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HJBM

Hans Journal of Biomedicine

2161-8976

Scientific Research Publishing

10.12677/hjbm.2024.143041

HJBM-88942

hjbm2024143_31530343.pdf

医药卫生

miRNA-494调控PI3K/AKT/mTOR通路促进滋养细胞增殖和迁移 MiRNA-494 Regulates the PI3K/AKT/mTOR Pathway to Promote Trophoblast Proliferation and Migration

刘

玉兰

² ¹ 何

凤屏

³ ¹ 郭

义红

³ ¹ 马

秋林

³ ¹ 张

咏梅

³ ¹ 陈

陆静

³ ¹ 刘

珮瑜

³ ¹ 范

舒舒

² ¹ 郭

艳乐

² ¹

东莞市妇幼保健院，生殖遗传研究所，广东东莞

汕头大学医学院附属粤北人民医院，产前诊断中心，广东汕头

null

12 06 2024

14 03 369 378

2014

This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

目的：探讨不明原因复发性流产(unexplained recurrent spontaneous abortion, uRSA)小鼠模型的微小RNA (miRNA)-494、PI3K/AKT/mTOR [哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)]的表达和促进胎盘滋养细胞增殖和迁移以及调节子宫和胎盘的炎症反应和免疫功能的作用机制。方法：首先构建了miRNA-494的过表达载体，实验分成miRNA-494模拟物组、模拟物对照组、miRNA-494抑制剂组、抑制剂对照组，采用脂质体2000分别转染miRNA-494模拟物(50 nmol/L)、miRNA-494抑制剂(50 nmol/L)，使用实时荧光定量PCR 技术检测miRNA-494模拟物组、模拟物对照组、miRNA-494抑制剂组、抑制剂对照组的表达水平；采用克隆形成实验检测各组胎盘滋养细胞的增殖能力，transwell法检测各组胎盘滋养层细胞的迁移程度，western blot检测各组胎盘滋养层细胞PI3K、AKT、mTOR的表达水平。结果：miRNA-494转染48 h后，miRNA-494模拟物组滋养细胞的miRNA-494表达水平明显高于模拟物对照组、抑制剂对照组和miRNA-494抑制剂组(P < 0.001)；反之miRNA-494抑制剂组滋养细胞的miRNA-494表达水平明显低于对照组和miRNA-494模拟物组(P < 0.001)；miRNA-494抑制剂组的PI3K/AKT和mTOR表达水平明显低于抑制剂对照组、模拟物对照组和miRNA-494模拟物组(P < 0.001)；miRNA-494抑制剂组的滋养细胞增殖和迁移比例显著低于抑制剂对照组、模拟物对照组和miRNA-494模拟物组(P < 0.01)。结论：miRNA-494通过调节Akt/PI3K/mTOR基因的表达，可以促进胎盘滋养细胞增殖和迁移，并参与调节子宫和胎盘的炎症反应和免疫功能，表明了调节miRNA-494的表达水平可能作为uRSA的预防和治疗的新靶点。 Objective: To investigate the expression of miRNA-494, PI3K/AKT/mTOR [mammalian target of rapamycin, mTOR] in a mouse model of unexplained recurrent spontaneous abortion (uRSA), and their mechanisms in promoting placental trophoblast proliferation and migration, as well as regulating inflammation and immune function in the uterus and placenta. Method: Firstly, an overexpression vector of miRNA-494 was constructed, and the experiment was divided into miRNA-494 mimic group, mimic control group, miRNA-494 inhibitor group, and inhibitor control group. Liposome 2000 was used to transfect miRNA-494 mimic group (50 nmol/L) and miRNA-494 inhibitor group (50 nmol/L), respectively. Real time fluorescence quantitative PCR technology was used to detect miRNA-494 mimic group, mimetic control group, and miRNA-494 inhibitor group The expression level of the inhibitor control group; The proliferation ability of placental trophoblast cells in each group was detected using clone formation experiments, the migration degree of placental trophoblast cells in each group was measured using Transwell method, and the expression levels of PI3K, AKT, and mTOR in placental trophoblast cells in each group were detected using Western blot. Result: After 48 hours of miRNA-494 transfection, the expression level of miRNA-494 in the trophoblast cells of the miRNA-494 mimic group was significantly higher than that of the mimic control group, inhibitor control group, and miRNA-494 inhibitor group (P < 0.001); On the contrary, the expression level of miRNA-494 in trophoblast cells in the miRNA-494 inhibitor group was significantly lower than that in the control group and miRNA-494 mimic group (P < 0.001); The expression levels of PI3K/AKT and mTOR in the miRNA-494 inhibitor group were significantly lower than those in the inhibitor control group, mimic control group, and miRNA-494 mimic group (P < 0.001); The proportion of trophoblast proliferation and migration in the miRNA-494 inhibitor group was significantly lower than that in the inhibitor control group, mimic control group, and miRNA-494 mimic group (P < 0.01). Conclusion: miRNA-494 can promote the proliferation and migration of placental trophoblasts by regulating the expression of Akt/PI3K/ mTOR genes, and participate in regulating the inflammatory response and immune function of the uterus and placenta, indicating that regulating the expression level of miRNA-494 may serve as a new target for the prevention and treatment of uRSA.

miRNA-494，PI3K/AKT/mTOR，滋养细胞，不明原因复发性流产, miRNA-494 PI3K/AKT/mTOR Trophoblast Cell Unexplained Recurrent Spontaneous Abortion

摘要

关键词

miRNA-494，PI3K/AKT/mTOR，滋养细胞，不明原因复发性流产

MiRNA-494 Regulates the PI3K/AKT/mTOR Pathway to Promote Trophoblast Proliferation and Migration<sup> </sup>

Yulan Liu¹, Fengping He^2*, Qiulin Ma², Yongmei Zhang², Hong Zeng², Lujing Chen², Peiyu Liu², Yihong Guo², Shushu Fan¹, Yanle Guo¹

¹Prenatal Diagnosis Center, Shantou University School of Medicine Affiliated Yuebei People’s Hospital, Shantou Guangdong

²Institute of Reproductive Genetics, Dongguan Maternal and Child Health Hospital, Dongguan Guangdong

Received: Apr. 15^th, 2024; accepted: May 31^st, 2024; published: Jun. 12^th, 2024

ABSTRACT

Objective: To investigate the expression of miRNA-494, PI3K/AKT/mTOR [mammalian target of rapamycin, mTOR] in a mouse model of unexplained recurrent spontaneous abortion (uRSA), and their mechanisms in promoting placental trophoblast proliferation and migration, as well as regulating inflammation and immune function in the uterus and placenta. Method: Firstly, an overexpression vector of miRNA-494 was constructed, and the experiment was divided into miRNA-494 mimic group, mimic control group, miRNA-494 inhibitor group, and inhibitor control group. Liposome 2000 was used to transfect miRNA-494 mimic group (50 nmol/L) and miRNA-494 inhibitor group (50 nmol/L), respectively. Real time fluorescence quantitative PCR technology was used to detect miRNA-494 mimic group, mimetic control group, and miRNA-494 inhibitor group The expression level of the inhibitor control group; The proliferation ability of placental trophoblast cells in each group was detected using clone formation experiments, the migration degree of placental trophoblast cells in each group was measured using Transwell method, and the expression levels of PI3K, AKT, and mTOR in placental trophoblast cells in each group were detected using Western blot. Result: After 48 hours of miRNA-494 transfection, the expression level of miRNA-494 in the trophoblast cells of the miRNA-494 mimic group was significantly higher than that of the mimic control group, inhibitor control group, and miRNA-494 inhibitor group (P < 0.001); On the contrary, the expression level of miRNA-494 in trophoblast cells in the miRNA-494 inhibitor group was significantly lower than that in the control group and miRNA-494 mimic group (P < 0.001); The expression levels of PI3K/AKT and mTOR in the miRNA-494 inhibitor group were significantly lower than those in the inhibitor control group, mimic control group, and miRNA-494 mimic group (P < 0.001); The proportion of trophoblast proliferation and migration in the miRNA-494 inhibitor group was significantly lower than that in the inhibitor control group, mimic control group, and miRNA-494 mimic group (P < 0.01). Conclusion: miRNA-494 can promote the proliferation and migration of placental trophoblasts by regulating the expression of Akt/PI3K/mTOR genes, and participate in regulating the inflammatory response and immune function of the uterus and placenta, indicating that regulating the expression level of miRNA-494 may serve as a new target for the prevention and treatment of uRSA.

Keywords:miRNA-494, PI3K/AKT/mTOR, Trophoblast Cell, Unexplained Recurrent Spontaneous Abortion

This work is licensed under the Creative Commons Attribution International License (CC BY 4.0).

http://creativecommons.org/licenses/by/4.0/

1. 引言

在复发性流产中uRSA占60%，uRSA患者并发早期妊娠胚胎丢失(early pregnancy embryo loss, EPEL)，是当今临床诊治的难点，更是严重危害妇女生育功能和临床迫切需要解决的的疑难杂症 [ 1 ] 。由于uRSA病因不清楚，其发病机制也未阐明，所以目前缺乏诊断EPEL的标志物和有效的治疗手段 [ 2 ] [ 3 ] 。在正常妊娠过程中，母胎免疫耐受达到平衡状态；如果该平衡被打破，则发生EPEL、uRSA和妊娠期高血压等病理性妊娠疾病 [ 4 ] 。近年来研究miRNAs在免疫细胞的生长发育、免疫应答的调控等过程中发挥重要的作用，尤其是miRNA参与调节胚胎发育其作用机制及调节特点在免疫系统中具有独特性 [ 3 ] 。miRNA在胚胎发育过程中起着关键作用，其失调也会导致不良妊娠事件的发生。在不同的组织中观察到miRNA整体表达量的差异，这表明精确的miRNA剂量控制在生物体发育中都发挥重要作用 [ 5 ] 。microRNAs (miRNAs)是一类长度为18~24个核苷酸的非编码小分子RNA，对多种生物学过程具有重要的调控作用。miRNA参与了机体的各种的生理病理过程，如生长、发育、分化、炎症、肿瘤的发生 [ 6 ] 。近期研究表明。miRNA-494在妊娠胎盘功能调节和母体对胎儿免疫反应中起关键作用，miRNA-494异常表达导致流产等不良妊娠结局 [ 7 ] 。miRNA的表达异常可能参与不良妊娠和妊娠并发症的进展，并作为母-胎对话的重要媒介参与调控巨噬细胞极化，进一步驱动蜕膜巨噬细胞炎症反应和免疫功能。已有研究表明，在不良妊娠疾病中，miRNA可通过影响NF-κB、STAT1等信号转导调控蜕膜巨噬细胞极化表型及其功能，从而参与疾病的发生发展 [ 8 ] 。miRNA参与调控滋养细胞增殖、迁移、侵袭、凋亡、血管生成，以及在复发性流产的发生机制中发挥重要作用 [ 4 ] [ 5 ] 。

最新研究显示mTORC1在小鼠蜕膜基质细胞和滋养细胞中均有表达，mTOR基因通过上游Akt/PI3K基因调控参与uRSA的发病机制 [ 9 ] [ 10 ] 。本文拟研究miRNA-494促进滋养细胞增殖和迁移，是否对Akt/PI3K/mTOR基因表达有调控影响胎盘功能的作用，为miRNA-494在uRSA中的作用提供新的理论基础。

2. 材料与方法 2.1. 实验材料 2.1.1. 细胞

滋养层细胞系HTR-8/SVneo购自上海帛科生物公司。

2.1.2. 主要试剂及设备

胎牛血清(FBS)、RPMI-1640培养基购自上海帛科生物公司；DMEM培养基购自上海雅吉；引物及探针由广州市锐博生物公司提供；cDNA Synthesis Kit、SYBR Green PCR Master Mix Kit、Applied Biosystems ABI 7500均购自美国Applied Biosystems公司；Lipofec-tamine 2000购自福州奕澜瑞生物公司；RIPA裂解液、0.25%的胰蛋白酶、电化学发光检测试剂盒购自上海爱必信；BCA蛋白检测试剂盒购自美国赛默飞；Annexin V-FITC/PI凋亡检测试剂盒购自北京Biosea；FL流式细胞仪购自美国贝克曼库尔特；TLR4、NF-κB、白介素6 (IL-6)、TNF-α鼠抗兔单克隆抗体和辣根过氧化物酶酶标二抗均购自英国Abcam公司；miRNA-146、TLR4、NF-κB、TNF-α、IL-6和内参GAPDH引物及探针由广州市锐博生物有限公司提供；miRNA-146抑制剂(50 nmol/L)、miRNA-146抑制剂对照、miRNA-146模拟物(50 nmol/L)和miRNA-146模拟物均购自上海生工基因公司。

2.2. 实验方法 2.2.1. 滋养细胞培养

HTR-8/SVneo滋养细胞株培养于含10%灭活胎牛血清、100 U/mL青霉素的RPMI-1640培养基中，放置于CO₂培养箱，5% CO₂，37.0℃和100%湿度条件下培养。稳定传代后收集对数生长期细胞进行后续实验。

2.2.2. miRNA-494过表达载体的构建

首先利用Primer 5.0设计包含miRNA-494前体序列的上下游引物，再在上游引物的5'端前面加上保护碱基和BamH頂每切位点序列(CGGGATCC)，下游引物的5'端加上保护碱基和EcoR頂每切位点序列(CGGAATTC)。引物委托上海生工生物有限公司进行合成，其序列见表1所示：

Table 1 Target genes and primer sequence

基因	引物序列
miRNA-494	上游：5'-TGAAACATACACGGGATCCACTC-3'
	下游：5'-GCGAGCACAACGGAATTCACGAC-3'
TLR4	上游：5'-CGCTTTCACCTCTGCCTTCACTACAG-3'
	下游：5'-ACACTACCACAATAACCTTCCGGCTC-3'
NF-κB	上游：5'-CTGAACCAGGGCATACCTGT-3'
	下游：5'-GAGAAGTCCATGTCCGCAAT-3'
IL-6	上游：5'-GAGAAGGGGGACCAACTCAG-3'
	下游：5'-GGTCTGTTGGGAGTGGTATCC-3'
TNF-α	上游：5'-CACACAAGTCTCCGCTAT-3'
	下游：5'-GTGCGATCCAGTCGCG-3'
GAPDH	上游：5'-TGCACCACCAACTGCTTAGC-3'
	下游：5'-GGCATGGACTGTGGTGATGAG-3'

表1. 目标基因及引物序列

2.2.3. qRT-PCR检测miRNA-494和miRNA-494转染和分组

采用qRT-PCR检测miRNA-494的表达，在碾碎组织中加入Trizol以提取血清中总RNA，使用逆转录试剂盒进行反转录，其间严格按说明书进行操作。miRNA-494正向和反向引物见表1，内参U6引物及探针由广州市锐博生物有限公司提供，U6正向引物为CGCTTCGG CAGCACATATACTA，反向引物为AACGCTTCACGAATTTGCGTAC。

miRNA-494转染：VSMC消化离心后以合适密度接种到培养板，24 h后细胞融合到60%~70%时按照Lipofec-tamine2000说明书进行操作，分成miRNA-494模拟物组和模拟物阴性对照组、miRNA-494抑制剂组和抑制剂阴性对照组，分别转染miRNA-494模拟物(50 nmol/L)、miRNA-494抑制剂(50 nmol/L)，模拟物阴性对照组和抑制剂阴性对照组加入同等剂量lipofectamine2000及PBS，5 h后换成完全培养基，荧光显微镜观察转染效率。

2.2.4. 酶联免疫吸附法

采用酶联免疫吸附法(ELISA)检测血清IL-6和TNF-a的表达水平，在Thermo Scientific Varioskan LUX全自动酶标仪上进行操作，IL-6和TNF-aL的ELISA试剂盒由广州市锐博生物科技有限公司提供。

2.2.5. 化学发光技术

在小鼠模型的尾静脉取1~3 ml外周血，放置不抗凝干燥管，常温下离心10~15分钟，收取血清检测雌激素、孕激素等。采用Cobas 601化学发光仪检测血清雌激素和孕激素水平，购自罗氏(中国)投资有限公司。

2.2.6. 滋养细胞生长活力的实验

采用MTT [3-(4，5-二甲基噻唑-2-基)-2，5-二苯基-2-四唑溴化[3-(4,5)-dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumromide，MTT]比色法测定滋养细胞生长的活力。miRNA-494转染滋养细胞接种于密度为1 × 10³个细胞/孔。磷酸盐缓冲盐水冲洗两次后，10 µL MTT溶液稀释为5 mg/mL，加到每一孔中。将培养皿在37℃下培养10分钟4 h，然后添加150 µL的二甲基亚砜，置摇床上低速震荡10分钟后，使结晶物充分溶解。在酶联免疫检测仪OD 490 nm处测量各孔的吸光值。

2.2.7. 滋养细胞增殖和凋亡实验

采用细胞克隆实验检测滋养细胞增殖能力，在生长期细胞加入0.25%的胰蛋白酶，消化后吹打为单个细胞，实验评价滋养细胞的增殖能力。取对数生长期的细胞，用0.25%的胰蛋白酶消化并吹打为单个细胞，离心后计数，取需要的细胞量(200~500个/孔)重悬于对应的培养基，接种于细胞培养皿里。于37℃、5% CO₂及饱和湿度的细胞培养箱内培养2~3周，在显微镜下能观察细胞克隆的情况。

滋养细胞凋亡实验使用Annexin V-FITC/PI凋亡检测试剂盒(北京Biosea生物技术有限公司)进行滋养细胞凋亡检测、鉴定和定量滋养凋亡细胞。将miRNA转染的细胞接种在密度为1 × 10⁵个细胞/孔的6孔板中，用冷磷酸盐缓冲盐水洗涤处理细胞两次，并将其重新悬浮在含有10 µL膜联蛋白V和5 µL PI的200 µL结合缓冲液中。将贴壁细胞和漂浮细胞结合，按照说明操作，使用FL流式细胞仪(美国贝克曼库尔特)测量百分比，以区分凋亡细胞(膜联蛋白V阳性和PI阴性)和坏死细胞(膜联蛋白V和PI阳性)。

2.2.8. 滋养细胞迁移实验

采用transwell小室实验评估胎盘滋养细胞的迁移能力。按照1 × 10⁵/ml的比例将滋养细胞悬液加入 transwell上室内，每孔100 μl在下室中加入200 μl含20% FBS的DMEM培养基，放入37℃、5% CO₂细胞培养箱中孵育12 h，用PBS清洗后，加入甲醛固定20 min，2.5%的结晶紫染色15 min，用棉签轻轻拭去小室内侧细胞，在倒置显微镜下观察记录小室内5个视野内的细胞数量。

2.2.9. western blotting检测

采用western blotting检测Akt、PI3K、mTOR、NF-κB、IL-6和TNF-a蛋白的表达水平，抗体购置Abcam。采用超敏ECL化学发光液试剂盒(上海爱必信生物科技有限公司)进行发光显影，用Imagequant TL软件分析蛋白条带。

2.3. 统计学分析

SPSS21.0软件进行统计学数据分析，计量资料采用 ± s表示，两组比较采用t检验，计数资料用例或百分率表示，采用Spearman分析小鼠模型miRNA-494表达与Akt、PI3K和mTOR水平的相关性，P < 0.05为差异有统计学意义。

3. 结果 3.1. 两组性激素E2、P、miRNA-494、IL-6、TNF-a水平比较比较

与对照组相比，uRSA模型组的雌二醇(E2)水平显著下降[92.27 ± 10.29vs.36.59 ± 7.22(P < 0.001)]，孕激素(P)水平也下降，66.31 ± 8.78 vs. 13.53 ± 3.12) (P < 0.001)]。

与对照组比较，uRSA小鼠的miRNA-494表达水平下调分别为[1.51 ± 0.47 vs. 5.83 ± 1.86 (P < 0.001)]。uRSA组炎症因子白介素(IL6)肿瘤坏死因子(TNF)-a的表达水平明显升高，与对照组比较分别为：IL-6为20.13 ± 5.79 vs. 109 ± 18.73 (P < 0.001)，TNF-a 13.27 ± 5.79 vs. 86.74 ± 13.85 (P < 0.001)，见表2。miRNA-494表达水平显著下调[(1.51 ± 0.47)%比(5.83 ± 1.86)%, P < 0.001]；BeWo细胞转染miRNA-494mimics 24 h后的细胞增殖的百分数显著稿于对照组[(17.2 ± 3.2)%比(5.1 ± 1.8)%]和miRNA-494 inhibitors组[(17.2 ± 3.2)%比(2.5 ± 0.7)%]，

Table 2 Comparison of serum miRNA-494, IL-6, and TNF-a levels between two groups ( x ¯ ± s

组别	例数(n)	miRNA-494相对表达量	IL-6 (ng/ml)	TNF-a (ng/ml)
对照组	10	5.83 ± 1.86	20.13 ± 5.79	13.27 ± 5.79
uRSA组	10	1.51 ± 0.47	109 ± 18.73	86.74 ± 13.85
T	-	10.431	59.146	48.689
P	-	0.001	0.000	0.000

表2. 两组血清miRNA-494、IL-6、TNF-a水平比较( x ¯ ± s )

3.2. miRNA-494过表达载体的构建

实验所克隆的miRNA-494前体大小为560 bp，扩增的目的片段与预期大小一致，且条带单一、无杂带。提取质粒经测序比对，结果与预期相符，无杂峰干扰，所提取的质粒经核酸蛋白浓度测定仪检测质粒浓度高、质量好，可用于细胞转染实验。

3.3. miRNA-494分别与IL-6、TNF-a进行相关分析

uRSA患者血清中miRNA-494表达水平明显下调，IL-6和TNF-a表达水平也升高。miRNA-494分别与IL-6和TNF-a呈负相关，分别为r = −0.683，r = −0.721，均为P < 0.01。

3.4. miRNA-494在滋养细胞的表达

结果显示，miRNA-494模拟物组在滋养细胞中呈高水平表达，miRNA-494抑制物组在滋养细胞中呈低水平表达，见图1(a)。

3.5. miRNA-494过表达水平与滋养细胞增殖能力

细胞克隆实验显示，miRNA-494模拟物增加滋养细胞的克隆形成，提示上调miRNA-494过表达具有促进滋养细胞增殖的能力；相反miRNA-494抑制物抑制滋养细胞的克隆形成，抑制滋养细胞的增殖，详见(图1(b))。

3.6. miRNA-494与滋养细胞生长活力

MTT结果显示，在miRNA-494模拟物(100 nmol/L)的作用下提高滋养细胞生长活力、且呈高水平表达(P < 0.001)；相反miRNA-494抑制物(100 nmol/L)的作用下抑制滋养细胞生长活力(P < 0.01) (见图1(c))。

3.7. miRNA-494的表达与滋养细胞凋亡

与对照组相比，miRNA-494模拟物组显著降低细胞凋亡率(P < 0.001)，miRNA-494抑制剂组显著增加细胞凋亡率(图1(d))。

3.8. miRNA-494过表达与滋养细胞迁移能力

Transwell实验结果显示，HTR-8/SVneo细胞经miRNA-494转染后，miRNA-494模拟物组的滋养细胞生长和迁移能力显著高于对照组和miRNA-494抑制剂组(图1(e)和图1(f))；miRNA-494过表达促进滋养细胞的生长和迁移(P < 0.001)，相反，下调miRNA-494表达水平抑制滋养细胞的生长和迁移(P < 0.01)，见图1(e)和图1(f)。

(a)：miR-494模拟物、抑制剂通过PCR检测滋养细胞和对照组miRNA-494的表达水平*P < 0.01，#P < 0.01；(b)：miR-494模拟物、抑制剂通过克隆实验检测滋养细胞增殖；*P < 0.05，#P < 0.01；(c)：miR-494模拟物、抑制剂通过MTT检测滋养细胞的生长活性；*P < 0.001，#P < 0.01；(d)：采用使用Annexin V-FITC/PI凋亡检测颗粒细胞凋亡的百分率；*P < 0.001；(e)：转染miRNA-494促进滋养细胞生长能力，mimic control为模拟物对照组滋养细胞的生长结果；miR-494 mimic组为HTR-8/SVneo细胞经miRNA-494模拟物转染后的滋养细胞的生长结果；inhibitor control为抑制剂对照组滋养细胞的生长结果，miR-494 inhibitor为HTR-8/SVneo细胞经miRNA-494抑制物转染后的的滋养细胞生长结果；(f)为HTR-8/SVneo细胞经miRNA-494模拟物和抑制物转染后的滋养细胞迁移能力，* P < 0.001，#P < 0.01。

图1. miRNA-494对滋养细胞增殖、生长、迁移和凋亡的影响

3.9. miRNA-494过表达对Akt、PI3K和mTOR蛋白表达的影响

采用westrn blotting检测Akt、PI3K和mTOR蛋白，与对照组比较，miRNA-494过表达可显著提高Akt、PI3K和mTOR的表达水平(P < 0.01, P < 0.001)，反之可显著降低Akt、PI3K和mTOR的表达水平(图2(a)~(c))，且miRNA-494水平与Akt和PI3K和水平分别呈正相关(r = 0.699, r = 0.736, P < 0.01)，与mTOR水平也呈正相关(r = 0.725, P < 0.01)。

4. 讨论

人类胎盘与胎儿和母体环境相连接，滋养层位于胎盘绒毛表面，与母体血液直接接触，在滋养层细胞表达并被称为“滋养层细胞”的独特miRNAs在正常和病理妊娠期间可能表现出特殊功能 [ 11 ] 。胎盘miRNAs包装在外泌体能够向母体和/或胎儿组织传递独特的信号 [ 12 ] ，在建立和维持母-胎免疫耐受机制。近年来，在生殖领域中miRNA对疾病发生发展的调节作用越来越受重视，生理状态下miRNA参与调控内膜容受性建立、滋养层细胞侵袭、母–胎交互对话等胚胎植入过程中的重要生物学行为来维持妊娠 [ 8 ] 。妊娠期间滋养细胞来源的外泌体转运miRNA并诱导单核细胞募集和分化来调节母体免疫，而巨噬细胞外泌体也通过携带外源性miRNA参与调控滋养层细胞的生物学功能，从而实现母–胎对话 [ 5 ] 。

mTOR基因定位于人类染色体1p36.2，其表达产物哺乳动物雷帕霉素靶蛋白是一种丝氨酸/苏氨酸蛋

图2. miRNA-494与Akt、PI3K和mTOR蛋白表达分析

白激酶，通过磷酸化信号通路将下游效应器调控大量的生命过程，包括转录、翻译和核糖体的生物组合等，是调控细胞生长、分化、增殖、血管形成的关键通路；mTOR通路影响胚胎干细胞及早期胚胎发育，并在小鼠囊胚的活化与胚胎着床中起到至关重要的作用 [ 13 ] ；已有研究证实mTOR在小鼠蜕膜基质细胞和滋养细胞中均有表达，在胚胎种植、生长和发育过程中发挥极其重要的作用 [ 14 ] 。mTOR/RPS6KB1通路调控滋养细胞蛋白翻译，mTOR基因缺陷的囊胚外层滋养细胞功能异常。本文细胞实验显示，miRNA-494过表达上调mTOR蛋白促进滋养细胞增殖和分化，提示mTOR在早期妊娠维持中发挥重要的作用，但是有关miRNA-494调控Akt/PI3K/mTOR信号通路与人类妊娠和胚胎方面等研究目前未见报道。

miRNA的异常改变也与不良妊娠的发生发展有关。来源于M1巨噬细胞的细胞外小泡递送miR-146a-5p和miR-146b-5p，通过靶向TRAF6抑制复发性自然流产中滋养层的迁移和侵袭，miR-146a-5p和miR-146b-5p低水平表达会导致不良妊娠结局 [ 15 ] 。miR-206的异常表达通过调节Wnt信号传导诱导先兆子痫和不良妊娠的发生 [ 16 ] 。MiR-148a-3p可能通过调节HOXC8而导致复发性植入失败的蜕膜化缺陷和不良妊娠结局 [ 17 ] 。Liu等人证实miRNA-100-5p下调并激活JAK/STAT信号通路促进子痫前期和不良妊娠事件的进展 [ 18 ] 。本文的研究发现，miRNA-494过表达提高Akt/PI3K和mTOR的表达水平，同时也影响雌激素和孕激素水平升高，反之，抑制剂组 miRNA-494表达水平的下调、Akt/PI3K和mTOR表达水平下调，雌激素和孕激素水也下降，提示miRNA-494低水平表达与雌激素和孕激素水下降有密切关系。本文认为，uRSA患者由于miRNA-494低表达水平和雌、孕激素水平紊乱，促使体内的Akt/PI3K和mTOR水平下调，诱发母胎界面氧化应激和炎症反应，推测miRNA-494可能是一个妊娠免疫适应的关键调节因子，对免疫巨噬细胞的募集起到至关重要的作用。可见，miRNA-494在免疫系统和炎症反应中发挥重要的调节作用，其调节特点在免疫系统中具有独特性。本研究前期成功构建了miRNA-494的过表达载体，与模拟物对照组和抑制剂对照组比较，过表达miRNA-494明显提高滋养细胞生长活力，miRNA-494抑制物组显著降低滋养细胞生长活力 [ 19 ] ；同时，过表达miRNA-494也加快滋养细胞增殖速度、降低滋养细胞凋亡率，反之滋养细胞增殖速度减慢和增加滋养细胞凋亡率。体现出miRNA-494在生殖细胞中发挥的调节作用，其机制可能是miRNA-494异常表达诱导胎盘滋养细胞过早衰老和凋亡，导致不良妊娠结局 [ 20 ] 。

本文进一步研究miRNA-494与炎症因子IL-6和TNF-a的研究。过表达miRNA-494下调 Il-6和TNF-α的表达水平，它们之间显示负性相关，由此可见miRNA-494可能通过mTOR通路参与抑制炎症因子表达、并对母胎界面的免疫反应具有一定的调控作用，正如与mTOR相关的通路，如信号转导因子和转录激活因子3 signal transducer and activator of transcription 3 (STAT3)信号通路也参与免疫细胞活化和胎盘形成，STAT3表达和功能异常影响胎盘形成，STAT3基因rs1053023多态性与uRSA风险相关 [ 21 ] ，可能是通过Akt/PI3K/mTOR信号通路发挥调控作用，其机制可能与下游的炎症因子参与免疫反应有密切关联。miRNA-494、miRNA-155和miRNA-124均从不同方式参与介导母–胎界面STAT1/STAT3对胎盘免疫细胞的调节功能，均佐证本文的实验结果。

综上所述，本研究成功构建了miRNA-494的过表达载体，并发现miRNA-494低表达可能参与早期流产和不良妊娠的进展。miRNA-494通过促进Akt/PI3K/mTOR基因的表达，参与调节子宫和胎盘的炎症反应和免疫功能，可以减少不良妊娠事件的发生，表明了调节miRNA-494的表达水平可能作为uRSA的预防和治疗的新靶点。

基金项目

1) 广东省基础与应用基础研究基金区域(粤莞)联合基金培育项目，编号(2021B1515140035)和(2022A1515140097)；

2) 广东省韶关市卫生计生科研项目编号(Y23077)。

文章引用

刘玉兰,何凤屏,郭义红,马秋林,张咏梅,陈陆静,刘珮瑜,范舒舒,郭艳乐. miRNA-494调控PI3K/AKT/mTOR通路促进滋养细胞增殖和迁移MiRNA-494 Regulates the PI3K/AKT/mTOR Pathway to Promote Trophoblast Proliferation and Migration[J]. 生物医学, 2024, 14(03): 369-378. https://doi.org/10.12677/hjbm.2024.143041

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