The Role Of E3 Ubiquitin Ligase CHIP In Gastric Cancer And The Discussing Of Its Mechanism

Objective This study is to clarify the role of E3 ubiquitin ligase CHIP in the AGS human gastric cancer cells and explore the internal mechanism of those effects.Methods The expression of CHIP in gastric cancer tissues was detected by q RT-PCR,Western blot and IHC assays. Establishing a stable CHIP-overexpressing cell line infected by lentivirus supernatants in the AGS human gastric cancer cells. The changes of cell growth, migration ability and invasion ability after CHIP overexpressing in the AGS cells were detected by Real-time x CELLigence system. The cell cycle analysis and cellular DNA content measurement were examined by flow cytometry. The differences of proliferation and apoptosis between AGS-h CHIP cells and the AGS-control cells were detected by Ki-67 and TUNEL assays. The migration ability of the two established cells was also observed by scratch healing assay. The activities of MMP-2 and MMP-9 in AGS cells after CHIP overexpressing were dectected by gelatinase zymography. The apoptotic associated genes’ m RNA and protein expression of the AGS-control and AGS-h CHIP cells were dectected by q RT-PCR and Western blot analysis. The expression of proteins of AKT and TRAF-NF-κB signaling in the two established cell lines were detected by Western blot.Results CHIP expression in human gastric caner tissues was clearly lower than that of paired normal gastric mucosa at both m RNA and protein levels. The expression level of CHIP in gastric cancer tissue was negatively correlated with the differentiation status and the TNM stages of the gastric cancer patients. The plasmids of MSCV-GFP-h CHIP and MSCV-GFP were stablely transfected into the AGS human gastric cancer cells respectively,and we successfully got the experimental group CHIP overexpressing AGS-h CHIP cells and the control group AGS-control cells. The cell growth of AGS-h CHIP cells was slower than AGS-control cells. The abilities of migration and invasion of AGS-h CHIP cells were markedly decreased than that of AGS-control cells. And the scratch healing assay also showed that the migration ability of AGS-h CHIP cells was weaker than that of AGS-control cells. The cell cycle analysis showed that there were no obvious differencesbetween AGS-control and AGS-h CHIP cells in any of the three phases. The proliferation ability of AGS-h CHIP cells was decreased than that of AGS-control cells. The apoptosis of AGS-h CHIP cells was increased obviously than AGS-control cells. The expression of ITGB1 gene at both the m RNA and protein level was significantly decreased in the AGS-h CHIP cells than AGS-control cells. The expressions of MMP-2 and MMP-9 at the protein level were significantly lower in AGS-h CHIP cells than that of AGS-control cells,and the activities of MMP-2 and MMP-9 were also significantly lower in AGS-h CHIP cells than AGS-control cells by using gelatinase zymography. The expression of the Bcl-2 gene at the m RNA level was markedly decreased in the AGS-h CHIP cells compared to that of the AGS-control cells, but no significant changes were observed in other apoptosis-associated genes including Bax, Bim, Mcl-1, Bcl-xl, c IAP1 and A20 at m RNA level. CHIP overexpression led to a clear reduction in the expression levels of p-AKT(Ser473) and p-AKT(Thr308). The expression of Rel A, p50, Rel B and TRAF-2 were clearly decreased in the AGS-h CHIP cells compared to that in the AGS-control cells.Conclusion CHIP plays an important role of cancer suppressor gene in human gastric caner, and it can be used as a reference index of cancer invasion, metastasis and prognosis in patients. The cell growth, migration and invasion abilities of AGS-h CHIP cells were markedly slower than that of the AGS-control cells. CHIP overexpression in AGS cells led to increased apoptosis and decreased cellular proliferation, likely due to the inhibition of Bcl-2 expression and the suppressed phosphorylation of AKT signaling. CHIP negatively regulates the TRAF-2-NF-κB signaling, which results to the decreased expression of target genes of NF-κB subunits such as ITGB1, MMP-2 and MMP-9, might be one of the important mechanisms of CHIP acts as a tumor suppressor in the gastric cancer cells. CHIP is as a novel molecular targeted therapy of gastric cancer, application of CHIP overexpression technology can become a potential means of gastric cancer gene therapy.