The Study Of The Regulation Of NECL2 Expression In Lung Cancer

In previous study of National Laboratory of Medical Biology,we have discovered a new cDNA clone whose structure is similar with those of nectin family,so named nectin-like molecule 2,that is NECL2.NECL2 belongs to the immunoglobulin superfamily and is widely expressed in multiple tissues. The structure of NECL2 is:a signal peptide,three immunoglobulin domains, a transmembrane region and a short cytoplasmic region.Up to now NECL2 has the following functions:Ca²⁺-independent cell-cell adhesion,synapse formation and tumor suppression.Down regulation or silent of NECL2 expression is found in many types of tumors.Others' work showed that in those tumors exhibiting loss of heterozygosity,inactivating mutation was rarely found,whereas promoter methylation was observed with high incidence and was correlated with down regulation of NECL2 expression. Furthermore,expression level of NECL2 was restored after treating cancer cell lines with 5-Aza-2-deoxycytidine.These results suggest that promoter methylation is perhaps the cause of down regulation of NECL2 expression. Considering the importance of NECL2 in neural development and tumor suppression,and the correlation between promoter methylation and down regulation of NECL2 expression,we studied the regulation of NECL2 expression in lung cancer cell lines which we are interested with.First,we detected the expression pattern of NECL2 in one human normal brain tissue and three lung cancer cell lines(A549,NCI-H446,Calu-3).Based on the prediction result which shows that there is a CpG island in 5' non-coding region,we know promoter methylation can lead to the silent of NECL2 in lung cancer cell lines using bisulfite sequencing and 5-AzaC treatment.Then,we used bioinformatics method to analyze the 5' non-coding region of human NECL2.The result showed that there are six conserved nucleotide sequences within the 4Kb region just upstream the human/mouse NECL2 translation start site.We also predicted the possible cis-acting elements.Next,we extracted the genome from normal brain tissue and cloned a 1.7Kb fragment of human NECL2 5' non-coding region.Based on the result of bioinformatics analysis,we generated a series of promoter deletion constructs.With dual luciferase reporter assay,we deduced that the possible core promoter for human NECL2 is from -68 to -329,designated ATG as+1.Bioinformatics analysis also showed that there are three putative Spl binding sites within the core promoter of human NECL2.Therefore we cloned the complete coding region of Sp1 and obtained an eukaryocyte expression plasmid of Sp1.We co-transfected this plasmid with the core promoter-reporter plasmid to test the possible effect of Sp1 on human NECL2 promoter transcription activity.With dual luciferase reporter assay,we found that over-expression of Sp1 in lung cancer cell lines does not affect the human NECL2 promoter transcription activity significantly.Finally,we treated cultured A549 and NCI-H446 cell lines with DDP and detected the changes of cells by cell cytometry.The result showed DDP killed lung cancer cells possibly by destroying cell cycle and inducing cell apoptosis, this can provide some clues for further study.In conclusion,we validated promoter methylation of NECL2 could lead to the silent of NECL2 in lung cancer cells by bisulfite sequencing and 5-AzaC treatment;with dual luciferase reporter assay,we deduced the possible core promoter for human NECL2 for the first time.We also analyzed the effect of Sp1 transcription factor on human NECL2 promoter transcription activity.These work provide basis for further elucidation of the regulation of human NECL2 expression.