[Short hairpin RNA silences Pin1 and affects proliferation and apoptosis in HeLa cell line].
OBJECTIVE
To study the effect of short hairpin RNA (shRNA) on the expression of Pin1 mRNA and protein and its influence on the proliferation and apoptosis in HeLa cell lines.


METHODS
The recombinant plasmid pSIREN-Pin1 expressing Pin1-targeted shRNA was constructed and then transfected into cervical cancer cell lines by lipofectamine (HeLa/p-shRNA), the control vector pSIREN-Con (HeLa/p-Con) and culture media (HeLa) as control, respectively. The Pin1 mRNA and protein expression were detected by RT-PCR and immunoblotting. The proliferation of cells after transfection was detected by methyl thiazolyl tetrazolium (MTT) and colony formation in soft agar. Flow cytometry (FCM) was used to test the apoptosis of cells marked with fluorescein isothiocyanate (FITC)-annexin V.


RESULTS
After transfected with pSIREN-Pin1 for 48 h, the expression of Pin1 mRNA and protein in HeLa cells were 0.19 +/- 0.05 and 0.33 +/- 0.14 respectively. Compared with HeLa/p-Con cells (0.84 +/- 0.16, 0.79 +/- 0.17) and HeLa cells (0.89 +/- 0.11, 0.81 +/- 0.15), the difference was significant respectively (P < 0.05). The proliferation was suppressed significantly in HeLa/p-shRNA cells. The colony ratios are as follows: HeLa/p-shRNA, (12 +/- 3)%; HeLa/p-Con, (20 +/- 5)%; HeLa, (24 +/- 4)% (P < 0.05). The apoptosis ratio was (24.3 +/- 5.7)% in HeLa/p-shRNA cells. It was significantly higher than that in HeLa/p-Con cells (5.0 +/- 1.4)% and HeLa cells (1.8 +/- 0.4)% (P < 0.05).


CONCLUSIONS
RNA interference through Pin1-targeted shRNA can effectively inhibit the expression of target gene, and might be potentially useful in gene therapy of Pin1 related cervical cancers. Silencing Pin1 gene can suppress the proliferation and promote the apoptosis in HeLa cells.
Li
Hong-yu
HY
Molecular Cancer Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhu
Tao
T
Zhou
Jin-hua
JH
Xu
Qian
Q
Wang
Shi-xuan
SX
Bai
Xiang-yang
XY
Lu
Yun-ping
YP
Ma
Ding
D
D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D049109:Cell Proliferation; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000737:chemistry; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D019696:Peptidylprolyl Isomerase / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods
D008055:Lipids; C086724:Lipofectamine; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C000606846:PIN1 protein, human; D019696:Peptidylprolyl Isomerase