Exosomes-packaged C-terminal Met Fragments Promote The Progression Of Gastric Cancer

Objective:1.The purpose of this research is to investigate the role of Hp in the progression of gastric cancer.We detected the expression of Met protein in exosomes from Hp infected gastric cancer,and explored the effect of exosomal Met protein on the proliferation and invasion of gastric cancer and its possible molecular mechanisms.2.We further investigated exosomal Met expression levels in the plasma of H.pylori negative gastric cancer patients and healthy controls through western blot analysis.Our present study was designed to investigate the existence of exosomal Met protein in the plasma of gastric cancer patients with a variety of gastric cancer presentations(Hp infection,TNM stage,and lymph node metastasis)and to compare its exosomal Met expression levels to those found in control volunteers with no diagnosis of cancer.In addition,we aim to provide clues and ideas for exploring new molecular markers for the diagnosis of gastric cancer.3.Methods:1.The expression differences of Met protein in GES1,AGS,SGC-7901,MGC-803,HGC-27,and BGC-823 were detected using western blot analysis.Consequently,we chose the AGS cells for further studies of the co-incubation of H.pylori with gastric cancer cells.Then the expression of Met protein and mRNA of Hp infected AGS cells were detected using western blot analysis and qRT-PCR,respectively.2.The exosomes of AGS cells were collected and identified by transmission electron microscopy.After co-culture with Hp and AGS,qRT-PCR was used to detect the expression of rab-related genes(RAB1 A,RAB5,RAB7,RAB27A,and RAB27B)in AGS cells.3.The expression of Met protein in exosomes from AGS cell or Hp infected AGS cells were detected by Western blot.The Met antibodys to different Met domains(full-length,cytoplasmic domains,and ectodomians)were used to detect the expression of Met protein in AGS cells and Hp-infected AGS cells.In addition,the expression of Met protein in exosomes and extracellular supernatants of Hp-infected AGS cells was detected by Western blot.4.CCK-8 and Matrigel invasion assays were used to detect the proliferation and invasion of AGS cells treated with PBS and various exosomes(AGS Exos、Hp-AGS Exos;Hp-AGSshCtrl Exos、Hp-AGSshMetl Exos、Hp-AGSshMet2 Exos).5.The expression of EGFR,STAT3,p-STAT3,MAPKAPK2,P-MAPKAPK2,ERK 1/2、p-ERK 1/2 in the AGS cells treated with PBS and various exosomes(AGS Exos、Hp-AGS Exos;Hp-AGSshCtrl Exos、Hp-AGSshMet1 Exos、Hp-AGSshMet2 Exos)were detected by Western blot.6.Plasma samples were collected from patients with gastric cancer and healthy controls,Western blots were used to detect exosomal Met protein expression in the plasma of gastric cancer patients with a variety of gastric cancer presentations.Results:1.The expressions of Met in these 3 gastric cancer cells lines were higher than gastric epithelial cell GES-1,especially in AGS cells.A significantly decrease of the full-length Met signal intensity was observed at 12h and 24h of co-incubation compared to that of AGS cells alone.QRT-PCR assays showed that there were no statistically significant differences in mRNA level of Met between H.pylori infected AGS cells and control groups.2.RAB1 A,RAB7,RAB27A,and RAB27B were up-regulated after infection of H.pylori in a time-dependent manner.The expression of Rab5A gene was down-regulated after 3 hours of H.pylori infection.3.Increased secretion of exosomes containing C-terminal Met fragment and ectodomain shedding of Met protein is induced by H.pylori infection.4.Exosomal C-terminal Met fragment promotes the proliferation and invasion of AGS cells in vitro.5.Exosomal C-terminal Met fragment enhances EGFR protein expression and leads to phosphorylation of MAPKAPK2 and ERK1/2 signaling in AGS cells.6.Exosomes carrying C-terminal Met fragment in patient-derived plasma may be an ideal biomarker for early detection of gastric cancer and an indicator of lymph node metastasis.Conclusions:Overall,we found that Hp treatment could promote the release of exosomes containing C-terminal Met fragment from AGS cells.Our current study showed that C-terminal Met fragments,detectable in exosomes from H.pylori infected AGS cells,promoted proliferation and invasion of tumor cells in vitro.In addition,Exosomal C-terminal Met fragment from patient plasma may be an ideal biomarker for early detection of gastric cancer and an indicator of lymph node metastasis.