Effects of miR?144 on proliferation, apoptosis and cisplatin resistance by targeting MYCN in pediatric neuroblastoma
10.3760／cma.j.issn.0253?3766.2019.07.006
- VernacularTitle:miR-144通过靶向MYCN对小儿神经母细胞瘤增殖凋亡及顺铂耐药的影响机制
- Author:
Huijuan LIU
1
;
Guiling LI
;
Pingchong LEI
Author Information
1. 河南省人民医院儿科
- Keywords:
MiR?144;
MYCN;
Pediatric neuroblastoma;
Proliferation;
Apoptosis;
Resistance
- From:
Chinese Journal of Oncology
2019;41(7):516-521
- CountryChina
- Language:Chinese
-
Abstract:
To investigate the effects and mechanisms of miR?144 on proliferation, apoptosis and cisplatin ( DDP ) resistance of neuroblastoma cells. Methods Real?time fluorescence quantitative PCR ( RT?qPCR ) was used to detect the mRNA expressions of miR?144 and MYCN in neuroblastoma cell lines, including SH?SY5Y and SK?N?SH, and human umbilical vein endothelial cells HUVEC. The miR?negative control, miR?144 mimics, si?negative control, si?MYCN, miR?144 mimics and pcDNA, miR?144 mimics and pcDNA?MYCN co?transfected SH?SY5Y cells were described as miR?NC, miR?144, si?NC, si?MYCN, miR?144+pcDNA and miR?144+pcDNA?MYCN group, respectively. The half maximal inhibitory concentration ( IC50 ) and cell proliferation were detected by 3?( 4, 5?dimethyl?2?thiazolyl)?2,5?diphenyl?2H tetrazolium bromide ( MTT) assay. The protein expressions of MYCN, p21, cyclin D1, Bax, Bcl?2 were analyzed by western blot.Cell apoptosis was detected by flow cytometry.The cell fluorescence activity was detected by double luciferase reporter gene assay.Results Compared with HUVEC cells, the expressions of miR?144 in neuroblastoma cells SH?SY5Y and SK?N?SH significantly decreased, while the mRNA and protein expression of MYCN significantly increased. The IC50 of DDP was 9.16 μg／ml in SH?SY5Y cells. The absorbance value in 490nm ( A490 value) of miR?144 group was 0.30 ± 0.03, significantly lower than 0.46±0.03 of miR?NC group. The cell apoptotic rate of miR?144 group was 26.94%± 2.01%, significantly higher than 9.68%±0.52% of miR?NC group. The IC50 value of DDP in miR?144 group was 2.95±0.26, significantly lower than 9.23±0.61 of miR?NC group. The expressions of p21, cyclin D1, Bax, Bcl?2 in miR?NC and miR?144 group were 2.67±0.19, 0.41±0.04, 2.12±0.21, 0.18±0.01 and 1.01± 0.07, 1.00 ± 0.06, 1.00 ± 0.05, 1.00 ± 0.06, respectively, with statistical significance ( all P＜0.05). Knockdown of MYCN showed the similar effects with those of miR?144 overexpression in SH?SYSY cells. MiR?144 significantly inhibited the fluorescence activity of ectopic MYCN expressing cells and negatively regulated the expression of MYCN. Overexpression of MYCN can reverse the effects of miR?144 on proliferation inhibition, apoptosis promotion and sensitization of SH?SY5Y cells to DDP. Conclusion MiR?144 inhibits proliferation, promotes apoptosis and enhances the sensitivity of neuroblastoma cells to DDP through targeting MYCN, which provides a potential treatment for neuroblastoma.
