Impacts of lncRNA MALAT1 on apoptosis, autophagy of granulosa cells and PI3K/Akt/mTOR pathway in polycystic ovary syndrome

doi:10.11958/20240332

Abstract

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

CLC Number:

R711.75

Figures/Tables 7

References 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^＊＊