Fetal growth restriction (FGR) is defined as a condition in which the fetus is fail to reach its potential for growth and development. The etiology of FGR is multifactorial, such as maternal causes, fetal causes, and causes involving placental insufficiency. Currently the incidence rates of FGR is the highest over the last decades and it is likely to grow, which becomes a public health challenge in both developed and developing countries20. As a newly discovered non-coding RNA, researchers all over the world pay great attention on circRNA. Nowadays, there are various methods which can be used to examine circRNA profiling, such as RT-qPCR, microarrays and NGS. Each method has its advantages and limitations; however, NGS have better sensitivity and capability of generating quantification. Neoteric bioinformatic approaches combined with biochemical enrichment strategies have allowed people to study circRNAs comprehensively, and many studies have proved circRNAs play an important role in human diseases11. Thousands of circRNAs have been identified as predictive biomarkers and have the potential to be therapeutic targets for therapy21, 30. Up to now, there are few reports on the relationship between circRNAs and FGR.
Maass et al. reported that some circRNAs of placenta might be related to pregnancy complications, such as fetal growth restriction, preeclampsia, HELLP syndrome, and diabetes22. Bai et al.1found some circRNAs, which were differentially expressed in placental tissue, contributed to the pathogenesis of preeclampsia. A placental villi circRNA screening from the Tang et al.29 identified 55 upregulated circRNAs and 59 downregulated circRNAs between GDM patients and normal pregnancies. CircRNAs can be used as miRNA sponges to regulate genes expression. Wang et al.29 provided hsa-circ-0000848 modulate the trophoblast cell function via the sponging of hsa-miR-370-3p. Nowadays, most studies of FGR focused on maternal causes. However, those placenta samples with maternal or fetal conditions leading to FGR were excluded from the study. According to our study, circRNAs may act as suitable biomarkers for uterine–placental perfusion, placental maldevelopment and insufficiency.
In this study, after all maternal and fetal causes were excluded from our samples, we characterized placenta-specific circRNAs in FGR. Firstly, by NGS, 18 placenta-specific circRNAs (hsa-circ-0005078, hsa-circ-0072697, hsa-circ-0084748, hsa-circ-0007440, hsa-circ-0005286, hsa-circ-0003288, hsa-circ-0035897, hsa-circ-0103279, hsa-circ-0005204, hsa-circ-0005939, hsa-circ-0006427, hsa-circ-0137008, hsa-circ-0006222, hsa-circ-0005238, hsa-circ-0088213, hsa-circ-0086190, hsa-circ-0002590, hsa-circ-0000972) were identified. Thus far, hsa-circ-0137008 was reported to suppress the malignant phenotype in colorectal cancer cells33. Other 17 circRNAs were reported for the first time.
Among the differentially expressed circRNAs in FGR, the qRT-PCR validation results of hsa-circ-0005238 attracted our attention. Hsa-circ-0005238 was notably decreased in the placentas of women with FGR, which has been not reported in pregnancy. Hence, we analyzed the functional role of hsa-circ-0005238 in trophoblast cells by migration, invasion, and apoptosis analysis. Our results showed that hsa-circ-0005238 overexpression inhibited, while its knockdown promoted apoptosis in trophoblast cells. Moreover, hsa-circ-0005238 overexpression promoted, while its knockdown repressed cell migration and invasion in trophoblast cells. During the pregnancy, placental trophoblasts cells move upstream along the arterial wall, replace the endothelium, and disrupt the muscular lining28. Only then, the trophoblast invasion during human placentation enables fetus to derive nutrition from mater. Therefore, we conjectured that hsa-circ-0005238 may play an important role in the pathogenesis of FGR. Nevertheless, its mechanisms were needed to be confirm by further experiments.
Based on the regulatory model of circRNAs as microRNAs sponge, the inhibitory effect of miRNAs on the target genes then disappears, and the expression of the target gene enhanced. This process is referred to as the competitive endogenous RNA (ceRNA) mechanism28. Firstly, we predicted potential targets of hsa-circ-0005238 by bioinformatic analysis and identified hsa-miR-370-3p as a potential target miRNA of hsa-circ-0005238. MiR-370-3p was revealed as a tumor promoter in breast cancer23 and gastric cancer32. It was pointed out that miR-370-3p was participate in fetal adrenal developmental programming5. However, how miR-370-3p are involved in FGR is unclear until now. Further, we found hsa-miR-370-3p was increased in the placentas of women with FGR. Moreover, hsa-circ-0005238 knockdown increased hsa-miR-370-3p expression in HTR-8 cells. Then hsa-circ-0005238 was confirmed as a sponge for hsa-miR-370-3p by luciferase reporter, AGO2 RIP assays and rescue experiment. MiRNAs inhibit target mRNA translation or participate in the degradation of target mRNA. In our work, hsa-miR-370-3p overexpression decreased CDC25B expression in HTR-8 cells. In addition, luciferase reporter proved CDC25B severd as a target mRNA for miR-370-3p in trophoblast cells. CDC25B is a member of phosphatases and key gene for entry into mitosis. When CDC25B is overexpressed, cells enter into mitosis before the usual time and show spindle abnormalities17. In contrast, depletion of CDC25B delays mitotic entry17. Lam17 reported a unique Chinese girl who presented with intrauterine growth retardation as well as adolescent delayed development and she was diagnosed as homozygous non-sense variant in the CDC25B gene by whole-exome sequencing analysis. It is suggested that CDC25B may be potentially therapeutic for FGR. Regrettably, we did not make a profound study the downstream pathway of the hsa-circ-0005283/ miR-370-3p /CDC25B axis, which can be investigated in the future.