Study On The Function And Mechanism Of LncRNA H19 Affecting Angiogenesis In Gastric Cancer

Objective:This study aimed to using bioinformatics methods to analyze the expression of lncRNA H19 in tissue samples of gastric cancer(GC)patients,and detect its correlation with CD31,angiogenesis marker.Then preliminarily explored the relationship between H19 expression and the prognosis of GC patients.Subsequently,we investigated the effect of conditioned medium(CM)from gastric cancer(GC)cells on the proliferation,migration and tube formation of human umbilical vein epithelial cells(HUVEC)by silencing H19 in MKN45 and SGC7901 GC cells.The potential molecular mechanism of H19 on GC angiogenesis was preliminarily explored.Methods:1.The expression of lncRNA H19 and angiogenesis marker CD31 in GC patients tissue samples were collected using bioinformatic methods.Patient survival data was included as well.We analysed the expression of H19 between gastric cancer tissues and noncancerous tissues.Next,we explore the relationship between H19 expression level and the prognosis of GC patients.Furthermore,the correlation between H19 and angiogenesis marker CD31 was analyzed.2.RT-qPCR was used to compare the expression of H19 in gastric cancer cells MKN45,SGC7901 and normal gastric epithelial cells GES-1.Then H19 was silenced in MKN45 and SGC7901 GC cells by infection with lentiviruses,including empty vector group(sh-NC)and interference H19 group(sh-H19).RT-qPCR was performed to detect the silencing efficiency.The conditioned medium from two groups of GC cells was collected to treat HUVECs.CCK-8 assays,wound healing assays and the tube formation assays were respectively used to detect the proliferation,migration and the tube formation capacity of HUVECs.3.RT-qPCR was performed to exam the expression of angiogenesis-related factors including VEGFA、PDGFB、PGF、MMP9 and THBS1 in sh-NC and sh-H19 GC cells.Western blot further explored the expression of VEGFA protein in GC cells after H19 silencing.We used recombinant VEGFA(rVEGFA)to treat HUVECs.Then,the differences in proliferation,migration and tube formation ability of HUVECs in sh-NC group,shH19 group and sh-H19-rVEGFA group were compared.4.Western blot was used to detect the EZH2 protein expression between the sh-NC group and sh-H19 group.RNA immunoprecipitation(RIP)experiment investigated the direct interaction between lncRNAH19 and EZH2.5.GSK126,an inhibitor of EZH2,was used to treat gastric cancer cells.RT-qPCR and Western blot experiments were used to detect mRNA and protein expression of VEGFA between the control group and GSK126 group.The conditioned medium from GC cells in each group was used to treat HUVECs to analyse the proliferation,migration and tube formation ability in HUVECs.Results:1.The expression of H19 was higher in GC tissues than that in noncancerous gastric tissues(P=0.0378),and its expression was related to the level of CD31(P=0.0261).GC patients with high level of H19 predicted an unfavorable prognosis.2.Compared with normal gastric mucosal epithelial cells,H19 expression was significantly up-regulated in MKN45 and SGC7901 gastric cancer cells.After lentivirus infection,the expression of H19 in MKN45 and SGC7901 gastric cancer cells was significantly inhibited.The conditioned medium from GC cells knocked down H19 significantly inhibited the proliferation,migration and tube formation ability of HUVECs..3.In MKN45 and SGC7901 GC cells,after silencing H19,the expression of VEGFA,PDGFB,PGF and THBS1 was significantly reduced in both two cell lines.Western blot showed that the protein levels of VEGFA in MKN45 and SGC7901 cells were remarkably decreased after H19 knocked down.Furthermore,rVEGFA treatment attenuated the inhibitory effects of conditioned medium from sh-H19 GC cells on the proliferation,migration and tube formation ability of HUVECs.4.RIP assay found that H19 could directly bind with EZH2 in GC cells.Western blot showed that the protein expression of EZH2 was decreased in sh-H19 GC cells.5.Treatment with GSK126,an inhibitor of EZH2,reduced the mRNA and protein expression of VEGFAin MKN45 and SGC7901 cells.In addition,the conditioned medium from GC cells treated with GSK126 significantly down-regulated the proliferation ability,migration rate and tubule formation ability of HUVEC.Conclusions:Overexpression of H19 in GC patient tissue specimens is positively associated with poor prognosis and tumor angiogenesis marker CD31.LncRNA H19 promotes gastric cancer angiogenesis by activating EZH2 to induce VEGFA expression.This study provides new strategy and molecular targets for the clinical targeted therapy of gastric cancer.