[Effects of dnmt1 gene silencing on cell cycle, proliferation, and apoptosis of gastric cancer cell line AGS].
OBJECTIVE
Dnmt1, a major DNA methyltransferase gene, is highly expressed in many cancers and lowly expressed in normal adult cells, therefore, its overexpression is closely related to tumorigenesis. This study was to assess effects of dnmt1 gene silencing on cell cycle, proliferation, and apoptosis of gastric cancer cell line AGS.


METHODS
The eukaryotic expression plasmid pshRNA-dnmt1, containing the sequence of short hairpin RNA (shRNA) targeting dnmt1, was constructed and transfected into AGS cells. PBS-treated cells and pTZU6+1-transfected cells were set as control. The expression levels of dnmt1 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Cell cycle was analyzed by flow cytometry; cell survival was analyzed by MTT assay; cell apoptosis was evaluated by AO/EB double staining, electron microscopy, and TUNEL.


RESULTS
pshRNA-dnmt1 targeting dnmt1 was successfully constructed, and confirmed by sequencing. Relative to control, 24, 48, and 72 h after transfection of pshRNA-dnmt1, the inhibitory rates of dnmt1 protein levels in AGS cells were 28.24%, 68.54%, and 81.47%, respectively, and those of dnmt1 mRNA levels were 21.63%, 52.97%, and 72.06%, respectively. The growth of AGS cells was suppressed 72 h after transfection: S phase cells were reduced from (36.58+/-1.76)% to (18.54+/-6.59)% (P<0.05), and G(2)/M phase cells were increased from (6.18+/-0.32)% to (18.53+/-1.42)% (P<0.05). Cell survival rates were 79.49%, 51.63%, and 39.16%, respectively, 24, 48, and 72 h after transfection of pshRNA-dnmt1. A lot of apoptotic and necrosis cells were observed after transfection.


CONCLUSIONS
The recombinant plasmid pshRNA-dnmt1 can efficiently and specifically inhibit the expression of dnmt1 gene and the proliferation of AGS cells, and induce cell apoptosis. It provides evidence for gene therapy of human cancers.
Chen
Dao-Rong
DR
Department of Gastroenterology, The First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400016, P. R. China. cdrcdrcn@yahoo.com.cn
Wang
Pi-Long
PL
Huang
Ai-Long
AL
Zhang
Bing-Qiang
BQ
D017209:Apoptosis; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000096:biosynthesis / Q000235:genetics*; D020868:Gene Silencing; D006801:Humans; D010957:Plasmids; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013274:Stomach Neoplasms / Q000378:metabolism / Q000473:pathology*; D014162:Transfection
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human