| Objective: Study the expression level and significance of RAGE in gastric cancertissues, adjacent tissues and normal gastric tissues and explore the relationship betweenthe RAGE protein levels and the characters of the patients; Then study the Changes of themetastasis related gene AKT, PCNA, mmp-2and protein in gastric cancer cells afterinhibited RAGE gene, and study the apoptotic index and cell cycle dynamics change ofgastric cancer cell after the RAGE gene was inhibited; Last study the affect of RAGE onthe adherence of H. pylori, and H. pylori infection on the expression of RAGE to gastricepithelial cell, and study the synergy of RAGE and H. pylori to gastric epithelial cell.Methods:180cases gastric cancer tissue, adjacent tissues and30cases normal gastrictissues were detected by immunohistochemical (IHC) assay. Combined with clinical data,analyses the relationships between the RAGE protein expression levels and the clinicalpathological characteristics of gastric cancer; Through contructing the recombinantlenti-virtl expression vector of gastric cancer cells SGC-7901, blocking the expression ofRAGE signal. Gastric cancer cells SGC-7901were divided into2groups: theexperimental group and the control group. The experimental group was administratedwith Lv-shRAGE, and the control group with nothing (NC).1) RAGEmRNA andAKTmRNA expressions of both groups were detected using Q-PCR, and their proteinexpressions were detected using western biot (WB).2) Q-PCR and WB were used todetect the PCNAmRNA and protein expressions, and cell active assay (MTT) were usedto detect the proliferative activities.3) Q-PCR and WB were used to detect theMMP-2mRNA and protein expressions, and transwell were used to detect the cellinvasive.4) Flow cytometry instrument was used to detect the apoptotic index and cellcycle dynamics change of both groups; Finally, through the WB method and the CFUcounting method to observe the mutual influence between RAGE and HP and to studydetermine the relationship between the two factors and gastric cancer:1) RAGE-positive MKN74cells and RAGE-negative MKN74cells were pretreated, prior infection withRAGE ligands (HMGB1and AGE), and their protein expressions were detected usingwestern blot (WB). Next adding bacterial suspension, the H. pylori adhesion to gastricepithelial cell were tested in the CFU counting method.2) Adding bacterial suspensioncaused a significant decrease in RAGE protein in dose and time-depentdant manner, thecontrol group with no adding bacterial suspension MKN74.3) Experiment were dividedinto4groups: the adding100μM bacterial suspension group, the adding RAGE ligandsgroup, the adding bacterial suspension and RAGE ligands group and the MKN74withnothing group. The interleukin-1βmRNA and interleukin-8mRNA were detected usingRT-PCR. Result: The first part:1) Expressions of RAGE protein were found in cancertissue, adjacent tissues and normal gastric tissues. The positive rate is70.0%(126/180) ingastric cancer tissues,45.0%(81/180) in adjacent tissues,40.0%(12/30). The differencewas statistically significant (P=0.0000).2) There is highest positive rate in gastric cancertissues, and the difference was statistically significant (P=0.0000, P=0.0000). There is nosignificant difference between adjacent tissues and normal gastric tissues (P=0.7551).3)According to histological grade, the positive rate is57.8%(33/57) in high differentiatedgastric cancer,66.7%(32/48) in moderately differentiated gastric cancer,81.3%(61/75) inlow differentiated gastric cancer. The poorer differentiated gastric cancer the higherpositive rate of RAGE protein expression, and the difference was statistically significant(P=0.0213).4) According to clinical staging (TNM staging), the positive rate is62.3%(53/85) in T1and T2stage,76.8%(73/95) in T3and T4stage. The poorer stagegastric cancer the higher positive rate of RAGE protein expression, and the differencewas statistically significant (P=0.0342).5) The positive rate is78.0%(103/132) withlymph node metastasis in gastric cancer,47.9%(23/48) with no lymph node metastasis ingastric cancer. There is a higher RAGE protein expression in the patients with lymphnode metastasis in gastric cancer. The difference was statistically significant (P=0.0002).6) The positive rate is65.7%(94/143) without distant metastasis in gastric cancer,86.5%(32/37) with distant metastasis in gastric cancer. The difference was statisticallysignificant ((P=0.0242). The second part:1) The expressions of RAGEmRNA andAKTmRNA were lower in the Lv-shRAGE group than the NC group, and the sameresults were obtained using PCR and WB (P＜0.05).2) The expressions of PCNAmRNAwas lower in the Lv-shRAGE group than the NC group, and the same results wereobtained using PCR and WB (P＜0.05). The proliferative activities was lower in theexperimental group than the control group (P＜0.05).3) The expressions of MMP-2mRNA was lower in the Lv-shRAGE group than the NC group, and the sameresults were obtained using PCR and WB (P＜0.05). The cell invasive was lower in theexperimental group than the control group (P＜0.05).4) The apoptotic index was higherin the Lv-shRAGE group than the NC group,(P＜0.05), and the cell cycle in theexperimental group remained in G0/G1compared with the control group. The last part:1)RAGE-positive MKN74cells were pretreated, prior infection, with RAGE ligands(HMGB1and AGE-BSA). The increments observed of RAGE protein expressions,ranging from53%-79%as described in WB, and adherence increments ranged between65%and70%, no effects were observed when RAGE-negative MKN45cells werepretreated with both RAGE ligands(P＜0.05). The increments in adhesion wereparalleled by increased levels of RAGE protein expressions. The results depicted werequite similar by WB and CFU counting.2) WB showed that the H. pylori increasedRAGE protein expression dose-dependent. Compared with control group, the incrementsof H. pylori dose were paralleled by increased levels of RAGE protein expressions, themost increments at250μM. In time course study, RAGE protein expressions were thehighest at48houses after adding bacterial suspension.3) The interleukin-1βmRNA andinterleukin-8mRNA were the lowest in the MKN74with nothing group and the highest inthe the adding bacterial suspension and RAGE ligands group. Conclusion: Theexpression of RAGE protein is the highest among gastric cancer tissues, adjacent tissuesand normal gastric tissues, which remend that RAGE proteinmay be associated with theoccurrence and development of gastric cancer,The expression of RAGE protein issignificantly different in histological grade, invasion depth, lymph node metastasis anddistant metastasis; RAGE-targeting silence can inhibited the proliferation of gastriccancer cells SGC-7901, When the expression of RAGE was reduced, the mRNA andprotein of AKT, PCNA and MMP-2were reduced, too; RAGE contributes to the adhesionof H. pylori to gastric epithelial cells and H. pylori infection produced an increase inRAGE expression, RAGE and H. pylori infection may also contribute to trigger aproinflammatory response, as assessed by the detection of both interleukin-1βmRNA andinterleukin-8mRNA, which may induced gastric inflammation and the occurrence ofgastric cancer. |
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