In this study, we enrolled 30 primary EC patients, previously untreated, and 15 control patients with healthy endometrium (HE) operated due to other gynecological pathologies (leiomyoma). The endometrium of these patients was histopathologically confirmed to be normal. To ensure that material obtained for miRNAs expression analysis contained only EC or HE tissue, we used Laser Capture Microdissection (LCM) to precisely dissect only specific fragments of tissue. From each FFPE sample of EC, only tumor tissue was dissected using LCM. In HE samples, only glandular endometrial tissue was dissected (Fig. 1). From the collected material we have determined the level of: miR-21-3p, miR-23b, miR-34a-5p, miR-96-5p, miR-125b-5p, miR-134-5p, miR-146-5p, miR-150-5p, miR-181b-5p, miR-182-5p, miR-199a-3p, miR-200b-3p, miR-211-3p, miR-221-3p, miR-410-3p, and miR-451a.
MiR-23b, miR-125b-5p, miR-199a-3p, miR-221-3p, and miR-451a are downregulated in EC
The analysis of 16 miRNAs revealed that the levels of expression of certain miRNAs were significantly downregulated in EC compared to HE. miR-23b was downregulated 4.54 times, (Fig. 2a, p<0.0001), miR-125b-5p was downregulated 7.15 times (Fig. 2b, p=0.0005), miR-199a-3p was downregulated 11.11 times (Fig. 2c, p<0.0001), miR-221-3p was downregulated 4.54 times, (Fig. 2d, p=0.0029), and miR-451a was downregulated 17.24 times (Fig. 2e, p<0.0001). In contrast, there were no statistically significant differences between the expression of miR-21-3p, miR-34a-5p, miR-96-5p, miR-134-5p, miR-146-5p, miR-150-5p, miR-181b-5p, miR-182-5p, miR-200b-3p, miR-211-3p, and miR-410-3p in EC compared to HE (Fig. 3a-k, p>0.05).
MiR-34a-5p and miR-146-5p are upregulated in EC1 compared to EC2
Next, we analyzed the differences in miRNAs expression profiles between EC1 and EC2. Out of 30 EC samples used in this study, 18 samples were EC1 type and 12 samples were EC2. The expression of two miRNAs was upregulated in EC1 compared to EC2. These were miR-34a-5p (Fig. 4a, upregulated 5.43 times, p=0.031) and miR-146-5p (Fig. 4b, upregulated 3.50 times, p=0.0479). There was no differences in expression of miR-21-3p, miR-23b, miR-96-5p, miR-125b-5p, miR-134-5p, miR-150-5p, miR-181b-5p, miR-182-5p, miR-199a-3p, miR-200b-3p, miR-211-3p, miR-221-3p, miR-410-3p, and miR-451a between EC1 and EC2 specimens (Fig. 5a-n, p>0.05).
MiR-23b, miR-125b-5p miR-199a-3p, miR-221-3p are downregulated in TCGA cohort
To confirm our findings, we analyzed the expression of investigated miRNAs in The Cancer Genome Atlas (TCGA) on the cohort of 418 EC tissues and 32 HE controls using OncomiR database 19. The levels of miR-23b (Fig. 6a, downregulated 1.87 times, p<0.0001), miR-125b-5p (Fig. 6b, downregulated 2.34 times, p<0.0001) miR-199a-3p (Fig. 6c, downregulated 1.26 times, p=0.0067), and miR-221-3p (Fig. 6d, downregulated 1.45 times, p=0.0110) were significantly downregulated in EC compared to HE. There were no differences in expression levels of miR-451a in EC compared to HE (Fig. 6e, p>0.05).
Decreased miR-23b expression is associated with worse survival
Further, we checked whether the expression of downregulated miRNAs in the EC tissue correlated with the survival of patients. We analyzed the patients' survival and miRNAs level in TCGA EC cohort using OncoLnc 20. We found that decreased expression of miR-23b was associated with worse survival (Fig. 7a, p=0.0203). The decreased expressions of miR-125-5p, miR-199a-3p, miR-221-3p, or miR-451a were not correlated with the survival of EC patients (Fig. 7b-e, p>0.05).