circRNA circ_0055724 Inhibits Trophoblastic Cell Line HTR-8/SVneo's Invasive and Migratory Abilities via the miR-136/N-Cadherin Axis

Abstract

Preeclampsia (PE) is one of the major causes of morbidity and mortality in pregnancy. According to recent research, circular RNAs (circRNA) may act as sponges for microRNAs (miRNAs) and modulate gene expression. Low expression of hsa_circ_0055724 (circ_0055724) in PE tissues was recently reported in literatures. However, its mechanism and function have not been reported. Therefore, we were committed to investigating the role and mechanism of circ_0055724 in PE. Our study first verified the low expression of circ_0055724 in PE tissues. Overexpression or knockdown of circ_0055724 enhances/weakens the trophoblast cell survival, migration, and invasion. Furthermore, CircInteractome predicted the binding sites of circ_0055724 and miR-136, while Starbase predicted miR-136 targeted N-cadherin. Luciferase reporter gene assay confirmed that circ_0055724 directly interacts with miR-136 and miR-136 directly interacts with N-cadherin. More results indicated that high expression of miR-136 and low expression of N-cadherin appeared in PE. Increased expression of circ_0055724 resulted in decreased miR-136 but increased N-cadherin expression. Hence, circ_0055724 and N-cadherin were positively correlated, while circ_0055724 and miR-136 had a negative correlation. In terms of mechanism, circ_0055724 may induce the expression of N-cadherin and regulate the proliferation, migration, and invasion of trophoblast cells through decreasing miR-136, which can be a promising biomarker for early diagnosis and prognosis of patients with PE.

Figure 1

circ_0055724 was downregulated in placental tissues from pregnant women. The expression level of circ_0055724 in 40 normal tissues and 40 PE tissues was detected by qRT-PCR. The difference was statistically significant (p < 0.001).

Figure 2

Effects of circ_0055724 on trophoblast proliferation, invasion, and migration. (a) Circ_0055724 was overexpressed in HTR-8/SVneo cells, and the overexpression efficiency was detected by qRT-PCR. (b) Knockdown of circ_0055724 in the HTR-8/SVneo cells and the qRT-PCR method was used to detect knockdown efficiency. (c, d) Represent the results of the CCK-8 method that was used to detect the light absorption values of HTR-8/SVneo cells at 450 nm wavelength at 0 h, 24 h, 48 h, 72 h, and 96 h in different groups. (e, f) Represent the number of migrating HTR-8/SVneo cells detected by Transwell assay after overexpression or silencing of circ_0055724, respectively. (g, h) Represent the number of invasive cells after overexpression or silencing of circ_0055724, respectively. ^∗∗p < 0.001.

Figure 3

miR-136 was a target of circ_0055724 in trophoblasts. (a) Binding site of hsa_circ_0055724 on miR-136 was predicted by CircInteractome, an online bioinformatics tool. (b) Luciferase reporter gene assay revealed the inhibitory ability. (c) Interaction between biotinylated hSA_circ_0055724 and miR-136 was shown by RNA pull-down assay. (d) Represents the expression level of miR-136 in hsa_circ_0055724 cells detected by qRT-PCR after overexpression or knockdown of hsa_circ_0055724, respectively. (e) Represents the expression level of miR-136 in 40 normal tissues and 40 PE placental tissues detected by qRT-PCR. (f) Correlation analysis of hsa_circ_0055724 and miR-136 in PE placental tissue showed negative correlation. ^∗∗p < 0.001.

Figure 4

N-cadherin is a direct target of miR-136. (a) The binding sites of N-cadherin (gene CDH2) and miR-136 was predicted by Starbase, an online bioinformatics tool. (b) Represents the results of luciferase reporter gene experiment carried out in HTR-8/SVneo cells. (c) Compared with miR-NC, overexpression of miR-136 could inhibit luciferase activity in cells, and the inhibition effect disappeared after mutation of the predicted N-cadherin binding site. mRNA and protein expression levels of N-cadherin in miR-136 knockdown or overexpressed HTR-8/SVneo cells were detected by qRT-PCR and WB methods. mRNA and protein expression levels of N-cadherin were increased or decreased by miR-136 knockdown or overexpression, respectively. qRT-PCR and WB methods were used to detect the expression levels of N-cadherin mRNA and protein in HTR-8/SVneo cells in different groups (vector, circ_0055724, circ_0055724+ miR-136). (d) Overexpression of hsa_circ_0055724 increased the expression levels of N-cadherin mRNA and protein, and the expression levels of N-cadherin partially decreased after cotransfection with miR-136. (e) Represents the mRNA expression of N-cadherin in 40 normal tissues and 40 PE tissues detected by qRT-PCR. (f) Correlation analysis of hsa_circ_0055724, miR-136, and N-cadherin expression in PE. ^∗∗p < 0.001.

Figure 5

circ_0055724 mediated trophoblast invasion and migration via the miR-136/N-cadherin axis. (a) The WB method was used to detect the low level of N-cadherin in HTR-8/SVneo cells. (b) The CCK-8 method was used to detect the light absorption values of HTR-8/SVneo cells at 450 nm at 0 h, 24 h, 48 h, 72 h, and 96 h in different groups. (c) The clonal formation experiment was conducted to count the number of colonies and thereby estimate the colony forming capacity of the HTR-8/SVneo cells. (d) Cell migration ability of HTR-8/SVneo cells in different groups was detected by Transwell assay. (e) Invasion ability of HTR-8/SVneo cells in different groups is represented and determined by Transwell assay. ^∗∗p < 0.001; ^∗∗∗p < 0.0001.