[Effect of eukaryotic expression plasmid DNA methyltransferase gene on methylation status and transcription level of DNA mismatch repair genes in human colon cancer cell line].
OBJECTIVE
To analyze the effect of eukaryotic plasmids containing sense or antisense DNA methyltransferase (Dnmt1) genes on the methylation status and transcription level of DNA mismatch repair (MMR) genes and microsatellite instability (MSI) in human colon cancer cell line.


METHODS
Human colorectal cells of the SW1116 line were cultured. Recombinant plasmids containing sense Dnmt1 (HMT) or antisense Dnmt1 (THM) gene, pCMV-HMT and pCMV-THM, were constructed. Then pCMV-HMT, pCMV-THM, and pcMV blank plasmid were transfected into SW1116 cells respectively by using lipofectAMINE. The expression of Dnmt1 protein was examined by Western blotting. The transcription levels of hMLH1 and hMSH2 genes were detected by using real-time (RT-PCR). The status of methylation in promoters of hMLH1 and hMSH2 genes were examined with methylation specific PCR (MSP). The MSI of DNA in SW1116 cells was evaluated by silver-stained polyacrylamide gel electrophoresis.


RESULTS
Both the expressions of the hMLH1 and hMSH2 gene mRNAs were remarkably decreased in the SW1116-HMT cells in comparison with those in the untransfected cells. The expression of hMSH2 gene mRNA in the SW1116-THM cells was remarkably increased in comparison with that in the untransfected cells. No significant difference in the expressions of the hMLH1 and hMSH2 gene mRNAs was found between the SW1116 cells transfected with blank pCMV and the untransfected SW1116 cells. MSP showed that the methylation level in the regions of hMLH1 and hMSH2 promoters was remarkably increased in the SW1116 cells transfected with sense Dnmtl plasmid. However, in the SW1116 cells the hMSH2 promoter region was changed from partially-methylated into de-methylated, and the hMLH1 promoter region remained non-methylated. MST test showed that extra bands indicating MSI were seen only in the D2S123 groups.


CONCLUSIONS
Dnmt1 regulates the expression and methylation status of MMR genes and affects MSI in human colon cancer cell line SW1116.
Shanghai Institute of Digestive Diseases, Renji Hospital, Shanghai Second Medical University, Shanghai 200001, China.
Rong
R
Lu
Jing-yuan
JY
Fang
Hong-yin
HY
Zhu
Ying-xuan
YX
Chen
Zhong-hua
ZH
Cheng
En-ling
EL
Li
D048868:Adaptor Proteins, Signal Transducing; D020137:Base Pair Mismatch; D002352:Carrier Proteins; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D019175:DNA Methylation; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism; D006801:Humans; D018895:Microsatellite Repeats; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics*; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D011484:Protein-Arginine N-Methyltransferases / Q000096:biosynthesis / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011484:Protein-Arginine N-Methyltransferases; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein