lncRNA HAND2-AS1通过调节miR-21表达对子宫内膜异位症患者子宫内膜基质细胞迁移和侵袭的抑制作用

doi:10.13481/j.1671-587X.20230323

摘要/Abstract

摘要：

目的探讨子宫内膜异位症（EMT）患者子宫内膜组织中长链非编码RNAs（lncRNAs）HAND2-AS1对异位子宫内膜（EC）组织中子宫内膜基质细胞（ESCs）增殖、迁移和侵袭能力的影响，并阐明其可能机制。方法收集30例EMT患者（EMT组）的EC组织和30例健康育龄女性（对照组）的子宫内膜组织，分离子宫内膜组织获取ESCs。采用脂质体转染法转染EMT患者EC组织的ESCs，并将ESCs分为pcDNA组（转染pcDNA空质粒）、HAND2-AS1组（转染HAND2-AS1过表达质粒）、mimic NC组（转染mimic NC）、miR-21 mimic组（转染miR-21 mimic）、pcDNA+mimic NC组（联合转染pcDNA和mimic NC）、HAND2-AS1+mimic NC组（联合转染HAND2-AS1过表达质粒和mimic NC）、pcDNA+miR-21 mimic组（联合转染pcDNA空质粒和miR-21 mimic）和HAND2-AS1+miR-21 mimic组（联合转染HAND2-AS1过表达质粒和miR-21 mimic），另设未转染的细胞为空白对照组。采用实时荧光定量PCR（RT-qPCR）法检测2组研究对象子宫内膜组织和ESCs中HAND2-AS1 mRNA和miR-21表达水平，采用Pearson相关系数分析其相关性。采用生物信息学软件Starbase预测lncRNA HAND2-AS1与miR-21的靶向作用关系，荧光素酶基因报告实验进行验证。CCK-8法检测各组ESCs增殖活性，Transwell小室实验检测ESCs的迁移和侵袭数。结果 2组研究对象年龄、体质量指数（BMI）和血清中卵泡刺激素（FSH）、黄体生成素（LH）及催乳素（PRL）水平比较差异均无统计学意义（P<0.05）。与对照组比较，EMT组患者血清中雌二醇（E2）水平升高（P<0.05），EMT组患者EC组织中HAND2-AS1 mRNA表达水平降低（P<0.05），miR-21表达水平升高（P<0.05）；与对照组比较，EMT组ESCs中HAND2-AS1 mRNA表达水平降低（P<0.05），miR-21表达水平明显升高（P<0.01）。Pearson相关系数分析，对照组研究对象HAND2-AS1与miR-21表达水平无明显相关性（r=0.34，P>0.05），EMT组患者ES组织中和ESCs中HAND2-AS1与miR-21表达水平呈负相关关系（r=－0.57，P<0.05）。RT-qPCR法检测，与空白对照组比较，HAND2-AS1组ESCs中HAND2-AS1 mRNA表达水平明显升高（P<0.01），miR-21 mimic组ESCs中miR-21表达水平明显升高（P<0.01），HAND2-AS1+mimic NC组ESCs中miR-21表达水平降低（P<0.05），pcDNA+miR-21 mimic组ESCs中miR-21表达水平明显升高（P<0.01）；与pcDNA+ miR-21 mimic组比较，HAND2-AS1+miR-21 mimic组ESCs中miR-21表达水平降低（P<0.05）。lncRNA HAND2-AS1与miR-21存在潜在结合位点；双荧光素酶基因报告实验，与mimic NC组比较，miR-21 mimic组HAND2-AS1-WT中荧光素酶活性明显降低（P<0.01）。与空白对照组比较，HAND2-AS1+mimic NC组ESCs增殖活性、迁移和侵袭数均明显降低（P<0.05或P<0.01），pcDNA+miR-21 mimic组ESCs增殖活性、迁移和侵袭数升高（P<0.05）；与pcDNA+miR-21 mimic组比较，HAND2-AS1+miR-21 mimic组ESCs增殖活性、迁移和侵袭数均降低（P<0.05）。结论 HAND2-AS1在EMT患者EC组织和ESCs中表达明显降低，其可通过靶向抑制miR-21表达下调EC组织中ESCs的增殖、迁移和侵袭，从而参与EMT的发生发展。

关键词: 子宫内膜异位症, 异位子宫内膜, 长链非编码RNA, HAND2-AS1, miR-21, 子宫内膜基质细胞, 细胞迁移, 细胞侵袭

Abstract:

Objective To discuss the effect of long non-coding RNA （lncRNA） HAND2-AS1 in endometrium tissue of the patients with endometriosis （EMT） on the proliferation， migration and invasion of the endometrial stromal cells （ESCs） in ectopic endometrium（EC）tissue，and to clarify the possible mechanism. Methods The EC tissue of 30 patients with EMT （EMT group） and the endometrium tissue of 30 healthy women of childbearing age （control group） were collected；the endometrium tissue was isolated，and the ESCs were collected. The ESCs in EC tissue of the EMT patients were transfected with liposome transfection method，and the ESCs were divided into pcDNA group （transfected with pcDNA empty plasmid），HAND2-AS1 group（transfected with HAND2-AS1 over-expression plasmid），mimic NC group（transfected with mimic NC），miR-21 mimic group（transfected with miR-21 mimic），pcDNA+mimic-NC group（transfected with pcDNA and mimic NC）， HAND2-AS1+mimic NC group（transfected with HAND2-AS1 over-expression plasmid and mimic NC）， pcDNA+miR-21 mimic group （transfected with pcDNA empty plasmid and miR-21 mimic），and HAND2-AS1+miR-21 mimic group（transfected with HAND2 AS1 over-expression plasmid and miR-21 mimic），and the cells without any transfection were used as blank control group.The expression levels of HAND2-AS1 mRNA and miR-21 in the endometrium tissue and ESCs of the subjects in two groups were detected by real-time fluorescence quantitative PCR（RT-qPCR） method；and the relationship between them was analyzed by Pearson correlation coefficient；bioinformatics software Starbase was used to predict the targeting relationship between lncRNA HAND2-AS1 and miR-21；the luciferase gene reporting assay was used to verify.The proliferation activities of the ESCs in various groups were detected by CCK-8 method；the numbers of migration and invasion cells were detected by Transwell chamber assay. Results There were no significant differences in the age， body mass index （BMI）， serum levels of follicle stimulating hormone （FSH）， luteinizing hormone （LH） and prolactin （PRL） of the subjects between two groups （P<0.05）. Compared with control group， the serum estradiol （E2） level of the patients in EMT group was increased （P<0.05）， the expression level of HAND2-AS1 mRNA in EC tissue of the patients in EMT group was decreased （P<0.05）， while the expression level of miR-21 was increased （P<0.05）. Compared with control group， the expression level of HAND2-AS1 mRNA in the ESCs in EMT group was decreased （P<0.05）， while the expression level of miR-21 was increased （P<0.01）.The Pearson correlation coefficient analysis results showed that there was no significant correlation between the expressions of HAND2-AS1 and miR-21 in the endometrium tissue and ESCs of the subjects in control group （r=0.34， P>0.05）， while there was a negative correlation between the expressions of HAND2-AS1 and miR-21 in the EC tissue and ESCs of the patients in EMT group （r=－0.57， P<0.05）. The RT-qPCR results showed that compared with blank control group， the expression level of HAND2-AS1 mRNA in the ESCs in HAND2-AS1 group was significantly increased（P<0.01），the miR-21 expression level in the ESCs in miR-21 mimic group was increased（P<0.01），the expression level of miR-21 in the ESCs in HAND2-AS1+mimic NC group was significantly decreased（P<0.05），and the miR-21 expression level in the ESCs in pcDNA+miR-21 mimic group was significantly increased（P<0.01）； compared with pcDNA+ miR-21 mimic group， the miR-21 expression level in the ESCs in HAND2-AS1+miR-21 mimic group was significantly decreased（P<0.05）.There was a potential binding site between lncRNA HAND2-AS1 and miR-21.The dual luciferase gene reporter assay results showed that compared with mimic NC group， the luciferase activity in HAND2-AS1-WT in miR-21 mimic group was significantly decreased（P<0.01）. Compared with blank control group， the proliferation activity of the ESCs， the numbers of migration and invasion ESCs in HAND2-AS1+mimic-NC group were significantly decreased （P<0.05 or P<0.01）， and the above indexes in pcDNA+miR-21 mimic group were significantly increased（P<0.05）； compared with pcDNA+miR-21 mimic group， the proliferation activity，the numbers of migration and invasion ESCs in HAND2-AS1+miR-21 mimic group were significantly decreased（P<0.05）. Conclusion The expressions of HAND2-AS1 in the EC tissue and ESCs of the EMT patients are significantly decreased， and it can down-regulate the proliferation， migration and invasion of the ESCs in EC tissue by targetly inhibiting the expression of miR-21， thus participats in the occurrence and development of EMT.

Key words: Endometriosis, Ectopic endometrium, Long non-coding RNA, HAND2-AS1, Mi21, Endometrial stromal cells, Cell migration, Cell invasion

中图分类号:

R711.2

苗卉,苗聪秀,李娜,韩晶. lncRNA HAND2-AS1通过调节miR-21表达对子宫内膜异位症患者子宫内膜基质细胞迁移和侵袭的抑制作用[J]. 吉林大学学报(医学版), 2023, 49(3): 733-741.

Hui MIAO,Congxiu MIAO,Na LI,Jing HAN. Inhibitory effect of lncRNA HAND2-AS1 on migration and invasion of endometrial stromal cells in patients with endometriosis by regulating expression of miR-21[J]. Journal of Jilin University(Medicine Edition), 2023, 49(3): 733-741.

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Group	Age(year)	BMI(kg·m^－2)	FSH[λ_B/(IU·L^－1)]	LH[λ_B/(IU·L^－1)]	PRL[ρ_B/(μg·L^－1)]	E2[ρ_B/(ng·L^－1)]
Control	31.09±0.80	23.70±0.70	5.98±0.31	5.45±0.77	16.55±3.61	35.21±3.53
EMT	31.39±1.04	22.67±0.86	7.74±0.85	5.29±0.69	21.66±3.39	51.59±5.08
P	0.82	0.39	0.13	0.89	0.33	0.03

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