Study On The Inhibitory Effect And Mechanism Of Ubenimex On Gastric Cancer Metastasis Via CD13/NAB1/MAPK Signaling Pathway

Objective: To observe the effect of Ubenimex on the migration and invasion of human gastric cancer(GC)cells and to explore the underlining mechanism by which Ubenimex inhibited the metastasis of GC.Methods: Human gastric cancer cells MGC-803,SGC-7901,BGC-823 and MKN-28 were treated with Ubenimex at different concentrations(0,0.12mg/m L,0.25mg/m L),and the migration ability of indicated cells was determined using wound healing assay.The effects of Ubenimex on the migration and invasion of MGC-803,SGC-7901,BGC-823 and MKN-28 cells were confirmed using transwell assay.Differentially expressed genes in MKN-28 cells after Ubenimex treatment was analyzed using m RNA microarray analysis.Putative functional genes forcing GC metastasis was screened using high-content screening(HCS),in which GC migration was suppressed obviously after NAB1 gene was silenced.Western blot analysis was used to detect the expression of CD13 and NAB1 in MGC-803,SGC-7901,BGC-823 and MKN-28 cells to identify the relationship between CD13 and NAB1 regulating shaft.In particular,MGC-803,SGC-7901,BGC-823 and MKN-28 cells were transfected with CD13 and NAB1 overexpression plasmid respectively via liposome transfection,and the mutual influence between each other in their expression was verified using Western blot assay.The protein expressions of p90 RSK,ERK1/2,RPS6 and Rab11,and EMT-markers in Ubenimex-treated GC cells were also detected using Western blot assay.Results: Ubenimex could inhibit the migration ability of MGC-803,SGC-7901,BGC-823 and MKN-28 cells.The data obtained from transwell assay showed that Ubenimex(0.12mg/m L or 0.25mg/m L)could remarkablely inhibit the migration ability of MGC-803,SGC-7901,BGC-823 and MKN-28 cells.The results acquired from Western blot assay indicated that Ubenimex could significantly upregulate the E-cadherin expression,but downregulate the protein level of N-cadherin and Vimentin.25 candidates that may be involved in the GC metastasis were screened,among which,NAB1 silence demonstrate most obvious effect in reducing migration ability GC cells.Western blot analysis showed that the protein expressions of CD13 and NAB1 in MGC-803,SGC-7901,BGC-823 and MKN-28 cells treated with Ubenimex were decreased(*P < 0.05),the expression of NAB1 protein was up-regulated(*P < 0.05)after CD13 was over expressed,while the expression level of CD13 protein was almost unaffected(*P < 0.05)after NAB1 was overexpressed,which indicated that NAB1 was the downstream molecule of CD13.Western blot analysis also showed that the protein expression level of p90 RSK,ERK1/2,RPS6 and Rab11 in GC cells treated with Ubenimex and transfected with NAB1 overexpression plasmid was significantly higher than that in the control group.Conclusion: Ubenimex attenuated the activity of NAB1 and regulate the activity of MAPK signaling pathway by targeting CD13,by which Ubenimex inhibited GC metastasis.