基于PI3K/Akt/mTOR信号通路探讨LINC00173对多囊卵巢综合征颗粒细胞自噬的影响

doi:10.11958/20240511

天津医药 ›› 2024, Vol. 52 ›› Issue (11): 1121-1126.doi: 10.11958/20240511

• 细胞与分子生物学 • 下一篇

基于PI3K/Akt/mTOR信号通路探讨LINC00173对多囊卵巢综合征颗粒细胞自噬的影响

赵元元(), 吴小华^△()

石家庄市第四医院（河北医科大学附属妇产医院）生殖中心，河北省母胎医学重点实验室，河北省生殖医学重点学科，石家庄市生殖健康与不孕不育研究所（邮编050011）

收稿日期:2024-04-28 修回日期:2024-06-21 出版日期:2024-11-15 发布日期:2024-11-12
通讯作者: △E-mail：wuxiaohua1965@163.com
作者简介:赵元元（1988），女，主管检验师，主要从事生殖内分泌方面研究。E-mail：summerzhaoyy@163.com
基金资助:
国家自然科学基金青年科学基金项目(82301850);河北省自然科学基金青年科学基金项目(H2022106020)

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

ZHAO Yuanyuan(), WU Xiaohua^△()

Center for Reproductive Medicine, the Fourth Hospital of Shijiazhuang (Gynecology and Obstetrics Hospital Affiliated to Hebei Medical University); Key Laboratory of Maternal and Fetal Medicine of Hebei Province; Provincial Key Medical Discipline of Hebei Province; the Institute of Reproductive Health and Infertility, Shijiazhuang 050011, China

Received:2024-04-28 Revised:2024-06-21 Published:2024-11-15 Online:2024-11-12
Contact: △E-mail：wuxiaohua1965@163.com

赵元元, 吴小华. 基于PI3K/Akt/mTOR信号通路探讨LINC00173对多囊卵巢综合征颗粒细胞自噬的影响[J]. 天津医药, 2024, 52(11): 1121-1126.

ZHAO Yuanyuan, WU Xiaohua. The effect of LINC00173 regulating autophagy of PCOS granulosa cells based on PI3K/Akt/mTOR signaling pathway[J]. Tianjin Medical Journal, 2024, 52(11): 1121-1126.

摘要/Abstract

摘要：

目的探讨长链非编码RNA 00173（LINC00173）对多囊卵巢综合征（PCOS）颗粒细胞（GCs）自噬的影响及其机制。方法选取行体外受精-胚胎移植（IVF-ET）的PCOS患者（PCOS组）和因输卵管因素行助孕治疗的非PCOS患者（对照组）各40例，采用实时荧光定量PCR（qPCR）法检测LINC00173在PCOS GCs中的表达。另择人卵巢颗粒细胞KGN为实验对象，根据是否过表达LINC00173或敲低LINC00173将KGN细胞分为Vector组和pcLINC00173组，siNC组和siLINC00173组。单丹磺酰尸胺（MDC）染色检测过表达LINC00173对KGN细胞自噬的影响，Western blot法检测KGN细胞微管相关蛋白1轻链3B（LC3B）、p62及磷脂酰肌醇3-激酶（PI3K）/丝氨酸/苏氨酸蛋白激酶B（Akt）/雷帕霉素靶蛋白（mTOR）信号通路相关蛋白磷酸化PI3K（p-PI3K）、磷酸化Akt（p-Akt）、磷酸化mTOR（p-mTOR）、PI3K、Akt及mTOR表达的影响。另设siNC组、siLINC00173组、siLINC00173+DMSO组和siLINC00173+LY294002组，检测LY294002对LC3B和p62蛋白表达的影响。结果 PCOS组LINC00173表达水平高于对照组（P<0.05）。LINC00173过表达后KGN细胞自噬体含量增加。与Vector组比较，pcLINC00173组自噬相关蛋白LC3B-Ⅱ/Ⅰ表达上调，p62表达下调，PI3K/Akt/mTOR信号通路相关蛋白p-PI3K/PI3K、p-Akt/Akt和p-mTOR/mTOR表达下调。与siNC组比较，siLINC00173组上述蛋白表达变化相反。siLINC00173+LY294002组LC3B-Ⅱ/Ⅰ蛋白表达水平高于siLINC00173组和siLINC00173+DMSO组，p62蛋白表达水平低于siLINC00173组和siLINC00173+DMSO组（P<0.05）。结论 LINC00173可诱导PCOS GCs自噬，其机制可能与抑制PI3K/Akt/mTOR信号通路有关。

关键词: 多囊卵巢综合征, RNA, 长链非编码, 自噬, LINC00173, 颗粒细胞

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

中图分类号:

R711.75

图/表 11

表1 siRNA的核苷酸序列

Tab.1 The sequences for siRNAs

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

表2 qRT-PCR引物

Tab.2 Primers for qRT-PCR

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

图1 GSE95728数据库分析PCOS患者和非PCOS患者差异表达的RNAs

Fig.1 GSE95728 database analysis of differentially expressed RNAs in PCOS patients and non PCOS patients

图2 qPCR检测KGN细胞中LINC00173过表达或干扰效率

Fig.2 qPCR analysis of the expression of LINC00173 after overexpression or interference efficiency in KGN cells

图3 2组KGN细胞MDC染色结果比较（×200）

Fig.3 Comparison of MDC staining between the two groups of KGN cells (×200)

图4 Western blot检测细胞LC3B-Ⅱ/Ⅰ和p62蛋白的表达

Fig.4 Western blot analysis of expression levels of LC3B-Ⅱ/Ⅰ and p62 between the two groups

表3 各组KGN细胞自噬相关蛋白的表达水平比较（n=3 $\bar{x} \pm s$）

Tab.3 Comparison of relative expression levels of autophagy related proteins in KGN cells between four groups

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^＊

表4 各组KGN细胞PI3K/Akt/mTOR信号通路蛋白表达水平比较（n=3，$\bar{x} \pm s$）

Tab.4 Comparison of relative expression levels of PI3K/Akt/mTOR signaling in KGN cells between the four groups

组别	p-PI3K/PI3K	p-Akt/Akt	p-mTOR/mTOR
Vector组	0.69±0.06	0.77±0.10	1.63±0.19
pcLINC00173组	0.30±0.01	0.11±0.01	0.85±0.08
t	11.105^＊	11.375^＊	6.553^＊
siNC组	0.46±0.16	1.47±0.16	0.15±0.04
siLINC00173组	1.43±0.08	2.73±0.14	0.82±0.03
t	9.392^＊	10.265^＊	23.210^＊

图5 Western blot检测各组KGN细胞p-PI3K、PI3K、p-Akt、Akt、p-mTOR、mTOR蛋白表达水平

Fig.5 Western blot analysis of p-PI3K, PI3K, p-Akt, Akt, p-mTOR and mTOR protein expression in KGN cells of each group

图6 Western blot检测各组细胞LC3B-Ⅱ/Ⅰ和p62蛋白的表达 A：siNC组；B：siLINC00173组；C：siLINC00173+DMSO组；D：siLINC00173+LY294002组。

Fig.6 Western blot analysis of expression levels of LC3B-Ⅱ/Ⅰ and p62 of each group

表5 各组KGN细胞自噬相关蛋白表达水平的比较（n=3，$\bar{x} \pm s$）

Tab.5 Comparison of relative expression levels of autophagy related proteins in KGN cells of each group

组别	LC3B-Ⅱ/Ⅰ	p62
siNC组	2.64±0.73	0.39±0.03
siLINC00173组	1.36±0.24^a	0.82±0.24^a
siLINC00173+DMSO组	0.92±0.12	0.81±0.20
siLINC00173+LY294002组	1.87±0.47^bc	0.40±0.14^bc
F	7.927^＊	5.978^＊

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基于PI3K/Akt/mTOR信号通路探讨LINC00173对多囊卵巢综合征颗粒细胞自噬的影响

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

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