Necl1 Protein Function And Mechanism Of Inhibition Of Proliferation And Migration Of Human Glioma

NECL1(Nectin-like molecule 1),also named as CADM3/IGSF4B/TSLL1/ SynCAM3,is a neural tissue-specific immunoglobulin-like cell-cell adhesion molecule.It is a single transmembrane protein,including three Ig-like domains as extracellular domain located at N terminus,one transmembrane motif and a short C terminal tail.Extracellular domain of NECL1 could form homo-or heterodimer with itself and other members in the family.C terminal tail could interact with both 4.1 protein family members and protein containing PDZ domain.Until now,it has been reported that NECL1 could play some role in synaptogenesis,myelination and cerebellum morphorlogenesis.Others had reported that NECL1 was abrogated from 13 human glioma cell lines comparing with normal tissue,which suggests that NECL1 may function in glioma generation.In our report,we applied Real time RT-PCR and immunohistochemistry to detect the expression of NECL1 in both glioma cell lines and primary glioma tissues.The result indicated that NECL1 was indeed lost in glioma cell lines and primary glioma tissues. For further study of the function of NECL1,we constructed recombinant adenovirus to induce NECL1 into glioma cell lines.The efficiency of infection was over 90%.Then flow cytometry and BrdU incorporation assay were employed to find that restoration of NECL1 in U251 would inhibit cell cycle process and DNA synthesis.It was also suggested that NECL1 could suppress tumor growth of U251 in nude mice.Additionally,the morphology of cells was changed when the U251 cells were cultured at high density with NECL1 re-expression.Wound line and Transwell assays were performed to study the function of NECL1 in U251 migration and invasion.The results indicated that NECL1 would inhibit cell migration and invasion in vitro.The result of Gelatin zymography assay suggested that restoration of NECL1 would influence the activity of MMP2 and MMP9.Therefore,we concluded that NECL1 may work as a potential tumor repressor in glioma generation.For studying the mechanism of NECL1 inhibiting cell proliferation and migration in glioma cell line,DNA gene biochip was applied to screen the different gene expression pattern of U251 with restoration of NECL1.204 differently expressed genes were found, including 184 down-regulated and 20 up-regulated.According to bioinformatic prediction and references report,we found the expression of several extracellular matrix proteins was changed,such as OPN,CCL2 and so on.Selecting OPN as the most important candidate, we examined the expression of OPN and its downstream molecules.The result was as expected.Then the conditional medium of U251 with NECL1 re-expression was collected and employed to treat cells.It was found that conditional medium could partly rescue the phenotype of U251 with NECL1 comparing with the wild type cells,which suggested some important factors influencing the function of NECL1 may exist in extracellular matrix.Besides extracellular molecules,we also try to look for the direct downstream signaling pathway proteins of NECL1.Yeast two-hybrid assay was performed to screen downstream interacting proteins of NECL1.Nine positive clones were obtained,including five existed proteins.Unfortunately,according to references,we did not think that there was any direction relationship between NECL1 with those candidate genes considering the spatio-temperal expression pattern of genes.Additionally,our preliminary data indicated that restoration of NECL1 in U251 would promote the active form of Cdc42 to shift to the inactive form,which suggested Cdc42 might work as one of downstream factors of NECL1 in U251.More data should be provided for further mechanism.