[Effect of DNA methyltransferase 1 gene silencing on proliferation and apoptosis of HeLa cells].
OBJECTIVE
To observe the effect of DNA methyltransferase 1 (DNMT1) gene silencing by RNA interfering technology on the proliferation and apoptosis of HeLa cells.


METHODS
Recombinant plasmid pshRNA-DNMT1-A, B and C were respectively transfected into HeLa cells by lipofectamine 2000, while cells transfected plasmid vector pSilencer3. 1-H1 and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and western blotting was used to detected the mRNA and protein expression of DNMT1 in HeLa cells transfected for 24, 48 and 72 hours. Cell counting kit-8 (CCK-8) assay was used to investigate the proliferation of the HeLa cells after transfection, while apoptosis was detected by flowcytometry (FCM) method.


RESULTS
Three DNMT1-targeted short hairpin RNA (shRNA) A,B and C were successfully inserted into the plasmid vector pshRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragments. The results indicated that both recombinant plasmid pshRNA-DNMT1-A and B could effectively knock down the expression of DNMT1 gene in human cervical cancer cells, of which pshRNA-DNMT1-B was the better choice. While no effect of pshRNA-DNMT1-C was seen. RT-PCR results showed that the relative mRNA expression of DNMT1 gene in HeLa cells transfected with pshRNA-DNMT1 for 24, 48 and 72 hours were 0.406 +/- 0.057, 0.191 +/- 0.036 and 0. 104 +/- 0.015, which were significantly lower than that in HeLa cells transfected by empty vector and non-transfected cells (0.520 +/- 0.020, 0.537 +/- 0.041, respectively, P < 0. 05). The western blotting analysis manifested that the relative expression of DNMT1 protein of HeLa cells transfected by pshRNA-DNMT1 for 24, 48 and 72 hours were 0.197 +/- 0.024, 0.075 +/- 0.015, 0.040 +/- 0. 013, which were significantly lower than that in transfected cells by empty vector and non-transfected cells (0.273 +/- 0.010, 0.283 +/- 0.016, respectively, P < 0.05). The CCK-8 results showed that the cell survival rates of HeLa cells transfected by pshRNA-DNMT1 for 24, 48, 72, 96 and 120 hours were 70.8%, 64.8%, 51.6%, 45.3% and 38.0%, there were statistically different compared with cells transfected by empty vector and non-transfected cells at different time-points (P < 0.01). The results of FCM indicated that the apoptosis rate of HeLa cells transfected with pshRNA-DNMT1 for 24, 48 and 72 hours were (17.7 +/- 1.3)%, (35.3 +/- 1.3)%, (47.6 +/- 1.6)%, which were significantly higher than empty vector transfected cells and non-transfected cells [(4.9 +/- 0.5)%, (5.1 +/- 0.7)%, respectively, P < 0.05].


CONCLUSIONS
DNMT1 can be successfully silenced by RNA interfering in cervical HeLa cells. Downregulation of DNMT1 can inhibit cervical cancer cells proliferation and induce cell apoptosis.
Zhao
Xian-lan
XL
Department of Obstetrics and Gynecology, The First Affiliated Hospital, Zhengzhou University, Zhengzhou, China.
Rao
Yan-ling
YL
Qiao
Yu-huan
YH
Zhang
Hui-li
HL
D017209:Apoptosis; D049109:Cell Proliferation; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human