LncRNA MALAT1对PCOS颗粒细胞凋亡、自噬和PI3K/Akt/mTOR通路的影响

doi:10.11958/20240332

摘要/Abstract

摘要：

目的探讨长链非编码RNA-肺腺癌转移相关转录子1（LncRNA MALAT1）对多囊卵巢综合征（PCOS）颗粒细胞的凋亡、自噬和磷脂酰肌醇-3-激酶（PI3K）/蛋白激酶B（Akt）/哺乳动物雷帕霉素靶蛋白（mTOR）通路的影响。方法 qRT-PCR检测PCOS卵巢组织、正常卵巢组织、人卵巢颗粒细胞KGN及正常卵巢上皮细胞IOSE80中LncRNA MALAT1的表达水平。体外培养KGN细胞，将KGN细胞分为control组、si-NC组、si-MALAT1组、pc-NC组、pc-MALAT1组、pc-MALAT1+740Y-P（PI3K激活剂）组。qRT-PCR检测各转染组细胞MALAT1 mRNA的表达水平；CCK-8检测细胞活力；流式细胞术检测细胞凋亡率；透射电镜观察KGN细胞中自噬小体；Western blot检测细胞B细胞淋巴瘤2（Bcl-2）、Bcl-2关联X蛋白（Bax）、微管相关蛋白Ⅱ轻链3（LC3Ⅱ）、微管相关蛋白Ⅰ轻链3（LC3Ⅰ）、Beclin1、p-PI3K、PI3K、p-Akt、Akt、p-mTOR、mTOR蛋白表达水平。结果 PCOS卵巢组织MALAT1 mRNA表达水平低于正常卵巢组织，KGN细胞中MALAT1 mRNA表达水平低于正常卵巢上皮IOSE80细胞；control组KGN细胞中可见少量的自噬小体；干扰MALAT1表达后KGN细胞OD₄₅₀值（24、48、72 h）、Bcl-2蛋白表达降低，细胞凋亡率、Bax、LC3Ⅱ/LC3Ⅰ、Beclin1、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR增高（P<0.05），自噬小体数量增多；过表达MALAT1后上述指标变化均逆转（P<0.05）；与pc-MALAT1组相比，pc-MALAT1+740Y-P组OD₄₅₀（24、48、72 h）值、Bcl-2蛋白表达降低，凋亡率、Bax、LC3Ⅱ/LC3Ⅰ、Beclin1、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR升高（P<0.05），自噬小体数量增多。结论过表达MALAT1可能通过抑制PI3K/Akt/mTOR信号通路，进而抑制PCOS颗粒细胞凋亡和自噬。

关键词: 多囊卵巢综合征, RNA，长链非编码, 粒层细胞, 细胞凋亡, 自噬, LncRNA MALAT1, PI3K/Akt/mTOR通路

Abstract:

Objective To investigate the impacts of long non-coding RNA-lung adenocarcinoma metastasis-associated transcripton-1 (lncRNA MALAT1) on apoptosis, autophagy and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/mammalian rapasin target protein (mTOR) pathway in granulosa cells of polycystic ovary syndrome (PCOS). Methods The expression levels of lncRNA MALAT1 in PCOS ovarian tissue, normal ovarian tissue, human ovarian granulosa cells (KGN) and normal ovarian epithelial cells IOSE80 were detected by qRT-PCR. KGN cells were cultured in vitro and grouped into the control group, the si-NC group, the si-MALAT1 group, the pc-NC group, the pc-MALAT1 group and the pc-MALAT1+740Y-P (PI3K activator) group. qRT-PCR was applied to detect the expression level of MALAT1 mRNA in cells of each transfection group. CCK-8 was applied to detect cell viability. Flow cytometry was applied to detect cell apoptosis rate. Transmission electron microscopy was applied to observe autophagosomes in KGN cells. Western blot assay was applied to detect expression levels of B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), microtubule-associated protein Ⅱ light chain 3 (LC3 Ⅱ), microtubule-associated protein Ⅰ light chain 3 (LC3Ⅰ), Beclin1, p-PI3K, PI3K, p-Akt, Akt, p-mTOR and mTOR proteins in cells. Results The expression level of MALAT1 mRNA in PCOS ovarian tissue was lower than that in normal ovarian tissue, and that in KGN cells was lower than that in normal ovarian epithelial IOSE80 cells. A small number of autophagosomes were observed in KGN cells in the control group. After interfering MALAT1 expression, OD₄₅₀ values (24, 48 and 72 hours) and the Bcl-2 protein expression of KGN cells were reduced, and the apoptosis rate, the expression of Bax, LC3 Ⅱ/LC3 Ⅰ, Beclin1, p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR proteins were increased (P<0.05). The number of autophagosomes increased. Changes of the above indexes were reversed after overexpression of MALAT1 (P<0.05). Compared with the pc-MALAT1 group, the OD₄₅₀ values (24, 48, 72 h), and the expression of Bcl-2 protein were obviously reduced in the pc-MALAT1+740Y-P group, while the apoptosis rate, number of autophagosomes, the expression of Bax, LC3 Ⅱ/LC3 Ⅰ, Beclin1, p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR proteins were obviously increased (P<0.05). The number of autophagosomes increased. Conclusion Overexpression of MALAT1 may inhibit apoptosis and autophagy in PCOS granulosa cells by inhibiting the PI3K/Akt/mTOR signaling pathway.

Key words: polycystic ovary syndrome, RNA, long noncoding, granulosa cells, apoptosis, autophagy, LncRNA MALAT1, PI3K/Akt/mTOR pathway

中图分类号:

R711.75

图/表 7

参考文献 15

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组别	OD₄₅₀
组别	24 h		48 h		72 h
control组	0.31±0.03		0.47±0.05		0.72±0.08
si-NC组	0.34±0.03		0.44±0.04		0.68±0.06
si-MALAT1组	0.18±0.02^ab		0.31±0.02^ab		0.45±0.05^ab
pc-NC组	0.29±0.03		0.48±0.04		0.67±0.07
pc-MALAT1组	0.45±0.04^c		0.66±0.07^c		0.91±0.08^c
pc-MALAT1+740Y-P组	0.26±0.03^d		0.38±0.04^d		0.62±0.06^d
F	51.621^＊＊		39.848^＊＊		29.155^＊＊
组别		MALAT1 mRNA		凋亡率/%
control组		1.07±0.09		16.41±1.76
si-NC组		1.04±0.08		18.24±2.35
si-MALAT1组		0.43±0.04^ab		40.67±3.58^ab
pc-NC组		1.12±0.10		16.84±1.62
pc-MALAT1组		1.86±0.23^c		8.27±0.84^c
pc-MALAT1+740Y-P组		1.82±0.21		21.52±1.83^d
F		85.308^＊＊		151.729^＊＊

组别	OD₄₅₀
组别	24 h		48 h		72 h
control组	0.31±0.03		0.47±0.05		0.72±0.08
si-NC组	0.34±0.03		0.44±0.04		0.68±0.06
si-MALAT1组	0.18±0.02^ab		0.31±0.02^ab		0.45±0.05^ab
pc-NC组	0.29±0.03		0.48±0.04		0.67±0.07
pc-MALAT1组	0.45±0.04^c		0.66±0.07^c		0.91±0.08^c
pc-MALAT1+740Y-P组	0.26±0.03^d		0.38±0.04^d		0.62±0.06^d
F	51.621^＊＊		39.848^＊＊		29.155^＊＊
组别		MALAT1 mRNA		凋亡率/%
control组		1.07±0.09		16.41±1.76
si-NC组		1.04±0.08		18.24±2.35
si-MALAT1组		0.43±0.04^ab		40.67±3.58^ab
pc-NC组		1.12±0.10		16.84±1.62
pc-MALAT1组		1.86±0.23^c		8.27±0.84^c
pc-MALAT1+740Y-P组		1.82±0.21		21.52±1.83^d
F		85.308^＊＊		151.729^＊＊

组别	Bcl-2	Bax	LC3Ⅱ/ LC3Ⅰ	Beclin1
control组	0.67±0.06	0.32±0.02	0.56±0.04	0.47±0.05
si-NC组	0.65±0.07	0.29±0.02	0.58±0.03	0.43±0.05
si-MALAT1组	0.31±0.04^ab	0.74±0.07^ab	1.17±0.13^ab	0.86±0.07^ab
pc-NC组	0.64±0.06	0.33±0.03	0.59±0.03	0.48±0.05
pc-MALAT1组	0.92±0.08^c	0.14±0.02^c	0.27±0.03^c	0.24±0.02^c
pc-MALAT1+ 740Y-P组	0.57±0.06^d	0.38±0.03^d	0.69±0.06^d	0.57±0.06^d
F	58.370^＊＊	182.582^＊＊	125.632^＊＊	91.266^＊＊

组别	Bcl-2	Bax	LC3Ⅱ/ LC3Ⅰ	Beclin1
control组	0.67±0.06	0.32±0.02	0.56±0.04	0.47±0.05
si-NC组	0.65±0.07	0.29±0.02	0.58±0.03	0.43±0.05
si-MALAT1组	0.31±0.04^ab	0.74±0.07^ab	1.17±0.13^ab	0.86±0.07^ab
pc-NC组	0.64±0.06	0.33±0.03	0.59±0.03	0.48±0.05
pc-MALAT1组	0.92±0.08^c	0.14±0.02^c	0.27±0.03^c	0.24±0.02^c
pc-MALAT1+ 740Y-P组	0.57±0.06^d	0.38±0.03^d	0.69±0.06^d	0.57±0.06^d
F	58.370^＊＊	182.582^＊＊	125.632^＊＊	91.266^＊＊

组别	p-PI3K/PI3K	p-Akt/Akt	p-mTOR/mTOR
control组	0.71±0.07	1.14±0.11	0.94±0.08
si-NC组	0.74±0.06	1.12±0.09	0.98±0.07
si-MALAT1组	1.18±0.12^ab	1.67±0.28^ab	1.47±0.16^ab
pc-NC组	0.76±0.07	1.09±0.10	1.01±0.10
pc-MALAT1组	0.35±0.04^c	0.72±0.06^c	0.68±0.06^c
pc-MALAT1+ 740Y-P组	0.82±0.07^d	1.26±0.15^d	1.07±0.08^d
F	73.680^＊＊	25.140^＊＊	41.574^＊＊