overexpressed ACSS2;the migration ability of ECA-109 was verified by Transwell before and after adding A-485 under normal and nutrient stress conditions;the relationship between the expression level of ACSS2and/or TGF-βand overall survival was analyzed by IHC and Image J Pro Plus software.Results:1.Compared to adjacent tissues,the expression of ACSS2 in ESCC was significantly increased and ACSS2 mainly expressed in the cytoplasm of tumor cells,and ACSS2 was strongly positive or high positive in the area where a large number of cells concentrated;the expression level of ACSS2 in ESCC was negatively correlated with MVD(R~2=-0.863,P<0.0001);the expression of ACSS2 was significantly up-regulated under the condition of nutrient stress and hypoxia,and the effect of nutrient stress on ACSS2 expression was greater than that of hypoxia under the condition of single factor;there was a correlation between expression of ACSS2 and the DFS and OS in ESCC tissues.2.Nutrient stress promoted the migration of tumor cells in a time-dependent manner,and the migration ability of tumor cells was significantly enhanced after 48-h nutrient stress conditions(P<0.0010),the invasion ability was significantly inhibited when ACSS2 targeted(P<0.001);overexpressing ACSS2 in KYSE-150 could significantly down-regulated E-cadherin(E-cad)and significantly up-regulated N-cadherin(N-cad),SNAI1,SLUG and ZEB-1 and so on,while after interference with ACSS2 in ECA-109,the expression of these proteins showed opposite changes;nutrient stress could up-regulate the expression of Vimentin,down-regulate the expression of E-cad at the same time,the above phenomenon could be effectively reversed by the addition of ACSS2 inhibitor;after cultured for 72 h under nutrient stress,VEGF-A(P<0.001),VEGF-C(P<0.001)and MMP-9(P=0.027)secreted by ECA-109 were significantly increased,but knockdown of ACSS2 expression could effectively block the changes of VEGF-A(P=0.209),VEGF-C P=0.086)and MMP-9(P=0.830).3.Under normal and nutrient stress conditions,compared the results of before and after interference ACSS2 treatment,the expression of IL-1β,IL-6,TGF-βwere showed to be regulated by ACSS2,and the expression peak of TGF-βwas the highest accrodding to CT value;the expressions of SNAI1,SLUG and Vimentin were increased in KYSE-150 overexpressing ACSS2,while the expression of E-cadherin was decreased,however,knockout of TGF-βby si RNA could effectively reverse the above phenomenon;the exocrine level of TGF-βdecreased significantly in KYSE-150 and ECA-109 with the addition of 5.5μM ACSS2 inhibitor for 48 h(P<0.001);when ECA-109 was added with 0.6 mg/ml TGF-βneutralizing antibody or10μM SB-431542(a TGF-βreceptor inhibitor)both for 48 h under nutrient stress,its invasion ability was significantly weakened;in 15 pairs of ESCC tissues,the expression of ACSS2 was positively correlated with TGF-β(R~2=0.4293,P=0.008),however,there was no significant correlation between them in adjacent tissues(P=0.1421).4.The acetylation expression of histone H3 was enhanced under nutrient stress,but the above phenomena could be reversed after si RNA-ACSS2 treatment;A-485inhibited TGF-βexpression on m RNA level in ECA-109 treated with nutrient stress,it could also reverse the phemonemon in KYSE-150 overexpressed ACSS2 where TGF-βincreased on protein level and exocrine level(P<0.001),of the invasion ability of ECA-109 was significantly decreased by adding A-485 under normal and nutrient stress;the overall survival of ESCC patients with high expression of ACSS2 and TGF-βwas significantly lower than that of patients with high expression of ACSS2and low expression of TGF-βor low expression of ACSS2 and TGF-βwas verified by retrospective analysis(P<0.0001).Conclusions:High expression of ACSS2 in ESCC tissues was closely related to the prognosis of patients.The up-regulation of ACSS2 significantly enhanced the invasion and migration of ESCC under nutrient stress;ACSS2 participates in the acetylation of histone H3 under nutrient stress and may be involved in EMT,invasion and metastasis through regulating TGF-β expression. |
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