Micro RNA -433 inhibits proliferation and invasion of glioma cells by targeting histone deacetylase 6
10.3760/cma.j.issn.1671-8925.2017.12.012
- VernacularTitle:微小RNA-433调控组蛋白去乙酰化酶6抑制胶质瘤增殖与侵袭
- Author:
Xiangsheng LI
1
;
Yanjuan MA
;
Lei HUI
;
Ruihua LIU
;
Shupeng ZHAO
;
Baozhe JIN
Author Information
1. 新乡医学院第一附属医院神经外科
- Keywords:
Micro RNA-433;
Histone deacetylase 6;
Glioma;
Proliferation;
Invasion
- From:
Chinese Journal of Neuromedicine
2017;16(12):1247-1254
- CountryChina
- Language:Chinese
-
Abstract:
Objective To detect the expression of micro RNA (miR)-433 and histone deacetylase 6 (HDAC6) in glioma tissues and investigate the effect of miR-433 on cell proliferation and invasion of human glioma cell line U251. Methods (1) Forty-two glioma samples, collected from patients accepted surgical resection and conformed by pathology in our hospital from January 2010 to December 2014, and 13 healthy brain tissues, collected from patients accepted surgery for craniocerebral trauma at the same time period, were used in our study; reverse transcription (RT)-PCR was used to detect the mRNA expression levels of miRNA-433 and HDAC6 in the glioma samples and brain tissues. (2) Human glioma cell line was routinely cultured and divided into blank control group, nonsense sequence control group and miRNA-433 mimics group;cells in the later two groups were transfected with nonsense sequences or miRNA-433 mimics, and cells in the blank control group did not give any treatment;the mRNA expression levels of miRNA-433, P21 and HDA C6 in these 3 groups were detected by RT-PCR;the cellular viability was measured by CCK-8 assay;flow cytometry was used to monitor the changes of cell cycle and apoptosis; cell invasion was evaluated by Transwell assay; HDAC6 protein expression was detected by Western blotting. (3) Wide-type (WT)HDAC63'-UTR and mutant type (MUT)HDAC63'-UTR luciferase report vectors were established; miR-433 mimics+WT HDAC63′-UTR and nonsense sequences+WT HDAC63'-UTR were transfected into the U251 cells, and dual-luciferase experiment was used to detect the fluorescence intensity of the cells; miR-433 mimics+MUT HDAC63'-UTR and nonsense sequences+MUT HDAC63'-UTR were transfected into the U251 cells, and dual-luciferase experiment was used to detect the fluorescence intensity of the cells. (4) U251 cells were divided into nonsense sequence control group, HDAC6 expression plasmids group and HDAC6 siRNA group, and nonsense sequences, HDAC6 expression plasmids or HDAC6 siRNA were transfected respectively; RT-PCR was used to detect the P21 and HDAC6 mRNA expressions and miRNA-433 expression; U251 cells were divided into miR-433 mimics group and miR-433 mimics+HDAC6 expression plasmids group, and miR-433 mimics or miR-433 mimics+HDAC6 expression plasmids were transfected, respectively, and one-5 d after that, CCK-8 was used to detect the cellular viability. Results (1) The miRNA-433 expressions gradually increased and HDA C6 mRNA expressions gradually decreased in the high-grade gliomas, low-grade gliomas and normal brain tissues, and significant differences were noted among each two groups (P<0.05);the miRNA-433 expression was negatively correlated with HDA C6 mRNA expression in the glioma tissues (r=0.829, P=0.000). (2) As compared with blank control group and nonsense sequence control group, miRNA-433 mimics group had significantly higher miRNA-433 and P21 mRNA expressions, cell percentage at G0/G1 stage, and apoptotic rate (P<0.05), and had statistically lower HDAC6 mRNA expression, cellular viability on 2-5 d of culture, number of transmembrane cells and HDAC6 protein expression (P<0.05). (3) The luciferase activity in cells from miR-433 mimics+WT HDAC63'-UTR group was significantly lower as compared with that in the nonsense sequences+WT HDAC63'-UTR group (P<0.05);the luciferase activity in cells from miR-433 mimics+MUT HDAC63'-UTR group and nonsense sequences+MUT HDAC63'-UTR group showed no significant differences (P>0.05). (4) The HDA C6 mRNA expressions were gradually increased, and P21 mRNA expressions were gradually decreased in the HDAC6 siRNA group, nonsense sequence control group, and HDAC6 expression plasmids group, with significant differences (P<0.05);on 2-5 d of culture, the cellular viability in the miR-433 mimics+HDAC6 expression plasmids group was significantly higher than that in the miR-433 mimics group (P<0.05). Conclusions The miRNA-433 expression level is low in human glioma tissues;miRNA-433 over-expression may inhibit the cell activity and promote cell apoptosis of glioma cell line U251 in vitro via inhibiting the HDAC6 expression.
