title: [Plasmid-mediated RNAi targeting hTERT inhibits telomerase activity in K562 cell line].
abstract: This study was aimed to investigate the effect of plasmid-mediated RNAi targeting hTERT on blocking hTERT gene and inhibiting telomerase activity in leukemia cell line K562. For inhibiting hTERT mRNA, three siRNA strands were chemosynthesized and transfected into K562 cells, two effective and specific siRNA strands were chosen. Then plasmid pSUPER-U6-Kan rhTERT-1, pSUPER-U6-Kan rhTERT-2 targeting hTERT mRNA were constructed and transfected into K562 cells by liposome. The expression of hTERT mRNA, telomerase activity and cell apoptosis were detected at 48 hours and 72 hours. The results showed that three chemosynthesized strands began to significantly inhibit target gene expression at 48 hours, but the inhibiting rates were different. The inhibiting effect disappeared after 72 hours. After plasmid pSUPER-U6-Kan rhTERT -1 (P-1 group) and pSUPER-U6-Kan rhTERT -2 (P-2 group) were transfected into K562 cells, the expressions of hTERT mRNA both decreased in the two groups. The expression of hTERT mRNA in P-1 group was 0.39+/-0.13 at 48 hours, 0.57+/-0.32 at 72 hours. The expression of hTERT mRNA in P-2 group was 0.55+/-0.20 at 48 hours, 0.88+/-0.23 at 72 hours. Telomerase activity in P-1 group was 0.42+/-0.07 at 48 hours, 0.31+/-0.08 at 72 hours; the telomerase activity in group P-2 was 0.49+/-0.27 at 48 hours, 0.39+/-0.03 at 72 hours while telomerase activities in both groups were significantly lower than that in negative control group (0.88+/-0.30, 0.88+/-0.32). At 48 hours, the apoptosis rates in P-1 group (18.39+/-3.08%) and P-2 group (15.5+/-3.59%) were significantly higher than that in negative control group (7.64+/-3.73%). At 72 hours the apoptosis rate in P-1 group (13.2+/-1.18%) and in P-2 group (12.86+/-3.09%) had no significant difference as compared with negative control group (8.07+/-0.19%). It is concluded that RNAi targeting hTERT inhibits the expression of hTERT mRNA, and therefore inhibits telomerase activity. The inhibiting effect is closely correlated with target site. The si-hTERT-1 effect is better than si-hTERT-2, while si-hTERT-3 almost has no effect at all. The effective time of plasmid-induced RNAi is obviously longer than that of chemosynthesized siRNA. The former is longer than 72 hours, while the latter is only 48 hours. After the telomerase activity is inhibited, cell apoptosis increases a little than control group at 48 hours. At 72 hours cell apoptosis has no difference with control group. It is supposed that some cells can be induced to differentiation when telomerase activity has been down regulated.
authors.affiliation: Department of Hematology, The First Affiliated Hospital, Dalian Medical University, Dalian 116011, Liaoning Province, China.
authors.forename: Mei-Feng
authors.initials: MF
authors.lastname: Li
authors.forename: Mei-Yun
authors.initials: MY
authors.lastname: Fang
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
mesh_terms: D017209:Apoptosis / Q000235:genetics*; D015536:Down-Regulation; D006801:Humans; D020014:K562 Cells; D010957:Plasmids / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; C509186:TERT protein, human; D019098:Telomerase