| There is an urgent need to screen and identify new biomarkers, which are warranted to provide more information on the tumor biology, chemotherapeutic effects, allowing a better prognostic and possibly predictive stratification of patients and reducing the mortality.Proteomic technologies and DNA microarray are providing the tools needed to discover and identify biomarkers associated with diverse diseases and biological processes. Nicotinamide N-methyltransferase (NNMT) was one of the potential tumor biomarkers identified recently by those technologies, in which 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 and may have a potential role for predicting response to radiation therapy or chemotherapy. All these functions need to be proven by studying the influence of NNMT on tumor cell.As colorectal cancer was one of the most common malignancies worldwide and is considered to be the second or third leading cause of cancer death, thus colorectal cancer was chosen in this study.Objective:1.To understand the influence of NNMT on the biological function of tumor cell;2.To explore the mechanisms of the influence of NNMT on tumor;3.To evaluate the relationship between NNMT and clinical pathological parameter of patients with colorectal cancer.Methods:1. Molecular clone techniques were used to construct NNMT protocaryon and eukaryotic expression plasmids.2. The recombinant glutathione S-transferase (GST)-NNMT and NNMT were produced and purified by gene engineering, and were used for immunization of mice and making hybridomas secreting Ag-specific monoclonal antibodies. Western-blot and Immunohistological stain were established to detect NNMT based on the prepared monoclonal antibodies.3. RT-PCR and Western-blot were used to screen low or no NNMT expression and high expression colorectal cancer cell strains.4. To construct NNMT high expression cancer cell models, the eukaryotic expression plasmids, pcDNA3.1/NNMT was transferred into low or no NNMT expression and high expression colorectal cancer strains; G418 was used to screen the positive cell strains.5.Based on the above constructed cell models and RNAi technique, the influence of NNMT on the biological function of tumor cells such as cell cycle, cell apoptosis, cell proliferation, invasion and immigration, and response to chemotherapy were studied. 6. The Affymetrix gene chips (Human Genome U133 plus 2.0 Array) were used for mRNA expression profiling of the colorectal strains with NNMT transferred or not.7. Immunohistological stain was used to detect NNMT in cancer tissue, and the relationship between NNMT and clinical pathological parameter of patients with colorectal cancer was evaluatedResults:1. NNMT cDNA was cloned from normal human liver tissue, and was inserted into protocaryon and eukaryotic expression plasmids, which named p GEX-4T-2/ NNMT and pcDNA3.1/NNMT respectively.2. Four strains of hybridoma cells secreting anti-human NNMT McAbs were obtained. McAbs secreted by all the strains have high specificity and reactivity. Western-blot and Immunohistological stain to detect NNMT based on the prepared monoclonal antibodies were set up.3. NNMT expression of colorectal cancer cells such as HT-29. SW480,COLO 320,SW620,HCT116 and HCE8693 were detected by RT-PCR and Western-blot. The COLO320 expressed the highest NNMT for both mRNA and protein level while the HCE8693, HT-29,,SW620 and HCT116 shoewd decreased in level respectively, with SW480 showing the lowest.4. NNMT highly expression cancer cell models, SW480 -pcDNA3.1/NNMT and COLO320-pcDNA3.1/NNMT, were constructed. These cell models were confirmed expressing highly NNMT mRNA and protein level by RT-PCR and Western-blot. NNMT mRNA and protein level can be detected in SW480-pcDNA3.1/NNMT while negative in wild or control SW480.There was a 1.62 fold increase of NNMT protein in COLO320-pcDNA3.1/NNMT when comparing with wild or control SW480.5. The results of cell growth curve showed:Both SW480-pcDNA3.1/NNMT and COLO320-pcDNA3.1/NNMT cell grow or proliferate faster than the control cells, the significance can be detected since the point of 72 hour of culture; and the difference of growth speed can be inhibited by RNAi of NNMT.6.The influnce of NNMTon response to chemotherapy showed that SW480-pcDNA3.1/NNMT was more resistant to 5-Fu when comparing with control cells, the IC50 of SW480-pcDNA3.1/NNMT cell was 16.8 mM; 8.8 mM and 9.2 mM for SW480-pcDNA3.1and SW480 respectively. However, no signifant change was detected when analyzing with Oxaliplatin.7. Matrigel and Transwelltest showed only SW480-pcDNA3.1/NNMT can go through the gel and membrance with 15-30 cell/low power microscope field.8.There was no significant change of cell cycle transition (G1,G2 and S phase) among SW480, SW480-pcDNA3.1/NNMT and SW480-pcDNA3.1 were detected.The same results were obstained in COLO320, COLO320-pcDNA3.1 and COLO320-pcDNA3.1/NNMT cell.9. Flow Cytometer(FCM)was used to analyse cell apoptosis.After treating with 5-FU for 24 hours,the cell percentage of LR in SW480-pcDNA3.1/NNMT was 9.12%, which was lower than 15.66% in SW480-pcDNA3.1 and 15.85% in SW480. Thus anti-apoptosis of NNMT can be drawn in SW480-pcDNA3.1/NNMT model. The same change trend of cell percentage of LR observed in COLO320-pcDNA3.1/NNMT model.10. There were 33 genes 2-fold upregulated or downregulated when comparing SW480-pcDNA3.1/NNMT with SW480-pcDNA3.1.The changed genes were related to metabolism,cell cycle, proliferation, apoptosis,signal transduction, and cell differentiation.The most of the upregulated genes were related to cholesterol synthesis and oxidoreduction.11. The diagnostic and prognostic function of NNMT protein in colorectal cancer was evaluated by analyzing 109 colorectal cancer tissues and 26 lymphnode through immunohistochemical staining for NNMT by using 2F8 antibodies.18 cases (16.5%) were positive, and the positive NNMT was mostly related to the poorly-differentaited adenocarcinoma; 12 (36.4%) NNMT positivity was significantly higher in poorly-differentaited adenocarcinoma cells when compared with the highly or moderately-differentaited adenocarcinoma, (P<0.01). No correlations or associations between NNMT and clinico-pathological parameters such as age, gender, tumor location,TNM abd Dukes grade were detected,(P>0.05).Conclusion:1. Through this study, we successfully obtained a couple of important tools for studying NNMT function. The tools were protocaryon and eukaryotic expression plasmids of p GEX-4T-2/NNMT and pcDNA3.1/NNMT; hybridoma cells secreting anti-human NNMT McAbs with high specificity and reactivity; Western-blot and Immunohistological stain based on the prepared antibodies, NNMT highly expression cancer cell models, SW480-pcDNA3.1/NNMT and COLO320-pcDNA3.1/NNMT; GST-NNMT and NNMT protein.2. NNMT expression was different in the tested colorectal cancer cells. The COLO320 expressed the highest NNMT for both in mRNA and protein level while SW480 showed the lowest. In cancer tissue, positive NNMT were mostly related to the poorly-differentiated adenocarcinoma when compared with the highly or moderately-differentiated adenocarcinoma. However, there were no correlations or associations between NNMT and clinico-pathological parametersdrawn out on age, gender, tumor location, TNM and Dukes grade.3. NNMT may be closely related to the biological function of tumour:it may improve cell growth or proliferation rate; influence the results of chemotherapy; enhance the ability for invasion and immigration; and play a role as anti-apoptosis.4. A total of 33 genes related to metabolism,cell cycle,proliferation, apoptosis, signal transduction, and cell differentiation were found upregulated or downre-gulated.The most of the upregulated genes were related to cholesterol synthesis and oxidoreduction.Those changed gene related mechanisms may explain the influence of NNMT on tumor cells, however, more work needs to be done to prove it. |
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