Ubiquitination and expression of metastasis associated 1 in gastric cancer MKN-45 cells

Abstract

Objective To observe the ubiquitination of metastasis associated 1 (MTA1) in gastric cancer cells and its effect on the expression of MTA1. Methods The non-transfected MKN-45 cells [Myc-MTA1 (-), HA-Ub (-)] served as the control group, and three experimental groups of MKN-45 cells were transfected with the myc tag of MTA1 plasmid [Myc-MTA1 (+ ), HA-Ub (-)], HA tagged ubiquitin plasmid [Myc-MTA1 (-), HA-U (+ )], or co-transfected with the two plasmids [Myc-MTA1 (+ ), HA-Ub (+ )]. 2 μg plasmid was transfected into each culture dish, and then the transfected cells were harvested after 24 h culture. MKN-45 cells in the experimental group were treated with proteasome inhibitor MG-132. The expression level of MTA1 was detected by immunoprecipitation (IP) and Western blotting. MTA1 expressing plasmid containing the Myc tag and HAtagged ubiquitin plasmid (HA-Ub) was co-transfected into cells, and the expression of Myc-MTA1 was detected. Flow cytometry was used to detect the apoptosis of MKN-45 cells induced by MG-132. Results (1) With the proteasome inhibitor MG-132 treatment of gastric cancer cell line MKN-45, MTA1 expression levels increased significantly in experimental group, there were (0.78±0.15) μg/ml and (1.37±0.33) μg/ml after 2 h and 6 h, compared with the control group [(0.28±0.09) μg/ml], the difference was statistically significant (t=2.134, 5.562, P<0.05). (2) After 6 h, the degree of ubiquitination in experimental group was (77.28±9.38)%, and the control group was (15.41±2.14)%, the difference was statistically significant (P<0.05). (3) In gastric cancer MKN-45 cells [Myc-MTA1 (+ ), HA-Ub (+ ) group, Myc-MTA1 can be strongly ubiquitin, the degree of ubiquitin was (64.59±7.61)%, compared with the other two groups, the difference was statistically significant (P<0.05). (4) MG-132 treatment of the experimental group and the control group of MKN-45 gastric cancer cells after 24 h of gastric cancer MKN-45 cells transfected with [Myc-MTA1(+ )、HA-Ub(+ )], the apoptosis ratio was (22.90±4.17)%, significantly higher than control group MKN-45 cell apoptosis ratio (8.25±1.04)%. C-poly adenosine diphosphate-ribose polymerase (c-PARP) levels showed that [Myc-MTA1 (+ ), HA-Ub (+ )] the degree of apoptosis was (29.77±5.04)%, significantly higher than the control group MKN-45 cells (13.10±3.28)% (t=1.845, 2.374, P<0.05). Conclusion MTA1 in human gastric cancer cells can be regulated through ubiquitination pathway. Its ubiquitination pathway plays an important role in the development and metastasis of gastric cancer. Key words: Gastric cancer; Metastasis associated 1; Ubiquitination