心肌梗死相关转录本对视网膜母细胞瘤影响机制的研究

蒋瑜; 孙先桃; 孙爽; 卢跃兵

doi:10.16766/j.cnki.issn.1674-4152.002670

心肌梗死相关转录本对视网膜母细胞瘤影响机制的研究

doi: 10.16766/j.cnki.issn.1674-4152.002670

郑州大学附属儿童医院河南省儿童医院郑州儿童医院眼科，河南郑州 450000

基金项目:

河南省医学科技攻关计划联合共建项目 LHGJ20190952

详细信息

通讯作者:
蒋瑜, E-mail: haiting2322@163.com

中图分类号: R739.72 R730.231
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- 被引次数: 0
出版历程
- 收稿日期: 2021-11-15
- 网络出版日期: 2022-11-30

Effect of myocardial infarction-associated transcripts on retinoblastoma

Department of Ophthalmology, Children' s Hospital of Zhengzhou University, Henan Children' s Hospital, Zhengzhou Children' s Hospital, Zhengzhou, Henan 450000, China

摘要

摘要: 目的探讨心肌梗死相关转录本(MIAT)靶向miR-145对视网膜母细胞瘤增殖和凋亡的影响。方法选取2015年9月—2020年9月在郑州大学附属儿童医院因视网膜母细胞瘤行眼球摘除患儿30例，收集患儿肿瘤组织及癌旁组织。培养人视网膜母细胞瘤细胞Y79，按照随机数字表法分为si-NC组、si-MIAT组、miR-NC组、miR-145组、si-MIAT+anti-miR-NC组、si-MIAT+anti-miR-145组。采用RT-qPCR检测细胞MIAT和miR-145的表达；CCK-8检测细胞增殖；流式细胞术检测细胞凋亡；Western blotting检测CyclinD1、p21、Bcl-2、Bax、p-PI3K、p-Akt蛋白表达；双荧光素酶报告检测MIAT与miR-145的靶向关系。结果与癌旁组织比较，视网膜母细胞瘤组织中MIAT表达显著上调(3.61±0.28 vs. 1.05±0.11)，miR-145表达显著下调(0.33±0.03 vs. 1.03±0.09，均P＜0.05)。与si-NC组比较，si-MIAT组细胞活力显著下调；细胞凋亡率显著上调；p-PI3K蛋白、p-Akt蛋白表达显著下调(均P＜0.05)。与miR-NC组比较，miR-145组细胞活力显著下调；细胞凋亡率显著上调(均P＜0.05)。与si-MIAT+anti-miR-NC组比较，si-MIAT+anti-miR-145组细胞活力显著上调；细胞凋亡率显著下调；p-PI3K蛋白、p-Akt蛋白表达显著上调(均P＜0.05)。结论抑制MIAT表达、上调miR-145表达可促进视网膜母细胞瘤凋亡，抑制增殖，其机制与PI3K/Akt通路有关。
- 长链非编码RNAMIAT /
- 微小RNA-145 /
- 磷脂酰肌醇-3激酶/蛋白激酶B信号通路 /
- 视网膜母细胞瘤细胞 /
- 增殖 /
- 凋亡
Abstract: Objective To explores the effect of lncRNA MIAT targeting miR-145 on the proliferation and apoptosis of retinoblastoma. Methods From September 2015 to September 2020, 30 children with retinoblastoma underwent enucleation in Children ' s Hospital of Zhengzhou University were selected, and tumor tissues and adjacent tissues were collected. Human retinoblastoma cells (Y79) were cultured and divided into si-NC, si-MIAT, miR-NC, miR-145, si-MIAT + anti-miR-NC and si-MIAT + anti-miR-145 groups according to the random number table method. RT-qPCR was used in detecting the expression levels of MIAT and miR-145 in the cells. CCK-8 was used in detecting cell proliferation, and flow cytometry was used in detecting cell apoptosis. Cyclin D1, p21, Bcl-2, Bax, p-PI3K and p-Akt protein expression levels were detected with Western blotting, and dual luciferase reporter was used in detecting the targeted relationship between MIAT and miR-145. Results Compared with adjacent tissues, the expression of MIAT in retinoblastoma tissue was significantly up-regulated, and the expression of miR-145 was significantly down-regulated (3.61±0.28 vs. 1.05±0.11, 0.33±0.03 vs. 1.03±0.09, all P < 0.05). Compared with the si-NC group, cell viability in the si-MIAT group was significantly down-regulated, mortality rate was significantly up-regulated, and p-PI3K protein and p-Akt protein expression were significantly down-regulated (all P < 0.05). Compared with the miR-NC group, cell viability in the miR-145 group showed significantly down-regulated, apoptosis rate was significantly up-regulated (all P < 0.05). Compared with the si-MIAT + anti-miR-NC group, cell viability was significantly up-regulated in the si-MIAT + anti-miR-145 group, cell apoptosis rate was significantly down-regulated, whereas the expression levels of p-PI3K and p-Akt proteins were significantly up-regulated (all P < 0.05). Conclusion The inhibition of MIAT expression and up-regulation of miR-145 expression can promote apoptosis and inhibit the proliferation of retinoblastoma. The mechanism is related to the PI3K/Akt pathway.
- Long non-coding RNA MIAT /
- MicroRNA-145 /
- Phosphatidylinositol-3 kinase/protein kinase B signalling pathway /
- Retinoblastoma cells /
- Proliferation /
- Apoptosis

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图 1 干扰MIAT表达对视网膜母细胞瘤细胞增殖和凋亡的影响

注：A为Western blotting检测增殖和凋亡相关蛋白表达；B为细胞凋亡图。

Figure 1. Effect of interfering MIAT expression on proliferation and apoptosis of retinoblastoma cells

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图 2 Western blotting检测增殖和凋亡相关蛋白表达

Figure 2. The expressions of proliferation and apoptosis related proteins detected by Western blotting

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图 3 MIAT与miR-145结合位点

Figure 3. binding site between MIAT and miR-145

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图 4 Western blotting检测增殖和凋亡相关蛋白表达

Figure 4. The expressions of proliferation and apoptosis related proteins detected by Western blotting

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图 5 PI3K/Akt信号通路相关蛋白表达图

Figure 5. Expression map of PI3K/Akt signaling pathway related proteins

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表 1 视网膜母细胞瘤组织与正常视网膜组织MIAT和miR-145表达量比较(x±s，n_i=30)

Table 1. Comparison of MIAT and Mir-145 expression in retinoblastoma tissues and normal retinal tissues(x±s, n_i=30)

组织来源	MIAT	miR-145
癌旁组织	1.05±0.11	1.03±0.09
视网膜母细胞瘤组织	3.61±0.28	0.33±0.03
t值	46.610	40.415
P值	< 0.001	< 0.001

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表 2 干扰MIAT表达对视网膜母细胞瘤细胞增殖和凋亡的影响(x±s，n_i=9)

Table 2. Effect of interfering MIAT expression on proliferation and apoptosis of retinoblastoma cells (x±s, n_i=9)

组别	MIAT	增殖(OD₄₉₀)			CyclinD1蛋白	p21蛋白	凋亡率(%)	Bcl-2蛋白	Bax蛋白
组别	MIAT	24 h	48 h	72 h	CyclinD1蛋白	p21蛋白	凋亡率(%)	Bcl-2蛋白	Bax蛋白
si-NC	1.00±0.10	0.66±0.06	0.76±0.07	0.85±0.08	0.85±0.08	0.22±0.02	8.64±0.55	0.89±0.09	0.29±0.02
si-MIAT	0.35±0.03	0.21±0.02	0.32±0.03	0.44±0.04	0.30±0.03	0.86±0.08	42.35±3.68	0.34±0.04	0.87±0.08
t值	18.678	21.345	17.332	13.752	19.312	23.283	27.179	16.753	21.101
P值	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

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表 3 miR-145过表达对视网膜母细胞瘤细胞增殖和凋亡的影响(x ±s，n_i=9)

Table 3. Effect of overexpression of miR-145 on proliferation and apoptosis of retinoblastoma cells (x ±s, n_i=9)

组别	miR-145	增殖(OD₄₉₀)			CyclinD1蛋白	p21蛋白	凋亡率(%)	Bcl-2蛋白	Bax蛋白
组别	miR-145	24 h	48 h	72 h	CyclinD1蛋白	p21蛋白	凋亡率(%)	Bcl-2蛋白	Bax蛋白
miR-NC	1.03±0.08	0.64±0.06	0.79±0.08	0.88±0.08	0.86±0.08	0.32±0.03	9.62±0.57	0.91±0.09	0.28±0.02
miR-145	3.49±0.31	0.20±0.02	0.36±0.04	0.45±0.05	0.30±0.03	0.90±0.09	45.38±4.36	0.37±0.03	0.89±0.09
t值	23.051	20.871	14.423	12.856	19.663	18.341	24.398	17.076	19.849
P值	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

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表 4 双荧光素酶报告实验(x±s，n_i=9)

Table 4. Double luciferase reporter assay(x±s, n_i=9)

组别	WT-MIAT	MUT-MIAT
miR-NC	1.01±0.09	1.00±0.09
miR-145	0.36±0.04	1.01±0.10
t值	19.799	< 0.001
P值	< 0.001	0.999

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表 5 抑制miR-145表达逆转了干扰MIAT对视网膜母细胞瘤细胞增殖和凋亡的影响(x±s，n_i=9)

Table 5. The expressions of proliferation and apoptosis related proteins detected by Western blotting(x±s, n_i=9)

组别	增殖(OD₄₉₀)			CyclinD1蛋白	p21蛋白	凋亡率(%)	Bcl-2蛋白	Bax蛋白
组别	24 h	48 h	72 h	CyclinD1蛋白	p21蛋白	凋亡率(%)	Bcl-2蛋白	Bax蛋白
si-MIAT+anti-miR-NC	0.29±0.02	0.41±0.04	0.55±0.05	0.25±0.02	0.84±0.08	48.09±5.29	0.26±0.02	0.92±0.09
si-MIAT+anti-miR-145	0.58±0.05	0.72±0.07	0.89±0.09	0.88±0.08	0.24±0.02	9.68±0.92	0.83±0.08	0.36±0.04
t值	16.155	11.535	9.907	22.920	21.828	21.460	20.737	17.058
P值	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

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表 6 PI3K/Akt信号通路相关蛋白表达(x±s，n_i=9)

Table 6. Expression of PI3K/Akt signaling pathway related proteins (x±s, n_i=9)

组别	p-PI3K蛋白	p-Akt蛋白
si-NC	0.90±0.10	0.89±0.09
si-MIAT	0.32±0.03^a	0.30±0.03^a
si-MIAT+anti-miR-NC	0.31±0.03	0.29±0.03
si-MIAT+anti-miR-145	0.88±0.08^b	0.91±0.09^b
F值	218.159	244.183
P值	< 0.001	< 0.001
注：与si-NC组比较，^aP＜0.05；与si-MIAT+anti-miR-NC组比较，^bP＜0.05。

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