Influence of I2PP2A gene silencing by RNA interference on proliferation and apoptosis of human acute promyelocytic leukemia cell line NB4-R1.
- VernacularTitle:RNA干扰I2PP2A基因表达对NB4-R1细胞增殖及凋亡的影响
- Author:
Yanfeng LIU
1
;
Pengcheng HE
1
;
Feng LIU
1
;
Xiaoyan CHENG
1
;
Mei ZHANG
1
Author Information
- Publication Type:Journal Article
- MeSH: Apoptosis; genetics; Caspase 8; metabolism; Cell Line, Tumor; Cell Proliferation; genetics; Drug Resistance, Neoplasm; Histone Chaperones; genetics; metabolism; Humans; Leukemia, Promyelocytic, Acute; metabolism; pathology; RNA Interference; Transcription Factors; genetics; metabolism; Tretinoin; pharmacology
- From: Chinese Journal of Hematology 2014;35(8):732-736
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo explore the effect of RNA interference of human I2PP2A gene on the proliferation and apoptosis of retinoic acid-resistant human acute promyelocytic leukemia (APL) cell line NB4-R1.
METHODSDesigned and constructed a RNA interference lentiviral vector I2PP2A-shRNA which targeted against I2PP2A gene, then transfected it into NB4-R1 via polybrene mediation. The I2PP2A expression levels before and after transfection were detected by qRT-PCR and Western blot, respectively. Meanwhile, the proliferation and apoptosis rates of each group were determined by CCK-8 and flow cytometry assay. The protein expressions of caspase-8 and PARP were detected by Western blot.
RESULTSBoth qRT-PCR and Western blot data showed the I2PP2A expression level was significantly downregulated in the transfection group. The I2PP2A mRNA expression level decreased by (70.0 ± 9.6)% and (64.0 ± 6.2)% respectively, compared with blank control and negative control group, and the I2PP2A protein expression level showed a consistent trend. CCK-8 proliferation assay indicated the NB4-R1 cell proliferation rate in I2PP2A-shRNA transfection group significantly reduced compared to blank control group (P<0.05). Flow cytometry results showed that NB4-R1 apoptosis rate in I2PP2A-shRNA transfection group increased by (6.30 ± 0.67) times and (6.04 ± 0.56) times, respectively (P<0.01). After inhibition of I2PP2A, the total caspase-8 and total PARP expressions decreased by (44.0 ± 3.1)% and (57.0 ± 4.0)%, respectively; Meanwhile, the cleaved caspase-8 (p43) and cleaved PARP (p89) increased by (36.0 ± 2.5)% and (45.0 ± 4.8)%, respectively compared with blank control group (P<0.05).
CONCLUSIONI2PP2A gene silenced by RNA interference could inhibit the proliferation and promote the apoptosis of NB4-R1, which may be regulated through caspase-8-induced exogenous apoptosis pathway.