3.3 correlation between miR-141-5p and hsa_circ_0063526 expression
RT-qPCR was used to determine the relative expression of miR-141-5p between endometriosis patients and the control group (Fig. 3a). We found the difference of means ± SEM was 0.5994 ± 0.08425 (P < 0.01). Pearson correlation analysis of the expression levels of hsa_circ_0063526 and miR-141-5p in the lesion tissue of endometriosis showed a negative correlation (r= -0.4267, p = 0.02, Fig. 3b). Pearson correlation analysis further indicated the interaction between hsa_circ_0063526 and miR-141-5p.
Due to the overexpression of hsa_circ_0063526, we constructed different siRNAs to knockdown hsa_circ_0063526. To determine the knockdown efficiency, three fluorescent small interfering RNAs were designed and synthesized. Fluorescent images showed successful transfection of siRNA into End1/E6E7 cells. Results showed that interference sequence 3 most significantly reduced the expression of hsa_circ_0063526 (fig.s1).
After transfection with si-hsa_circ_0063526, the expression level of hsa_circ_0063526 in End1/E6E7 cells in the si-hsa_circ_0063526 group was significantly decreased (P < 0.05, Fig. 3c), and the expression level of miR-141-5p was significantly increased (P < 0.05, Fig. 3d), compared with that in the Blank group and the si-NC group. The results showed that hsa_circ_0063526 could negatively regulate the expression level of miR-141-5p in End1/E6E7 cells.
3.4 Down-regulation of hsa_circ_0063526 can inhibit the proliferation, invasion, and migration of endometriosis cells
Migration, invasion, and proliferation of ectopic endometrial cells are essential pathological processes for the development of endometriosis. Therefore, 72 hours after transfection of si-hsa_circ_0063526, this study further explored the changes of cell migration and invasion and cell proliferation ability after down-regulation of hsa_circ_0063526. First of all, the ability to invasion by Transwell experiments, after End1 / E6E7 cell transfected with hsa_circ_0063526 siRNA, the cells entering the lower chamber was relatively lesser compared to the control group, cell invasion ability decreased. Difference between means ± SEM = 70.75 ± 10.99 (P < 0.05, Fig. 4). The ability of cell migration was tested by wound healing experiment, after End1 / E6E7 cell transfected hsa_circ_0063526 siRNA, the width of the cell scratch was wider than that of the control group. The ability of cell migration was decreased. Difference between means ± SEM=-333.3 ± 72.12 (P < 0.05, Fig. 5). Further, the effect of down-regulation of hsa_circ_0063526 on cell proliferation was detected by using EdU cell proliferation assay. 72 h after siRNA transfection, compared with the si-NC control group, the EdU test results showed that the proportion of End1/E6E7 cells in the mitotic stage was lesser after the down-regulation of hsa_circ_0063526, and the proliferation capacity of the cells was decreased after siRNA transfection. Difference between means ± SEM = 32.38 ± 6.639 (P < 0.05, Fig. 6). PCR and ELISA results showed that the expression of E-cadherin mRNA, an important epithelial marker of EMT, was up-regulated (P < 0.05, Fig. 7).
3.5 Down-regulation of miR-141-5p can rescue the proliferation, invasion, and migration of endometriosis inhibited by hsa_circ_0063526.
However, after co-transfected with si-hsa_circ_0063526 + inhibitor-miR-141-5p, the proliferation, invasion, and migration of endometriosis cells were rescued (P < 0.05, Fig. 4, 5, and 6). PCR and ELISA results showed that the expression of E-cadherin, an important epithelial marker of EMT, was down-regulated compared with the si-hsa_circ_0063526 group (P < 0.05, Fig. 7).
The above experimental results showed that knockdown hsa_circ_0063526 inhibited the development of endometriosis. Inhibition of microRNA-141-5p can rescue the inhibitory effect brought by knockdown of hsa_circ_0063526, that is to say, hsa_circ_0063526 promotes the development of endometriosis through sponging (inhibition) of microRNA-141-5p.
3.6 Comparison of miR-141-5p treatment and pathological changes
No adverse reactions were observed in mice treated with miR-141-5p. At the end of miRNA-141-5p treatment, mice were sacrificed by cervical dislocation and endometriosis lesions were collected. First, we evaluated the volume of the lesions between the miR-141-5p treatment group and the control group. All lesions were cystic. Lesions in miR-141-5p treatment groups were relatively small. (Figure8 a,b,c)
Besides, the pathological difference of the endometrial part is further compared by the histological area. The histological area of all lesions was observed under the microscope to exclude muscle and destructed parts. The histological area of endometriosis in abdominal lesions of the miR-141-5p treatment group was significantly smaller than the two control groups (p < 0.05, Figure 8 d-g).
3.7 Expression difference of endometriosis-related genes
The differences in gene expression related to endometriosis were detected by RT-qPCR. The expression of some genes known to promote the development of endometriosis decreased in the miR-141-5p group. Expression of N-cadherin, Vimentin, K-ras, MAPK-14, ER-α, ER-β, ZEB-1 was decreased in the miR-141-5p treatment group (P<0.05 Figure 9). The quantitative decrease in gene expression is 6.5-fold for K-Ras, 1.81-fold for MAPK14, 13.9 fold for ER-α, 97.2 fold for ER-β, 4.63 fold for N-Cadherin, 2.37 fold for Vimentin, 3.98-fold for ZEB-1 in 141 treated group compared to saline group. Expression levels of miR-141, E-Cadherin, ZO-1(Zona Occludens 1) were increased in the miR-141-5p treatment group (P<0.05, Figure 9). Expression levels of Notch, IL-6 was not statistically significant between treatment and control groups (P>0.05, Figure 9).
Immunohistochemistry results showed that the protein expression level of the miR-141-5p treatment group was significantly changed. The intensity of N-cadherin and vimentin in stromal cells decreased in the miR-141-5p treatment group(Figure 10).
3.8 Comparison of si-hsa_circ_0063526 treatment and pathological changes
No adverse reactions were observed in mice treated with si-hsa_circ_0063526 agomir. At the end of si-hsa_circ_0063526 agomir treatment, mice were sacrificed by cervical dislocation and endometriosis lesions were collected. We evaluated the volume of the lesions between the si-hsa_circ_0063526 agomir treatment group and the control group. All lesions were cystic. Lesions in si-hsa_circ_0063526 agomir treatment groups were relatively small. (Figure11a)
Besides, the pathological difference of the endometrial part is further compared by the histological area. The histological area of all lesions was observed under the microscope to exclude muscle and destructed parts. The histological area of endometriosis in abdominal lesions of the miR-141-5p treatment group was significantly smaller than the two control groups (p < 0.05, Figure 11b-e).
3.9 Endometriosis-related gene expression differences
RT-qPCR was used to detect the gene expression differences associated with endometriosis. Compared with the control group, some genes known to promote the development of endometriosis were decreased in the si-hsa_circ_0063526 agomir group. MAPK-14, K-ras, N-cadherin, Vimentin, ER-α, ER-β were decreased in the si-hsa_circ_0063526 agomir group (P < 0.05 Figure 12). The decrease folds of quantitative gene expression were 5.4 times of K-ras, 3.4 times of MAPK14, 7.5 times of ER-α, 5.2 times of ER-β, and 2.1 times of N-cadherin. The expression levels of E-cadherin and Zona occludens 1 (Zona Occludens 1) in the si-hsa_circ_0063526 agomir group were increased (P<0.05, Figure 12).
Immunohistochemical results showed that E-cadherin staining intensity was significantly increased in the si-hsa_circ_0063526 treatment group (Figure 13). The results are consistent with the overall research results, indicating that our research results have good stability and reliability.