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