Construction Of Nicotinamide N-methyltransferase Over-expressed SW480 Cell Model

Nicotinamide N-Methyltransferase (NNMT) is an S-adenosyl-L-methionine cytosolic enzyme that catalyzes the N-methylation of nicotinamide and other pyridines to form pyridinium ions. Now NNMT in both mRNA and protein levels were found over-expressed in a wide range of tumors. When comparing normal tissue with cancer tissue, abnormal expression of NNMT has been reported in tumors such as papillary thyroid cancers, glioblastoma, stomach adenocarcinoma, colon cancer, renal carcinoma, oral squamous cell carcinoma, and lung cancer. NNMT was also found increased in serum of patients with cancer. Those results indicate that NNMT may be closely related to the biological function of tumour, and may influence the results of chemotherapy, thus become a potential biomarker for diagnosis of tumor. All these functions need to be proven by studying the influence of NNMT on tumor cell.Object:To construct the eukaryotic expression plasmids of NNMT and establish NNMT over-expressed sw480 cell model.Methods:NNMT coding fragment was cut from pGEX-4T-2/NNMT plasmids which digested with the restriction enzyme BamHI/XhoI, and ligated to the plasmid pcDNA3.1-NNMT digested with the same restriction enzyme. The recombinant expression plasmids were named as pcDNA3.1-NNMT and identified by BamHI/XhoI restriction enzyme analysis and DNA sequencing.Then pcDNA3.1-NNMT were transferred into colon cancer cell line SW480. G418, RT-PCR and Western blot was used to screen and evaluate the positive cell clone. The influence of NNMT on the biological function of tumor cells such as cell cycle, cand cell proliferation wereanalysed by Flow Cytometer and cell growth curve respectively.Results:Restriction analysis and DNA sequencing confirmed the correct sequence and insertion site of the recombinant pcDNA3.1-NNMT; NNMT highly expression cancer cell models, SW480-pcDNA3.1/NNMT were constructed, NNMT mRNA and protein level can be detected in SW480-pcDNA3.1/NNMT while negative in wild or control SW480; The results of cell growth curve showed SW480-pcDNA3.1/NNMT cells grow or proliferate faster than the control cells; However, no significant difference was observed in cell cycle.Conclusion1. The plasmid pcDNA3.1-NNMT was constructed successfully2. The NNMT over-expressed sw480 cell model was constructed successfully3. High expression of NNMT speeded the growth of SW480, and made the growth curve shift to the left.