[Inhibition of malignant phenotype of HeLa cells by RNA interference of the telomerase activity].
OBJECTIVE
To investigate the biology of HeLa cells upon inhibition of human telomerase catalytic subunit (hTERT) gene by RNA interference in vitro.


METHODS
Four shRNAs (A, B, C and D) targeting hTERT gene were designed and prepared by in-vitro transcription. The expression of hTERT gene was evaluated by immunofluorescent staining and telomeric repeat amplification protocol (TRAP) ELISA (TRAP-ELISA), after transient transfection of shRNAs by lipid formulation. Through the initial selection, shRNA (B) was noticed as the most efficient one in down-regulating hTERT gene and therefore was chosen as the ultimate shRNA used in the experimental groups. Those transfected by non-silencing RNAi were chosen as the control groups. Cell spreading and migration were studied by microscopy and cell adhesion to fibronectin (FN) was assayed by cell counting kit-8 (CCK-8). Cell invasion was assessed by Boyden chamber assay.


RESULTS
Cell spreading study revealed that the rates of spreading cells in the experimental groups were (5.6 +/- 2.3)% at 30 min, and (26.3 +/- 6.1)% 2 h after the inoculation, respectively, whereas the rates of spreading cells in the control groups were (31.3 +/- 7.9)% and (79.4 +/- 4.8)%, respectively. There were significant differences between the two groups (P < 0.01). However, most of the cells in both groups became spreading after 24 h. Cell adhesion assay demonstrated that the rate of adhesion cells on FN in experimental groups was (67.2 +/- 2.8)%, less than that in control groups (83.7 +/- 5.4)% (P < 0.05). The relative migration distance was (27.1 +/- 6.2)% in the experimental group, lower than that of the control group (58.7 +/- 15.0)%. The invasion assay revealed that the invading cells were 75.7 +/- 14.5 in the experimental group, in contrast to 165.1 +/- 11.0 in the control group after 4 h incubation on matrigel. The difference between these two groups was significant (P < 0.05).


CONCLUSIONS
In vitro shRNA silencing of hTERT gene can down-regulate the telomerase activity, leading to an inhibition of the malignant phenotype of HeLa cells, including decreased ability of cell spreading and adhesion, reduction of cell migration, and declined invasive ability through Matrige assay.
Shi
Ying-ai
YA
The Key Laboratory of Patho-biology, Ministry of Education, Jilin University, Changchun 130021, China.
Zhao
Qiang
Q
Zhang
Li-hong
LH
Wang
Guang-lan
GL
Yu
Xiao-xia
XX
Li
Yu-lin
YL
Wu
Shan
S
D002448:Cell Adhesion; D002465:Cell Movement; D049109:Cell Proliferation; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D009361:Neoplasm Invasiveness; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology*; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D034741:RNA, Small Interfering; D019098:Telomerase