Preliminary Study Of The Effect Of ART1 On The Proliferation Of Esophageal Squamous Cell Carcinoma And Its Mechanism

Background and purposePrevious studies have shown that esophageal cancer has the characteristics of high incidence,high mortality and poor prognosis.Previous research by our group showed that arginine-specific mono-ADP-ribosyltransferase(ART1)has been confirmed to be associated with the development of tumors such as colon cancer and liver cancer,and arginine-specific mono-ADP ribosylation is the basis of poly-ADP ribosylation.In colon cancer,ART1 can regulate the proliferation of tumor cells by regulating RhoA and there is data showing that the expression of RhoA in esophageal cancer is higher than that in adjacent normal tissues.However,the relationship between ART1 and ESCC and its relationship with RhoA has not been reported in the literature.Therefore,this study was to investigate the effect of ART1 on the proliferation of ESCC,and at the same time,it also carried out a preliminary exploration of its mechanism.This study not only provides a basis for further exploration of the role of arginine-specific mono-ADP ribosylation in ESCC but also provides new ideas for the new direction of seeking therapeutic target for esophageal cancer.Methods1.Sixty cases of paraffin specimen of human ESCC(including well,medium and low differentiated ESCC and control group)were selected and the expression of ART1 was detected by immunohistochemistry.In addition,in the ECA109 and TE13 cell lines,the expression of ART1 was detected by immunofluorescence.2.CCK-8,EdU,and flow cytometry were used to detect the effect of ART1 on its proliferation after treatment with its specific inhibitor MIBG in ECA109 cells and TE13 cells.3.After the expression of ART1 was inhibited,the expression levels of mRNA of ART1 and RhoA were detected by Q-PCR.And the changes in protein expression of ART1,RhoA/ROCK1 signaling pathway and its downstream proliferation-related factors C-Myc,CyclinA and CyclinD1 were detected by Western Blot.Results1.The expression of ART1 in human ESCC and human ESCC cell lines.(1)The results of immunohistochemistry showed that ART1 was expressed in human ESCC tissue and control group,which located in the cytoplasm and cell membrane.Statistical analysis showed that the expression of ART1 in control group was significantly lower than that in cancer tissues(P<0.05).There was a difference in the expression of ART1 in different differentiated cancer tissues(P<0.05),but there was no difference in expression of different age,gender,lymph node metastasis and depth of invasion(P>0.05).(2)Immunofluorescence results showed that ART1 was expressed in both ECA109 cell line and TE13 cell line,and located in cell membrane and cytoplasm.2.Effect of ART1 on the proliferation in ECA109 cells and TE13 cells.(1)After ECA109 cells and TE13 cells were treated with the ART1 specific inhibitor MIBG respectively,the proliferation activity of cells detected by CCK-8 assay showed that the proliferation activity of ECA109 cells and TE13 cells decreased gradually with the increase of MIBG concentration,showing a concentration-dependent(P<0.05).(2)After ECA109 cells and TE13 cells were treated with the ART1 specific inhibitor MIBG respectively,the results of cell proliferation detected by EdU showed that the proliferation of ECA109 cells and TE13 cells in the MIBG-treated group was significantly lower than that of the control group(untreated group)(P<0.05).(3)After MIBG treatment of ECA109 cells and TE13 cells respectively,flow cytometry results show that ECA109 cells were arrested in S phase(P<0.05),while TE13 cells were arrested in G1 phase(P<0.05).3.Effect of ART1 on RhoA/ROCK1 signaling pathway-associated proteins in ESCC cells.(1)After ECA109 cells and TE13 cells were treated with the ART1 specific inhibitor MIBG respectively,the results of Q-PCR showed that the mRNA expression of ART1 and RhoA in the MIBG-treated group was significantly lower than that in the control group(untreated group)(P<0.05).(2)After ECA109 cells and TE13 cells were treated with the ART1 specific inhibitor MIBG respectively,western blot results indicated that the protein expression of ART1,RhoA/ROCK1,C-Myc,CyclinA and CyclinD1 in MIBG-treatment group was also lower than that in the control group(untreated group)(P<0.05).ConclusionsThe expression of ART1 in ESCC tumor tissues were significantly higher than that in control group,and its expression may be related to the degree of differentiation.It is suggested that arginine mono-ADP ribosylation may play an important role in the proliferation of ESCC,which may be achieved by regulation of RhoA/ROCK1 pathway and its downstream molecules,but the specific mechanism needs further study.