The Preliminary Inquiry Of The Mechanism Of Recombinant Lamprey LIP Molecule Affected Cancer Cells

Lampreys are primitive jawless vertebrates,which located between invertebrates and vertebrates.They provide a wealth of genetic information about the origin and evolution of organisms,and have a high research value.Lamprey has a unique immune system,variable lymphocyte receptor(VLR)related immune mechanisms,which provide a reliable basis for understanding adaptive immune systems.And we found a novel functional immune molecule called lamprey immune protein(LIP)in supraneural body,which has the cytotoxic activity in cancer cells.It presents the breakthough progress in studying the mechanism of anti-tumor and adaptive immune evolution and origin.To further explore the mechanism of killing tumor cells of LIP molecule,we used recombinant LIP(rLIP)which has the same activity with nature LIP,and explored the interaction of rLIP with various types of cell surfaces receptor molecules based on the cell membrane.Here,we used the small interfering RNA(siRNA),real-time quantitative PCR,LDH release assay,immunofluorescence methods and so on,and combined with(DeltaVision OMX)ultra-high resolution microscope,confocal microscope and other auxiliary equipments,the interaction of rLIP with various types of molecules on the cell membrane were observed.We found r LIP molecule has the strong cytotoxic activity with cancer cells(including suspension cells and adherent cells),and conbined with the cell membrane of cancer cells.So we have explored the interaction of rLIP with phospholipid molecules,important protein receptors,lipid raft and its components of the cell membrane in detail.These results show that some important protein receptors of common cytotoxic such as CD59 molecules,as well as some important cell membrane transport proteins such as PLSCR1,ABC transport and aminophospholipid transloease had no effect with the r LIP,but the rLIP could conbind with PS,which caused the cell death more serious.However,when the specific phosphatidylinositol-phospholipase C(PI-PLC)preincubated the cancer cells which could get rid of the GPI-APs of cell surfaces,it was found rLIP could not conbind with the lipid raft anymore and inhibited the cytotoxic activity of rLIP effectively at the same time.We also found when we knockdown the PIGN(GPI7 homologs)which can catalyst the glycan bind to the GPI anchor,the rLIP cytotoxic activity was weakened effectively.Therefore,we predict the glycan core of GPI-APs will play an important role in the rLIP cytotoxic activity because rLIP has the lectin domain.Our study is first to explore the mechanism of cytotoxic activity of recombinant lamprey LIP protein molecule,and explore the interaction of rLIP with various types of cell surfaces receptor molecules based on the cell membrane.And we found GPI-APs as recognition receptors play an important role in the mechanism of rLIP molecule.So as to further reveal the lamprey rLIP molecule specific receptor of cancer cell,and to explore the mechanism of intracellular pathways laid the foundation,as well as to explore the lamprey antitumor mechanism of the origin and evolution of adaptive immunity provide the new perspective.