The Mechanism Of Aberrant Expression Of AChE In Tn Antigen Positive Cells Origin From Human Colon Cancer Cell Line HT-29

Objective:To explore the expression of three different isoforms(AChE-T?ACNE-H?AChE-R)of AChE on HT-29-Tn+ cells separated by Tn antigen magnetic beads.The relationship between AChE and proliferation or apoptosis of HT-29 colon cancer cells and the mechanism of AChE differential expression were explored.To investigate the relationship between the expression of Tn antigen and AChE of HT-29 cells stimulated by TNF-a in vitro.Methods:The human colon cancer HT-29-Tn+ cell line purchased from ATCC was used to obtain the purified HT-29-Tn-cells and HT-29-Tn+ cells by immunomagnetic bead sorting.Flow cytometry was used to detect the percentage of Tn antigen expression on HT-29-Tn-cells and HT-29-Tn+ cells,respectively.HT-29 colon cancer cells were cultured in cell culture plates,adding different concentrations(0 ng/ml 10 ng/ml,20 ng/ml,30 ng/ml)of TNF-a,and incubated in 37 C cell incubator for 48h.The mean fluorescence intensity of Tn antigen on HT-29 cells was analyzed statistically.The AChE-T/AChE-H/AChE-R gene was amplified by qPCR and RT-PCR.Western-blot was used to detect the expression of AChE protein in HT-29-Tn-cells and HT-29-Tn+cells.ELISA assay was used to detect the secretion of AChE in the culture lysate and culture supernatant.CCK-8 and Annexin V-FITC/PI were used to detect the proliferation and apoptosis of HT-29-Tn-and HT-29-Tn+ cells treated by TNF-a,AChE and its inhibitors(neostigmine and Huperzine A).BSP method was used to detect the methylation status of AChE promoter in HT-29-Tn-cells and HT-29-Tn+ cells respectively.Results:1.The expression of AChE in HT-29-Tn+ cells decreasced,and the levels of AChE decreased to some extent in protein level.AChE-T,AChE-H and AChE-R both decreased in transcription level.There was hypermethylation of AChE-encoding gene promoter region in HT-29-Tn+ cells.Exogenous AChE can inhibit HT-29 cell proliferation and induced apoptosis.But AChE inhibitors(neostigmine,huperzine A)effect is opposite.2.Tn+ cells increase significantly during co-culture of TNF-a and HT-29 cells.The percentage of Tn+ cells increased with the concentration of TNF-a in a certain concentration range of TNF-a.TNF-a(range form Ong/ml to 30ng/ml)induces apoptosis in HT-29-Tn-cells in a dose-dependent manner,and the ability to induce apoptosis of HT-29-Tn+ cells is not obvious.When the concentration of TNF-a reached 50ng/ml,the apoptosis of HT-29-Tn-cells and HT-29-Tn+ cells increased rapidly,and the effect of inducing apoptosis of HT-29-Tn-cells was more obvious compared to HT-29-Tn+ cells.3.With the increase of TNF-a concentration,the transcriptional levels of AChE-T,AChE-H,AChE-R decreased and the expression of AChE decreased in HT-29 cells.Conclusion:1.High methylation of AChE promoter result in the low expression of AChE protein in HT-29-Tn+ cells cells.2.Exogenous AChE can induce apoptosis of HT-29-Tn+ and Tn-cells.3.The sensitivity of HT-29-Tn+ and Tn-cells to TNF-a is different;TNF-a is more prone to induce apoptosis of HT-29-Tn-cells.