Primary Analysis Of Novel NPCEDRG Gene In Tumor Suppression Function

Part 1 Investigation of NPCEDRG Gene Function in Nasopharyngeal Carcinoma with Tet-on SystemObjective:This research was to establish an ideal CNE2 cell line with tightly regulated and reversible expression of NPCEDRG for investigation in the function of NPCEDRG gene in nasopharyngeal carcinoma.Methods:Recombinant expression vector pRevTRE-NPCEDRG was constructed. Tet/CNE2 cell line was first established by transfecting regulating plasmid pRevTet-on into CNE2 cells with G418 screen.Then the response plasmid of recombinant pRevTRE-NPCEDRG was steadily transfected into Tet/CNE2 cells by Hygromycin screen.1000 ng/ml of doxycline(Dox) was used to induce the expression of NPCEDRG,and a cell clone sensitive to Dox was selected.The best-induced concentration was determinated with different concentration of Dox induction.Cell morphous,growth curves,clony formation rate and cell cycle distribution were detected after restoration of NPCEDRG expression with Dox induction.Results:Varying the concentration of Dox in the media could modulate the level of NPCEDRG expression in a dose-dependent manner within 0-3000ng/ml range, and 3000 ng/ml Dox could produce the best induction with little toxic effect.After the restoration of NPCEDRG expression,the degree of CNE2 differentiation was higher than ever,the growth capacity and clone formation potential of CNE2 cells in soft agar were significantly suppressed,and the cell percentage in G₀/G₁ phase increased,while percentage of cells entering the S and G₂ phase decreased.This data indicates that the abnormality of NPCEDRG expression is associated with nasopharyngeal carcinogenesis and that it might play an important role in inducing cell differentiation,controlling cell growth and regulating the cell cycle. Conclusions:1.An ideal CNE2 cell line with tightly regulated and reversible expression of NPCEDRG was successfully established for further studies on NPCEDRG gene.2.NPCEDRG protein could promote differentiation of CNE2 and suppresse its growth.And CNE2 cells were hold in G₀/G₁ phase after restoration of NPCEDRG. The malignant phenotype of CNE2 could be partly reversed via up-regulating the expression of NPCEDRG.Part 2 Search for The Functional Sites of NPCEDRG by Random MutationObjective:The goal of this research was to use a random mutation approach to search for the functional domains or sites of NPCEDRG gene with comparing wild NPCEDRG and its mutant.Methods:Based on the base mispairing,a mutant of NPCEDRG was produced by random mutation in PCR.The structure of the mutant protein was predicted with bioinformatics on-line.The ORFs' cDNAs of the wild NPCEDRG gene and its mutant were recombined into the pcDNA^TM3.1/myc-HisB vector with two tags of myc and 6×His following the target genes.Two recombinant expression vectors of mutational or wild NPCEDRG gene,and the pcDNA^TM3.1/myc-HisB vector were transfected respectively into CNE2 cells,then wild pcDNA^TM3.1/myc-HisB-NPCEDRG/CNE2 and mutational pcDNA^TM3.1/myc-HisB-NPCEDRG/CNE2 cell lines were established after two weeks' screen with 300μg/ml G418.Growth curves and cell cycle distribution were detected with expression of wild NPCEDRG and mutational NPCEDRG individually.Differential analysis between the wild NPCEDRG and its mutant was used to evaluate the importance of the mutational sites to the function of NPCEDRG.Results:The sequence of the NPCEDRG mutant revealed two altered codons, T²⁶⁰-C²⁶⁰ and T²⁸⁷-C²⁸⁷,which resulted in amino acid exchanges V⁷³-A⁷³ and M⁸²-T⁸².Two recombinant expression vectors of wild NPCEDRG and its mutant were successfully transfected into CNE2 cells.All of the results about the effects of wild NPCEDRG gene restoration expression on CNE2 cells in this part,were dramatically similar with those in part 1.That is,after the restoration expression of NPCEDRG or its mutational protein,the growth capacity was significantly suppressed,and the cell percentage in G₀/G₁ phase increased,while percentage of cells entering the S and G₂ phase decreased.And there was no difference of statistic meaning between wild and mutational pcDNA^TM3.1/myc-HisB-NPCEDRG/CNE2 cells.Conclusions:1.Two recombinant expression vectors,wild pcDNA^TM3.1/myc-HisB-NPCEDRG and mutational pcDNA^TM3.1/myc-HisB-NPCEDRG,were successfully constructed.2.Amino acid exchanges in NPCEDRG,V⁷³-A⁷³ and M⁸²-T⁸²,couldn't change its inhibitory role to NPC,and these sites shouldn't be necessary for NPCEDRG reacting in nasopharyngeal carcinoma.