The effect and mechanism of miR-15b gene interference on cerebral ischemia-reperfusion injury

doi:10.11958/20220433

Abstract

Abstract:

Objective To investigate the effect of microRNA-15b (miR-15b) gene interference on cerebral ischemia-reperfusion injury and its mechanism. Methods Astrocytes from cerebral cortex of newborn Wistar rats within 24 hours of birth were cultured and identified. Oxygen glucose deprivation/recovery method was used to treat the simulated cerebral ischemia-reperfusion injury in vivo, which was recorded as the model group. miR-15b interfering adenovirus vector and negative control vector were constructed and transfected into cells of the above model group respectively, which were recorded as the overexpression group, the silence group, the overexpression control group and the silence control group. Astrocytes from the cerebral cortex without special treatment were used as the blank control group. After 24 hours, the morphological changes of cells were observed by inverted phase contrast microscope in each group. The cell viability was detected by MTT assay, and the cell viability was detected by lactate dehydrogenase (LDH) leakage rate. The apoptosis rate was detected by flow cytometry. Real-time quantitative polymerase chain reaction was used to detect the expression levels of miR-15b, blymphoma-2 (Bcl-2), cysteine protease-3 (Caspase-3) and cysteine protease-9 (Caspase-9) messenger RNA (mRNA). The expressions of Bcl-2, Caspase-3 and Caspase-9 protein levels were detected by Western blot assay. Luciferase reporter gene experiment verified that miR-15b could target and regulate Bcl-2. Results Compared with the control group, the adherent cells decreased in the blank group, the overexpression control group and the silence control group, cells shrunk and became round, and the distribution was loose, cell viability and Bcl-2 mRNA and protein expression decreased, cell viability and apoptosis rate, and the expression levels of miR-15b, Caspase-3, Caspase-9 mRNA and protein increased (P<0.05). Compared with the blank group and the silencing control group, the number of adherent cells in the overexpression group decreased and was more sparsely distributed. It could be seen that cells floated in the culture medium, the cell survival rate and the expression of Bcl-2 mRNA and protein in the overexpression group decreased, the cell viability and apoptosis rate, and the expression of miR-15b, the expressions of Caspase-3, Caspase-9 mRNA and protein increased (P<0.05). Compared with the blank group and the silenced control group, the adherent cells in the silenced group increased, but still lower than the control group. The cells shrank slightly, the cell survival rate and the expression of Bcl-2 mRNA and protein increased in the silencing group, the cell viability and apoptosis rate, and the expression of miR-15b, the expressions of Caspase-3, Caspase-9 mRNA and protein decreased (P<0.05). Results of luciferase reporter gene experiments confirmed that miR-15b can target and regulate Bcl-2. Conclusion Overexpression of miR-15b can aggravate cerebral ischemia-reperfusion injury, presumably through the mitochondrial pathway to induce the deformation and apoptosis of damaged nerve cells.

Key words: reperfusion injury, hypoxia-ischemia, brain, neurons, RNAi therapeutics, microRNA-15b, B-lymphoma-2, Caspase-3, Caspase-9

CLC Number:

R329.21

Figures/Tables 8

References 18

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组别	存活率	细胞活力	凋亡率
对照组	95.62±6.39	1.85±0.33	4.55±0.81
空白组	29.51±5.84^a	52.27±9.02^a	40.26±7.04^a
过表达对照组	28.94±5.73^a	49.58±9.23^a	42.55±7.30^a
沉默对照组	26.45±6.86^a	51.14±9.55^a	41.93±7.55^a
过表达组	9.12±1.02^abc	77.25±11.78^abc	64.54±10.06^abc
沉默组	55.23±8.04^abd	27.36±5.15^abd	21.18±2.03^abd
F	76.586^＊＊	28.127^＊＊	28.700^＊＊

组别	存活率	细胞活力	凋亡率
对照组	95.62±6.39	1.85±0.33	4.55±0.81
空白组	29.51±5.84^a	52.27±9.02^a	40.26±7.04^a
过表达对照组	28.94±5.73^a	49.58±9.23^a	42.55±7.30^a
沉默对照组	26.45±6.86^a	51.14±9.55^a	41.93±7.55^a
过表达组	9.12±1.02^abc	77.25±11.78^abc	64.54±10.06^abc
沉默组	55.23±8.04^abd	27.36±5.15^abd	21.18±2.03^abd
F	76.586^＊＊	28.127^＊＊	28.700^＊＊

组别	miR-15b	Bcl-2	Caspase-3	Caspase-9
对照组	1.03±0.18	1.01±0.20	0.98±0.17	1.00±0.19
空白组	1.78±0.31^a	0.52±0.09^a	2.05±0.38^a	1.62±0.28^a
过表达对照组	1.85±0.34^a	0.50±0.09^a	2.01±0.35^a	1.59±0.26^a
沉默对照组	1.82±0.33^a	0.47±0.08^a	1.99±0.36^a	1.65±0.30^a
过表达组	2.98±0.52^abc	0.18±0.03^abc	3.26±0.57^abc	2.74±0.51^abc
沉默组	1.11±0.19^bd	0.87±0.16^abd	1.24±0.21^bd	1.07±0.16^bd
F	16.952^＊＊	30.145^＊＊	21.377^＊＊	14.108^＊＊

组别	miR-15b	Bcl-2	Caspase-3	Caspase-9
对照组	1.03±0.18	1.01±0.20	0.98±0.17	1.00±0.19
空白组	1.78±0.31^a	0.52±0.09^a	2.05±0.38^a	1.62±0.28^a
过表达对照组	1.85±0.34^a	0.50±0.09^a	2.01±0.35^a	1.59±0.26^a
沉默对照组	1.82±0.33^a	0.47±0.08^a	1.99±0.36^a	1.65±0.30^a
过表达组	2.98±0.52^abc	0.18±0.03^abc	3.26±0.57^abc	2.74±0.51^abc
沉默组	1.11±0.19^bd	0.87±0.16^abd	1.24±0.21^bd	1.07±0.16^bd
F	16.952^＊＊	30.145^＊＊	21.377^＊＊	14.108^＊＊

组别	Bcl-2	Caspase-3	Caspase-9
对照组	1.71±0.27	0.52±0.08	0.48±0.07
空白组	0.79±0.12^a	1.51±0.25^a	1.15±0.21^a
过表达对照组	0.76±0.12^a	1.55±0.28^a	1.22±0.22^a
沉默对照组	0.80±0.13^a	1.46±0.24^a	1.06±0.18^a
过表达组	0.30±0.08^abc	2.31±0.37^abc	1.85±0.31^abc
沉默组	1.26±0.18^abd	0.78±0.14^bd	0.51±0.12^bd
F	26.973^＊＊	20.052^＊＊	19.406^＊＊