The Effects And Clinical Significance On Heparanase Of Gastric Cancer Caused By Helicobacter Pylori

Objective:The morbidity and mortality of gastric cancer(GC)have always been high all over the world,especially in China.H.pylori is listed as a class I carcinogen of GC.Heparanase(HPA)is an endoglycosidase capable of degrading heparan sulfate(HS)in the extracellular matrix(ECM)and basement membrane(BM);thus,it is involved in tissue remodelling and cell migration,which leads to inflammation,angiogenesis,and tumour metastasis.The purpose of this study is to observe the expression of HPA in GC MKN-45 cells and gastric cancer tissue infected by H.pylori and to clarify the correlation between the expression of HPA and H.pylori infection and its mechanism.Methods:HPA mRNA and HPA protein expression were detected by RT-PCR and Western blot at different time points after H.pylori and MKN-45 cell were cocultured.Western blot were used to detect changes in levels of MAPK and NF-kB signal molecules at different time points after coculture of H.pylori and MKN-45.Before coculture of MKN-45 and H.pylori,MAPK inhibitor SB203580 was given to MKN-45 cell,then cocultured and detected NF-kB levels.Similarly,after MKN-45 cell was intervented by SB203580,RT-PCR and Western blot were used to detect the changes of HPA mRNA and HPA protein,CCK-8 method,Transwell method and scratch test were used to determine the proliferation,invasion and migration ability of gastric cancer cells.After siRNA interference technology was used to silence HPA expression,HPA mRNA and HPA protein expression were detected by RT-PCR and Western blot,at the same time,the CCK-8 proliferation test and Clone test were used to confirm the proliferation and clone formation ability of tumor cell.99 gastric cancer tissues were collected after operation of gastric cancer patients,H.pylori was detected by C-13 breath test before operation and immunohistochemistry of cancer tissue.HPA and MAPK expression were still tested using immunohistochemistry.The correlation among H.pylori infection,HPA and MAPK and the clinical pathological features of gastric cancer was analyzed by SPSS 22.0 software.Overall survival(OS)and relapse-free survival(RFS)of GC patients was estimated by Kaplan-Meier method.The independent and multiple factor of HPA and MAPK with prognosis were determined by COX proportional hazards models to further elucidate the clinical significance and prognostic value of HPA protein expression in gastric cancer,thus providing theoretical basis for the mechanism of H.pylori infection in gastric cancer.Results:It was demonstrated that HPA mRNA expression was highest 6 h post-infection and the expression of the HPA protein was highest 24 h post-infection in H.pylori-infected gastric cancer cells.Furthermore,it was demonstrated that H.pylori infections significantly enhanced the expression of MAPK and NF-kB in MKN-45 cells at the mRNA and protein levels.p-p38 MAPK increased at 30 minutes and peaked at 60 minutes,and p-p65NF-kB peaked at 240 minutes when H.pylori and MKN-45 were cocultured,then decreased little by little.SB203580,which is an inhibitor of MAPK,significantly decreased the expression of NF-kB in MKN-45 cells infected with H.pylori.When SB203580 was administered,the expression of HPA significantly decreased and the proliferation,invasion and migration ability of MKN-45 cells infected with H.pylori were corresponding decrease.After silencing HPA expressionin in MKN-45 cells infected with H.pylori,HPA mRNA and HPA protein were declining and the proliferation and clone formation ability of tumor cells also declined.Specimens from 99 patients with GC were included in this study.H.pylori infection was observed in 70 of the 99 patients with GC,accouting for 70.7%,significantly better than 40% of health checker,furthermore,H.pylori infection was related to lymph node metastasis.H.pylori infection was also significantly correlated with the expression of HPA and MAPK(C=0.244,P=0.012 and C=0.202,P=0.040,respectively),in addition,the expression of HPA was relevant to the expression of MAPK(C=0.221,P=0.024).HPA-positive gastric cancers showed poorer prognosis than HPA-negative cases(OS,P=0.02;RFS,P=0.06).In H.pylori-positive gastric cancer,HPA-positive case was also a significant factor to predict poor prognosis(OS,P < 0.01;RFS,P = 0.01).COX proportional hazards models showed that Gastric cancers positively stained with HPA,lymph node metastasis,tissue differentiation,depth of invasion had a tendency of the worst prognosis.Conclusion:H.pylori infection can significantly enhance HPA expression by activating MAPK signal pathway,thus promoting the proliferation,invasion and metastasis of GC;HPA can be used as a treatment target for gastric cancer infected by H.pylori.HPA protein positive expression indicates a higher mortality rate in GC patients,especially when infected by H.pylori.