Inhibitory Effect Of AAV-mediated PTEN-Long Protein Expression On Glioma

PTEN is a well-known tumor surppressor like P53.PTEN is a phosphatase,which has the dual funtion of lipid phosphatase and protein phosphatase.Its lipid phosphatase activity can inhibit tumorigenesis and development,and has been thoroughly studied.PTEN is mainly involved in the PI3K/Akt/mTOR signaling pathway,and its lipid phosphatase activity can dephosphorylate PIP3 to PIP2,then reduce the phosphorylation level of AKT to block this signaling pathway and inhibit the malignant proliferation of tumor cells.With the in-depth development of research on PTEN,researchers have discovered many translation variants which different from the classic PTEN,and named them as PTEN-L,PTEN-M,PTEN-N,PTEN-O,etc.The translation initiation site of these translation variants is located the upstream of start codon ATG of the classic PTEN.Among them,PTEN-L(PTEN-Long)not only has the similar tumor suppressive function as PTEN but also has the secretion characteristics,which in concerned by researchers.PTEN-Long can secrete from the donor cells into the blood circulation system,and enter the recipient cells again after circulating to distant tissues and organs.This property enables PTEN-Long to suppress tumor cells growth in a paracrine manner.In theory,we can use human-derived PTEN-Long protein for direct intravenous administration to enter almost all target cells,which has unique advantages for tumor treatment.The PI3K/Akt/mTOR signaling pathway,composed of three major factors,PI3K,Akt,and mTOR,which can regulate the effects of cell proliferation,differentiation,adhesion,and movement.It has been reported that the activity of this signaling pathway is significantly increased in glioma stem cells(GSCs),which plays roles in maintaining self-renewal ability and promoting their migration and invasion of GSCs.The premise of treatment for glioma is to be able to cross the blood-brain barrier(BBB).Gene therapy vectors play a crucial role in tumor therapy.Among them,adeno-associated virus(AAV)vectors have advantages in low pathogenicity,weak immunogenicity to the host,long-term stable expression,and extensive cell and tissue affinity,which make it as one of the most promising carriers,and have been widely used in clinical research.According to the tissue tropism of different serotypes of AAV,AAV2 can cross the blood-brain barrier and carry the gene of interest into the brain.We constructed AAV2-PTEN-Long vector to form a recombinant AAV virus for the treatment of gliomas,providing research basis for the clinical treatment of brain malignant gliomas.We constructed AAV2-PTEN-Long vector to form a recombinant AAV virus.At the cellular level and individual level,the recombinant AAV virus is used for treatment,to explore the inhibitory effect of recombinant AAV virus on glioma,and to study its potential value in the clinical treatment of glioma.Firstly,we cloned the target gene at the molecular level,successfully constructed a eukaryotic expression vector and packaged the rAAV virus.Secondly,we overexpressed PTEN-Long in U87MG,LNZ-308 and A172 glioma cell lines which lacking PTEN,and observed the effects on the downstream molecules of PTEN such as p-AKT,mTOR,etc.at the protein level by WB;Then we classified the cells into four groups:PTEN-Long overexpression group,empty vector group,blank control group and PTEN positive control group,and observed the effect of PTEN-Long on migration,invasion,cell cycle,cell apoptosis,proliferation and tumorigenicity of U87MG cells.Finally,at the individual level,the nude mice were subcutaneously and intracranially tumor-bearing.The AAV virus was injected through intraperitoneal and the tail vein to observe the size of the tumor and detect the inhibitory effect of PTEN-Long on gliomaIn a word,we have successfully packaged the AAV2 virus that overexpressed PTEN-Long and used the rAAV2 to infect U87MG,A172,LNZ-308 cells.When overexpression of PTEN-Long at the cellular level,it not only can suppressed the proliferation,migration,and invasion of tumor cells but also promote the apoptosis of tumor cells.At the individual level,injection of rAAV2 can also significantly inhibit tumor growth.Our virus rAAV2 can carry PTEN-L across the blood-brain barrier and inhibit the growth,migration and invasion of glioma cell line U87MG;...