Although the clinical pregnancy rate of in vitro fertilization-embryo transfer (IVF-ET) has reached approximately 50-60%, a portion of couples still suffer from recurrent implantation failure. Studies have demonstrated the pivotal role of lncRNAs in embryo implantation in mammals [7,9,20]. Based on the RNA-Seq results that have been obtained, we constructed a RIF related ceRNA network to propose potential diagnostic biomarkers or therapeutic targets. In total, 617 DEmRNAs, 69 DElncRNAs and 107 DEmiRNAs were identified, among which 381 mRNAs, 35 lncRNAs, and 66 miRNAs were upregulated, and 236 mRNAs, 34 lncRNAs, and 34 miRNAs were downregulated.
In general, the GO and KEGG analyses revealed that up-regulated genes enriched in regulation of transcription, DNA-templated and morphogenesis of an epithelium. Meanwhile, down-regulated genes enriched in cellular response to retinoic acid and canonical Wnt signaling pathway. The enriched pathways included fatty acid degradation and inositol phosphate metabolism. These biological events were involved in embryo implantation and identified by previous studies . The canonical Wnt signaling pathway, known as Wnt/β-catenin signaling pathway, is identified as a major signaling branch involved in the maintenance of endometrial receptivity . Moreover, the fatty acid degradation pathway is confirmed to be essential in endometrial stroma cell decidualization [23,24]. Inositol phosphate metabolism pathway may play an important role in regulating the estrogen-dependent activation of cellular events in the rat endometrium . Besides, the significant enrichment pathways of DElncRNAs were Inositol phosphate metabolism and Phosphatidylinositol signaling system, suggesting that inositol phosphate related pathways might be pivotal in the pathogenesis of RIF.
In addition, the PPI network was constructed and four high degree hub genes (GNG11, BDKRB2, PLCB1, THBS1) were identified. GNG11, also known as G protein subunit gamma 11, is an important component of the transmembrane signaling system . Ayusawa and colleagues has revealed that GNG11 modulates cellular senescence in normal human fibroblasts by oxidative stress . Their further study showed that increased GNG11 expression can induce reactive oxygen species (ROS) generation and abnormal nuclear morphology, thus inhibiting cell growth in SUSM-1 cells . Previous studies have demonstrated that oxidative stress and reactive oxygen species system is crucial in maintenance of endometrial receptivity [29,30]. Taken together, GNG11 may be a potential regulator in acquiring of endometrial receptivity. Bradykinin (BK) is a vasoactive peptide which participates in a variety of biological processes [31-33]. There are two subtypes of bradykinin receptor, BDKRB1 and BDKRB2, of which BDKRB2 has a high affinity with bradykinin. BDKRB2 has been reported to be involved in the epithelial-mesenchymal transition (EMT) process which is critical for multiple endometrial functions . PLCB1 has been identified to be essential for uterine preparation during embryo implantation and impaired expression of PLCB1 lead to embryo implantation failure in mice . Ramhorst et al displayed that vasoactive intestinal peptide (VIP) induces endometrial stromal cells decidualization and promotes angiogenesis by down-regulating thrombospondin-1 (THBS1) .
To investigate the role of lncRNAs in RIF, we constructed a lncRNA related ceRNA network. Three hub lncRNAs were PART1, PWRN1 and PGM5P3-AS1. PART1 and PWRN1 were confirmed to influence cell proliferation, apoptosis, migration and invasion in many malignant tumors [36,37]. These basic biological and pathological processes have also been proved to affect endometrial receptivity. Meanwhile, a study using bioinformatics approaches proposed that PGM5P3-AS1 may be associated with the progression of hepatocellular carcinoma . Furthermore, present study indicated three down-regulated miRNAs (hsa-miR-1207-5p, hsa-miR-134-5p, hsa-miR-1225-5p) and five up-regulated miRNAs (hsa-miR-30c-5p, hsa-miR-30b-5p, hsa-miR-145-5p, hsa-miR-21-5p, hsa-miR-196b-5p) in this ceRNA network. Hsa-miR-145 overexpression was found during the decidualization period in endometrium of recurrent implantation failure patients. Overexpressed hsa-miR-145 suppresses endometrial stromal cells decidualization through inhibition of Smad1 . Moreover, hsa-miR-145 affects embryo attachment by reducing the expression of IGF1R and PAI-1 in endometrial epithelial cells [40,41]. Additionally, miR-21 is highly expressed in mouse endometrial stromal cells at implantation sites and plays a key role in embryo implantation via regulating the target gene RECK . Referring to adenomyosis, overexpressed miR-21 can resume impaired decidualization through modulation of KLF12 and NR4A1 expression . Besides, the high level of miR-30 family in endometrial epithelial cells is essential for establishment of endometrial receptivity . These hub lncRNAs and miRNAs may regulate the development of RIF, whereas further investigations are still needed to validate the underlying mechanisms.
Above all, our study obtained 120 lncRNAs-miRNAs-mRNAs relationships in the ceRNA network. Numerous studies have identified that the lncRNAs-miRNAs-mRNAs regulation network functions in the pathogenesis of RIF and generation of receptive uterus [44-48]. The ceRNA network deserves more attention since it could contribute more to our understanding of embryo implantation. However, given that these results proposed in the study are only based on bioinformatic methods, in vitro experiments should be established to validate potential diagnostic and therapeutic targets for RIF.