长链非编码RNA-02075参与调节子宫内膜异位症内膜基质细胞糖酵解的实验研究

doi:10.12280/gjszjk.20230047

摘要/Abstract

摘要：

目的:比较子宫内膜异位症（endometriosis，EMs）患者在位和异位内膜中长链非编码RNA-02075（LINC02075）的表达水平，研究LINC02075对内膜基质细胞糖酵解的调节作用。方法:收集10例23~36岁EMs患者的在位和异位内膜，以及10例25~36岁接受宫内节育器（intrauterine device，IUD）置入手术的健康女性的子宫内膜，采用实时荧光定量聚合酶链反应（polymerase chain reaction，PCR）检测LINC02075的表达；培养异位内膜基质细胞，对其进行过表达LINC02075处理，以空白组作为对照，检测糖酵解相关酶的表达及细胞增殖、迁移和凋亡率。结果:LINC02075在内膜细胞的细胞核和细胞质中广泛表达。与正常内膜和EMs患者在位内膜比较，EMs患者异位内膜LINC02075的mRNA表达水平显著升高（P<0.05）。在异位内膜基质细胞中过表达LINC02075导致其葡萄糖的消耗显著增加（P<0.05），乳酸生成量差异无统计学意义（P>0.05），同时与糖酵解相关的酶如己糖激酶1（hexokinase 1，HK1）、丙酮酸脱氢酶激酶1（pyruvate dehydrogenase kinase 1，PDK1）、乳酸脱氢酶A（lactate dehydrogenase A，LDHA）、葡萄糖转运蛋白1（glucose transporter type 1，GLUT1）和醛缩酶A（aldolase A，ALDOA）的mRNA表达水平均呈上升趋势，其中LDHA mRNA表达水平升高，差异有统计学意义（P<0.05）；异位内膜基质细胞的增殖和凋亡率均增加、迁移力降低（均P<0.05）。结论:EMs患者异位内膜中LINC02075的过表达可能通过增强糖酵解机制使异位内膜细胞增殖、迁移能力降低。

关键词: 子宫内膜异位症, 细胞增殖, 瓦尔堡效应，肿瘤学, 有氧糖酵解, LINC02075

Abstract:

Objective: To compare the expression levels of long non-coding RNA-02075 (LINC02075) in the eutopic and ectopic endometrium from patients with endometriosis, and to investigate the possible roles of LINC02075 in the regulation of glycolysis in endometrial stromal cells. Methods: The samples of eutopic and ectopic endometrium were from 10 patients with endometriosis aged 23-36 years. The endometrium samples from 10 normal women accepting the surgery of intrauterine device placement aged 25-36 years were used as the control. The expression of LINC02075 was evaluated by RT-qPCR. The ectopic endometrial stromal cells were in vitro cultured. The overexpression of LINC02075 was induced by the lentivirus and adenovirus vectors. The glycolytic enzymes, cell proliferation, migration and apoptosis rates were tested. Results: LINC02075 was widely expressed in the nucleus and cytoplasm of endometrial stromal cells. The expression level of LINC02075 mRNA in ectopic endometrium (the endometriosis group) was significantly higher than that in normal endometrium (the control group) and eutopic endometrium (the endometriosis group) (all P<0.05). Overexpression of LINC02075 significantly increased glucose consumption (P<0.05), and increased lactate production (but P>0.05) in ectopic endothelial stromal cells, while the expression levels of glycolysis-related enzymes such as hexokinase 1 (HK1), pyruvate dehydrogenase kinase 1 (PDK1), lactate dehydrogenase A (LDHA), glucose transporter type 1 (GLUT1) and aldolase A (ALDOA) were also increased, among which the increased expression level of LDHA mRNA was statistically significant (P<0.05). Overexpressed LINC02075 also increased the proliferation and apoptosis and decreased the migration of endometrial stromal cells (all P<0.05). Conclusions: The abnormally elevated LINC02075 in ectopic endometrium of patients with endometriosis may promote proliferation and reduce migration of endometrial stromal cells by the enhanced glycolytic mechanism.

Key words: Endometriosis, Cell proliferation, Warburg effect, oncologic, Aerobic glycolysis, LINC02075

朱佩佩, 李凯全, 钟晨怡, 舒黎, 吴春香, 刘金勇, 侯振, 刁飞扬, 冒韵东. 长链非编码RNA-02075参与调节子宫内膜异位症内膜基质细胞糖酵解的实验研究[J]. 国际生殖健康/计划生育杂志, 2023, 42(4): 265-271.

ZHU Pei-pei, LI Kai-quan, ZHONG Chen-yi, SHU Li, WU Chun-xiang, LIU Jin-yong, HOU Zhen, DIAO Fei-yang, MAO Yun-dong. Effect of Long Non-Coding RNA-02075 on Glycolysis in Endometrium Stromal Cells of Endometriosis: A Preliminary Experimental Study[J]. Journal of International Reproductive Health/Family Planning, 2023, 42(4): 265-271.

图/表 5

表1 实时荧光定量PCR引物序列

引物名称	引物序列（5′-3′）
LINC02075	上游：CTGAGACCATCAACCCTATC
	下游：ACTTATTCAACTGGCACTCC
GLUT1	上游：GGCCAAGAGTGTGCTAAAGAA
	下游：ACAGCGTTGATGCCAGACAG
PDK1	上游：TGTGAAGATGAGTGACCGAGGAG
	下游：GGCAATCCATAACCAAAACCAG
HK1	上游：GCTCTCCGATGAAACTCTCATAG
	下游：GGACCTTACGAATGTTGGCAA
PKM	上游：ATGTCGAAGCCCCATAGTGAA
	下游：TGGGTGGTGAATCAATGTCCA
ALDOA	上游：ATGTCGAAGCCCCATAGTGAA
	下游：TGGGTGGTGAATCAATGTCCA
LDHA	上游：ATGGCAACTCTAAAGGATCAGC
	下游：CCAACCCCAACAACTGTAATCT
SDHA	上游：CGGCATTCCCACCAACTA
	下游：GCCCGACCAAAGACAACC
β-actin	上游：TCAAGATCATTGCTCCTCCTGAG
	下游：ACATCTGCTGGAAGGTGGACA

图1 LINC02075在EMs中的表达和亚细胞定位注:图A为采用实时荧光定量PCR检测3组内膜IINCO2075的相对表达量比较,以β-actin为对照.,EMs异位内膜组(n=10)INCO2075 mRNA的相对表达量高于正常子宫内膜组(n=10)和EMs在位内膜组(n=10);****P<0.001。图B为通过免疫荧光法鉴定人子宫内膜细胞的纯度,使用抗波形蛋白(vimentin)抗体(在子宫内膜基质细胞中特异性表达)和抗角蛋白(keratin )抗体(在子宫内膜上皮细胞中特异性表达)进行检测,红色为波形蛋白,绿色为角蛋白,蓝色为DAPI染色的细胞核(×200),标尺=100 pm。图C为通过免疫荧光的L.INCO2075探针显示LINCO2075的位置,探针18s和U6分别在细胞质和细胞核中稳定表达(×100),DAPI表示细胞核(蓝色),标尺=50 pum.

图2 LINC02075对葡萄糖代谢的影响注：图A为实时荧光定量PCR检测腺病毒包装的人LINC02075 cDNA转染异位子宫内膜基质细胞后，LINC02075在原代异位子宫内膜基质细胞中的相对表达量，β-actin为内参基因。图B为过表达LINC02075后，异位子宫内膜基质细胞的葡萄糖相对消耗量的变化，与对照组相比，过表达组葡萄糖消耗增加（P<0.05）。相对葡萄糖消耗量（mmol/L）=葡萄糖消耗量/活细胞数目（106个细胞）。图C为过表达LINC02075后，异位子宫内膜基质细胞的乳酸相对生成的变化，与对照组相比，过表达组相对乳酸生成量呈上升趋势，但差异无统计学意义（P>0.05）。相对乳酸生成量（mmol/L）=乳酸生成量/活细胞数目（106个细胞）。图D为通过PCR检测异位内膜基质细胞过表达LINC02075后其糖代谢相关酶的mRNA水平变化。*P<0.05，****P<0.000 1。HK1 己糖激酶1，PDK1 丙酮酸脱氢酶激酶1，LDHA 乳酸脱氢酶A，PKM 丙酮酸激酶，ALDOA 醛缩酶A，GLUT1 葡萄糖转运蛋白1，SDHA 琥珀酸脱氢酶A。

图3 LINC02075对子宫内膜基质细胞增殖和凋亡的影响注：图A为实时荧光定量PCR检测慢病毒包装的人LINC02075 cDNA和阴性对照分别转染异位子宫内膜基质细胞后，LINC02075在EMs中的相对表达量，内部对照采用β-actin。图B为CCK-8法检测腺病毒转染后24、48、72 h子宫内膜基质细胞的增殖情况，48、72 h过表达组的OD值显著高于对照组（P<0.05）。图C为Annexin-V/PI双染色检测细胞凋亡，流式细胞术根据Annexin-V和PI在细胞表面的荧光强度对细胞进行分布，Q4象限的细胞群代表早期凋亡细胞，Annexin-V阳性细胞的百分比表示细胞凋亡水平，结果表明，过表达LINC02075可促进子宫内膜基质细胞的凋亡（P<0.01）。*P<0.05，**P<0.01，***P<0.001，****P<0.000 1。

图4 LINC02075对异位内膜基质细胞迁移力的影响注：迁移实验中细胞通过Transwell迁移的代表性显微图（结晶紫染色法×100）和通过Transwell迁移的细胞的定量图，数据显示为每个视野中迁移细胞的平均数量。*P<0.05，**P<0.01。标尺=100 μm。空白组：异位子宫内膜基质细胞。阴性对照组：转染了阴性对照慢病毒的异位内膜基质细胞。过表达组：转染了慢病毒包装的人LINC02075 cDNA的异位内膜基质细胞。

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