The effect of LINC00173 regulating autophagy of PCOS granulosa cells based on PI3K/Akt/mTOR signaling pathway

doi:10.11958/20240511

Abstract

Abstract:

Objective To investigate the effect and mechanism of long non-coding RNA 00173 (LINC00173) on autophagy of granulosa cells (GCs) in polycystic ovary syndrome (PCOS). Methods A total of 40 PCOS patients and 40 patients (non-PCOS) treated with tubal factors who underwent in vitro fertilization-embryo transfer (IVF-ET) were selected. The expression levels of LINC00173 in GCs of PCOS were detected by qPCR. Human ovarian granulosa cells KGN were selected as the experimental subject. The cells were divided into the Vector group, the pcLINC00173 group, the siNC group and the siLINC00173 group based on their over-expression or interference with LINC00173. Dansylcadaverine (MDC) staining was employed to assess the impact of LINC00173 on autophagy of KGN cells. Western blot assay was conducted to investigate the effect of LINC00173 on autophagy and the PI3K/Akt/mTOR signaling pathway in KGN cells, focusing on expression levels of key pathway-related proteins including LC3B-Ⅱ/Ⅰ, p62, p-PI3K, p-Akt, p-mTOR, PI3K, Akt and mTOR. The effects of LY294002 on the expression of LC3B-Ⅱ/Ⅰ and p62 proteins were detected in the siNC group, the siLINC00173 group, the siLINC00173+DMSO group and the siLINC00173+LY294002 group. Results LINC00173 was significantly upregulated in GCs of PCOS patients (P<0.05). The overexpression of LINC00173 in KGN cells resulted in the presence of autophagosomes and autophagolysosomes in cytoplasm (P<0.05). Compared with the vector group, there was an increased expression of autophagy-related proteins LC3B-Ⅱ/Ⅰ and a decreased expression in p62 in the pcLINC00173 group (P<0.05). Additionally, there was a decreased expression of PI3K/Akt/mTOR signaling pathway-related proteins p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR. Conversely, in the siLINC00173 group, the expression of LC3B-Ⅱ/Ⅰ decreased while p62 expression increased compared to the siNC group (P<0.05), along with alterations in PI3K/Akt/mTOR signaling pathway-related proteins. Compared to the siLINC00173+DMSO group or the siLINC00173 group, PI3K/Akt/mTOR signaling pathway inhibitor LY294002 was found to alleviate the inhibitory effect of siLINC00173 on LC3B-Ⅱ/Ⅰ protein expression in KGN cells in the siLINC00173 group (P<0.05) and to reduce the impact of siLINC00173 on up-regulation of p62 protein expression (P<0.05). Conclusion Results reveal thatLINC00173 can induce autophagy activation in PCOS GCs, and the mechanism of the action may be related to the inhibition of PI3K/Akt/mTOR signaling pathway.

Key words: polycystic ovary syndrome, RNA, long noncoding, autophagy, LINC00173, granulosa cells

CLC Number:

R711.75

Figures/Tables 11

References 14

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基因名称	核苷酸序列（5'→3'）
siNC	上游：UUCUCCGAACGUGUCACGUTT
siNC	下游：ACGUGACACGUUCGGAGAATT
siLINC00173	上游：GGAACGUUCAGCGGAAUAUTT
siLINC00173	上游：AUAUUCCGCUGAACGUUCCTT

基因名称	核苷酸序列（5'→3'）
siNC	上游：UUCUCCGAACGUGUCACGUTT
siNC	下游：ACGUGACACGUUCGGAGAATT
siLINC00173	上游：GGAACGUUCAGCGGAAUAUTT
siLINC00173	上游：AUAUUCCGCUGAACGUUCCTT

基因名称	引物序列（5′→3′）	产物大小/bp
LINC00173	上游：GCATCCAGCTACCCAGACTC	133
LINC00173	下游：CCTGCAGCACGCAATTAGAC	133
β-actin	上游：CAGAGCAAGAGAGGCATCC	217
β-actin	上游：CTGGGGTGTTGAAGGTCTC	217

基因名称	引物序列（5′→3′）	产物大小/bp
LINC00173	上游：GCATCCAGCTACCCAGACTC	133
LINC00173	下游：CCTGCAGCACGCAATTAGAC	133
β-actin	上游：CAGAGCAAGAGAGGCATCC	217
β-actin	上游：CTGGGGTGTTGAAGGTCTC	217

组别	LC3B-Ⅱ/Ⅰ	p62
Vector组	0.89±0.03	1.33±0.12
pcLINC00173组	1.34±0.03	0.29±0.08
t	18.371^＊	12.490^＊
siNC组	1.99±0.34	0.25±0.05
siLINC00173组	0.35±0.02	1.09±0.06
t	8.340^＊	18.628^＊