Primary Analysis Of NPC Candidate Suppressor Gene STGC3 Function By Site-directed Mutagenesis

Objective To investigate suppressor function of a new NPC candidate gene STGC3 cloned recently, three mutant recombinants were constructed and transfected into CNE2 cell line. The biological activity of CNE2 cell was evaluated to analyse the structure-function relationship of the gene. The bases of the DNA, which might contribute to the activation and function of the suppressor gene STGC3, were observed by site-directed mutagenesis.Methods The structure of STGC3 was predicted by software on web and software. N-glycosylation site, protein kinase C phosphorylation site as well as Casein kinase II phosphorylation site was selected motifs which were previously determined. Three pairs of mutant primers aimed at changing one base of the motifs were designed by three different site-directed mutagenesis strategies which were overlap extension PCR, MutanBEST and Stratagene protocol. Three eukaryon expression mutant recombinants were constructed aiming at STGC3 gene one base changed at its cDNA C656G , C725T and T913G , all of which were transfected into CNE2 cell by liposome lipofectamine 2000. The stable pcDNA3.1^TM/myc-His B-STGC3 wild type and mutants transfectd CNE2 cell line were obtained by G418 screening. The mRNA and protein levels of STGC3 were detected by RT-PCR and Western blotting, while the cell morphology and the location of the protein in the cell was surveyed by immunohistochemistry. Growth curves, colony formation and cell cycle distribution were detected in CNE2 cell line with STGC3 gene high expression by FCM and MTT.Results The consequences of double restriction enzyme digestion and DNA sequencing demonstrated all pcDNA3.1^TM/myc-His B-STGC3 wild type and mutant vectors were constructed successfully. The stable CNE2 cell line contained these recombinants were established and verified by RT-PCR and Western blotting. Immunohistochemistry photo showed that STGC3 protein was expressed stablely. The results of growth curves, colony formation and FCM demonstrated that with the 43rd serine and 66th serine being changed, STGC3 gene partly lost in its proliferation suppressing activity on CNE2 cell (p>0.05), growing more slowly than that of CNE2 cells (P<0.05), while 129^th serine changing made no change of the function showed the same growing speed with wild type STGC3 gene transfected NPC cells (P>0.05). FCM analysis indicated wild type and Casein kinase II phosphorylation site with one base changing possessed the highest apoptosis rate (P<0.05). All mutants had little effects on the distribution of cell cycle (P>0.05).Conclusions:1. The restoration expression of STGC3 gene might down-regulate the proliferation ofNPC CNE2 cell.2. The 656^th and 725^th cytosine are two key structures of STGC3 gene supressorfunctions.3. The 913^rd thymine might contribute little to the STGC3 gene suppressor functions.