Mechanisms Of PBX1 Regulating Gastric Cancer Carcinogenesis And Progression And Nanoparticle Delivering AntimiR-21 For Gastric Cancer Therapy

Background and purpose:Gastric cancer is a common malignant tumor of digestive tract,with the incidence of fourth and the second leading cause of cancer death worldwide.Homeobox gene PBX was first identified as a potential oncogene in pre B cell leukemia.Although PBX1 has been shown to play an important role in cell development and tumorigenesis,the mechanism of it in gastric cancer is unclear.Therefore,the aim of this study was to investigate the mechanism of PBX1 in the occurrence and development of gastric cancer.An endogenous non coding small RNA(micro RNA,miRNA)plays an important role in post transcriptional regulation,but the therapeutic effect of micro RNA is less studied.Previous studies have confirmed that miR-21 is a oncogene in gastric cancer,the major target is phosphatase and tensin homolog deleted on chromosome ten gene(PTEN)and programmed cell death protein 4(PDCD4).Mi R-21 is a potential therapeutic target,and the biggest obstacle for its clinical use is the lack of effective gene carrier,the research aims to develop a safe and effective carrier for antimiRNA used in the treatment of gastric cancer.Methods:The expression of PBX1 in human gastric cancer was detected by qRT-PCR and immunohistochemistry.We observed the effects of PBX1 over expression and knockdown on the gastric cancer's proliferation,invasion and migration,the effects of PBX1 on subcutaneous tumor growth in nude mice.We designed PBX1 gene mutants to investigate the site of action in gastric cancer.Western blotting and immunofluorescence assay were used to detect the expression of epithelial and mesenchymal markers.The qRT-PCR assay was used to analyze the expression of tumor growth and angiogenic factors.For the development of a novel gene delivery vector,the Zeta potential and the particle size of the nanoparticles were measured by DLS.MTT methods was used to detect the toxicity of nano-carrier on gastric cancer cells.The fluorescence microscopy and flow cytometry analysis was used to determine the transfection efficiency.Quantitive RT-PCR was used to detect the expression of miR-21 and PTEN,PDCD4.Western blotting was used to detect the protein level.We observed the effects of NP/antimiR-21complex on the gastric cancer cells proliferation,migration and invasion.The antitumor effect in vivio was studied in the xenografts model of gastric cancer on the nude mice.Results:The immunohistochemistry analysis of 82 cases of gastric cancer specimens confirmed that the high expression of PBX1 in gastric cancer tissues,and low expression in the adjacent normal tissues(P=0.008),and its expression was correlated with the histological types and lymph node metastasis in gastric cancer.Overexpression of PBX1could promote the proliferation,invasion and migration ability in gastric cancer cell line BGC-823,and promote the tumor growth and metastasis in nude mice.In contrast,PBX1knockdown inhibited the proliferation,invasion and migration ability in gastric cancer cell line NCI-N87.At the same time,PBX1 could promote the epithelial mesenchymal transition(EMT)in BGC-823,the expression of E-cadherine was down regulated,the expression of N-cadherine,Vimentin and Snail were up-regulated.In addition,PBX1 can promote the expression of tumor growth and angiogenic factors,b FGF,NRG2 and MMP9.The 252nd residues(Phe²⁵²)on PBX1 was mutated to arginine and alanine,EMT changes caused by PBX1 was lost.A new gene dilivery vector composed of PEG-SS-PLA-SS-PEI tri-block copolymer was designed and synthesized.It could be self-assembled into nano micelle particles(NP).The average diameter is 68 nm.The average Zeta potential was39m V.When N/P=32,antimiR-21 can be fully condensed into the nano carrier to form a complex,and the high GSH content in gastric cancer cells can destroy the two sulfur bond in the carrier,which leads to the release of encapsulated antimiR-21.The in vitro assays showed that nanomicelles had no significant cytotoxicity on gastric cancer cells.The transfection efficiency of the system was similar to that of Lipofectamine^TM2000.The expression level of miR-21 in SGC7901 could be significantly reduced by NP/anti-miR-21transfected.Down regulation of miR-21 significantly induced apoptosis,inhibited the migration and invasion of gastric cancer cells and increased the expression of target genes,such as PTEN and PDCD4.More importantly,in the subcutaneous of nude mice model of gastric carcinoma,NP/antimiR-21 composite objects within the systemic administration can significantly inhibit the growth of tumor.And there was no obversily side effect on the function of liver and kidney of mice.The expression of the target gene PDCD4 and PTEN was increased in subcutaneous tumor.Conclusions:PBX1 plays an oncogenic role in gastric cancer,which can promote the proliferation,invasion and migration of gastric cancer cells.PBX1 can promote the tumor growth and peritoneal metastasis,and induce the change of EMT.The hydrophobic binding between PBX1 and HOX hexapeptide domain plays a key role in the process of EMT.PBX1 can regulate the expression of tumor growth and angiogenesis factors.The polymer micelles can condense the antimiR-21,with low cytotoxicity and passive targeting,NP/antimiR-21 complex can inhibit gastric cancer growth,invasion and migration,induce apoptosis of gastric cancer cells,inhibit gastric cancer growth in vivo with low liver and kidney toxicity.Therefore,the nano carrier delivering antimiR-21 was a perspective treatment for gastric cancer.