miR-34a/c induce caprine endometrial epithelial cell apoptosis by regulating circ-8073/CEP55 via the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways

microRNAs (miRNAs) and circular RNAs (circRNAs) are important for endometrial receptivity establishment and embryo implantation in mammals. miR-34a and miR-34c are highly expressed in caprine receptive endometrium (RE). Herein, the functions and mechanisms of miR-34a/c in caprine endometrial epithelial cell (CEEC) apoptosis and RE establishment were investigated. miR-34a/c downregulated the expression level of centrosomal protein 55 (CEP55) and were sponged by circRNA8073 (circ-8073), thereby exhibiting a negative interaction in CEEC. miR-34a/c induced CEEC apoptosis by targeting circ-8073/CEP55 through the regulation of the RAS/RAF/MEK/ERK and phosphoitide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathways. Positive and negative feedback loops and cross-talk were documented between the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways. miR-34a/c regulated the levels of RE marker genes, including forkhead box M1, vascular endothelial growth factor, and osteopontin (OPN). These results suggest that miR-34a/c not only induce CEEC apoptosis by binding to circ-8073 and CEP55 via the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways, but may also regulate RE establishment in dairy goats.     

Circ-8073 regulates CEP55 by sponging miR-449a to promote caprine endometrial epithelial cells proliferation via the PI3K/AKT/mTOR pathway

Circular RNAs (circRNAs) are a large class of endogenous non-coding RNAs that function as regulators in various cells and tissues. Here, the function and mechanism of circRNA8073 (Circ-8073) on endometrial epithelial cells (EECs) and the development of endometrial receptivity were investigated in dairy goats. Circ-8073 could bind to and inhibit miR-449a activity. Circ-8073 binding to the target site of miR-449a had a negative feedback relationship. Centrosomal protein55 (CEP55) was a direct target gene of miR-449a, and Circ-8073 could increase the expression levels of CEP55 by sponging miR-449a in EECs in vitro. Circ-8073/miR-449a/CEP55 could promote EECs proliferation via the PI3K/AKT/mTOR pathway. In addition, CEP55 could regulate the expression levels of vascular endothelial growth factor (VEGF) and forkhead box M1 (FOXM1) in EECs, which contributed to the development of endometrial receptivity. These findings showed that Circ-8073 regulated CEP55 by sponging miR-449a to promote EEC proliferation via the PI3K/AKT/mTOR pathway, suggesting that it could function as a regulator in the development of endometrial receptivity in dairy goats.     

GnRH agonist treatment regulates IL-6 and IL-11 expression in endometrial stromal cells for patients with HRT regiment in frozen embryo transfer cycles

To evaluate the effect of gonadotropin-releasing hormone agonist (GnRHa) pretreatment time on clinical outcomes of patients who underwent endometrial preparation in HRT cycles and the molecular mechanism in frozen-thawed embryo transfer (FET) cycles, we retrospectively chose 1143 cycles and separated four groups. Endometrial tissues were collected from 44 patients who were cancelled on the day of embryo transfer (there were 10 patients refused to collect endometrium) and were tested for endometrial receptivity marker mRNA and miR-124-3p expression. Furthermore, endometrial stromal cells (ESCs) were transfected to investigate the molecular mechanism. The clinical pregnancy rate and live birth rate were significantly high in group B. The endometrial expression of the IL-6 and IL-11 mRNAs was significantly increased in groups with GnRHa pretreatment compared with group A without the GnRHa pretreatment. Similar results were obtained for the endometrial receptivity markers LIF and integrin αvβ3. The groups treated with GnRHa exhibited a progressive and significant time-dependent decrease in the IL-6 and IL-11 mRNA. In contrast, the levels of LIF and integrin αvβ3 expression remained unaltered among group B-D. In addition, transfection of ESCs with miR-124-3p mimics significantly reduced levels of the IL-6 and IL-11 mRNAs and proteins. The luciferase report assay demonstrated that IL-6 and IL-11 is a target gene of miR-124-3p. The results showed that ultra-long GnRHa administration can improve outcomes, especially after 3 cycles of GnRHa pretreatment, and endometrial receptivity through IL-6 and IL-11 expression levels of ESC regulated by the miR-124-3p for patients with HRT, who underwent FET cycles.     

The lncRNA small nucleolar RNA host gene 5 regulates trophoblast cell proliferation,invasion, and migration via modulating miR-26a-5p/N-cadherin axis

Pre-eclampsia (PE) is a pregnancy-specific disease characterized by the occurrence of hypertension and proteinuria after two weeks of gestation. Long noncoding RNAs (lncRNAs) are emerging as key regulators in PE development. This study aims to investigate the role of lncRNA, small nucleolar RNA host gene 5 (SNHG5), in the pathogenesis of PE. The expression of SNHG5 was significantly downregulated in placental tissues from patients with severe PE compared normal controls. Overexpression of SNHG5 promoted trophoblast (HTR-8/SVneo) cell proliferation, invasion, and migration, and flow cytometry results showed that SNHG5 overexpression inhibited apoptosis and caused a decrease of cell population at the G ₀/G ₁ phase and an increase of cell population at the S phase, while knockdown of SNHG5 had the opposite effects. The interaction between SNHG5 and miR-26a-5p was predicted by bioinformatics analysis and confirmed by luciferase reporter assay and RNA immunoprecipitation, and miR-26a-5p was negatively regulated by SNHG5; miR-26a-5p expression was upregulated in PE placental tissues and was inversely correlated with SNHG5 expression. Furthermore, miR-26a-5p was predicted to target the 3′ untranslated region of N-cadherin, which was confirmed by luciferase reporter assay, and miR-26a-5p overexpression suppressed N-cadherin expression in HTR-8/SVneo cells. N-cadherin mRNA expression was downregulated in PE placental tissues and was positively correlated with SNHG5 expression. Both overexpression of miR-26a-5p and knockdown of N-cadherin suppressed HTR-8/SVneo cell invasion and migration, and also attenuated the effects of SNHG5 on the cellular functions of HTR-8/SVneo cells. In conclusion, our study suggested that SNHG5 promotes trophoblast cell proliferation, invasion, and migration at least partly via regulating the miR-26a-5p/N-cadherin axis.     

Upregulated lncRNA Gm2044 inhibits male germ cell development by acting as miR-202 host gene

Long non-coding RNAs (lncRNAs) have been found to participate in the regulation of human spermatogenic cell development. However, little is known about the abnormal expression of lncRNAs associated with spermatogenic failure and their molecular mechanisms. Using lncRNA microarray of testicular tissue for male infertility and bioinformatics methods, we identified the relatively conserved lncRNA Gm2044 which may play important roles in non-obstructive azoospermia. The UCSC Genome Browser showed that lncRNA Gm2044 is the miR-202 host gene. This study revealed that lncRNA Gm2044 and miR-202 were significantly increased in non-obstructive azoospermia of spermatogonial arrest. The mRNA and protein levels of Rbfox2, a known direct target gene of miR-202, were regulated by lncRNA Gm2044. Furthermore, the miR-202-Rbfox2 signalling pathway was shown to mediate the suppressive effects of lncRNA Gm2044 on the proliferation of human testicular embryonic carcinoma cells. Understanding of the molecular signalling pathways for lncRNA-regulated spermatogenesis will provide new clues into the pathogenesis and treatment of patients with male infertility.     

LncRNA DANCR promotes cervical cancer progression by upregulating ROCK1 via sponging miR-335-5p

Emerging evidence highlights the key regulatory roles of long noncoding RNAs (lncRNAs) in the initiation and progression of numerous malignancies. The lncRNA identified as differentiation antagonizing nonprotein coding RNA (DANCR) is a novel lncRNA widely involved in the development of multiple human cancers. However, the function of DANCR and its potential molecular mechanism in cervical cancer remain unclear. In this study, we discovered that DANCR was significantly elevated in cervical cancer tissues and cells, and was closely correlated with poor prognosis of cervical cancer patients. In addition, knockdown of DANCR inhibited proliferation, migration, and invasion of cervical cancer cells in vitro, indicating that DANCR functioned as an oncogene in cervical cancer. Moreover, we verified that DANCR could directly bind to miR-335-5p, isolating miR-335-5p from its target gene Rho-associated coiled-coil containing protein kinase 1 (ROCK1). Functional analysis showed that DANCR regulated ROCK1 expression by competitively binding to miR-335-5p. Further cellular behavioral experiments revealed that miR-335-5p mimics and ROCK1 knockdown reversed the effects of upregulated DANCR on proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of cervical cancer cells by rescue assays. In summary, this study demonstrated that DANCR promoted cervical cancer progression by functioning as a competing endogenous RNA (ceRNA) to regulate ROCK1 expression via sponging miR-335-5p, suggesting a novel potential therapeutic target for cervical cancer.     

Microarray expression profiles analysis revealed lncRNA OXCT1-AS1 promoted bladder cancer cell aggressiveness via miR-455-5p/JAK1 signaling

Bladder cancer (BCa) is one of the most prevalent cancers of the urinary system worldwide. Accumulating evidence suggests that long noncoding RNAs (lncRNAs) perform a vital function in the pathogenesis and progression of BCa. In the current study, we identified a novel lncRNA OXCT1-AS1 and investigated its role and potential mechanisms in BCa. The microarray results showed the expression of lncRNAs, microRNAs, and messenger RNAs between BCa primary tumor tissues and metastatic lymph nodes were significantly different. The quantitative polymerase chain reaction verification was performed to ensure the reliability of the screening results. The Cell Counting Kit 8 and transwell assay were used to assess the tumor cell proliferation and invasion abilities in vitro, respectively. The dual-luciferase activity assay was performed to investigate the potential mechanism of competing endogenous RNA network. lncRNA OXCT1-AS1, which elevated in metastasis lymph node, was significantly upregulated in BCa cell lines compared with SVHUC-1. We demonstrated OXCT1-AS1 inhibited miR-455-5p to decrease its binding to the JAK1 3′-untranslated region, which could upregulate the expression of JAK1 at the protein level, thus promoting BCa proliferation and invasion. Therefore, lncRNA OXCT1-AS1 could act as a potential biomarker and therapeutic target for patients with BCa.     

The decreased lncRNA ZEB2-AS1 in pre-eclampsia controls the trophoblastic cell line HTR-8/SVneo's invasive and migratory abilities via the miR-149/PGF axis

Pre-eclampsia (PE) is a pregnancy disease that causes maternal death and threatens the health of newborns. Accumulating evidence has revealed the essential roles of long noncoding RNAs (lncRNAs) in the progression of PE. The present investigation determined lncRNA ZEB2 antisense RNA 1 (ZEB2-AS1) expression in PE and looked into the potential role of ZEB2-AS1 in modulating trophoblastic cell functions. Quantitative real-time polymerase chain reaction evaluated gene expression. Western blot analyzed the placental growth factor (PGF) protein level. Cell counting kit-8 and Transwell invasion assays assessed the proliferative and invasive abilities of placental trophoblast cells, respectively. Wound healing assay determined cell migratory potentials. Dual-luciferase reporter assay assessed the targeting relationship among ZEB2-AS1, miR-149, and PGF. Downregulation of lncRNA ZEB2-AS1 was detected in placentas from patients with PE when compared with those from normal pregnancies. Moreover, ZEB2-AS1 upregulation markedly promoted proliferative, migratory, and invasive potentials in HTR-8/SVneo cells, while knockdown of ZEB2-AS1 had the opposite effects. The effects on HTR-8/SVneo cells mediated by ZEB2-AS1 was correlated with the miR-149/PGF axis. These findings indicate that ZEB2-AS1 contributes to PE progression by affecting cell proliferative and invasive capacities via the miR-149/PGF axis in HTR-8/SVneo cells. In sum, we identified that ZEB2-AS1 was a novel aberrantly expressed lncRNA in the placentas of PE patients and lncRNA ZEB2-AS1 modulated trophoblastic cell line HTR-8/SVneo's proliferative and invasive potentials via targeting the miR-149/PGF axis.     

Immunolocalisation of phosphorylated STAT3, interleukin 11 and leukaemia inhibitory factor in endometrium of women with unexplained infertility during the implantation window

Background  

Uterine receptivity and embryo implantation are critical in the establishment of pregnancy. The diagnosis of endometrial fertility requires more precise measurements of endometrial receptivity. Interleukin (IL-11) and leukemia inhibitory factor (LIF) are essential for murine implantation and signal via intracellular phosphorylation (p) of STAT3 in the endometrium. Both cytokines are present in the endometrium of women duiring the receptive window. Endometrial IL-11, IL-11 receptor alpha (IL-11Ralpha), LIF and pSTAT3 in women with primary unexplained infertility was compared to normal fertile women during the implantation window.     

长链非编码RNA的微RNA海绵作用与呼吸系统疾病

长链非编码RNA（long non-coding RNAs, lncRNAs）是一类由长度大于200个核苷酸组成的长链非编码序列。lncRNAs具有较长的序列,使得lncRNAs具有复杂的二级及三级结构,这也是lncRNAs结合DNA、RNA和蛋白质及其行使复杂功能的结构基础。MicroRNA（miRNAs)是长度在19到25个核苷酸之间的非编码单链RNA分子,是目前研究最多的小分子非编码RNA。而lncRNAs通过结合或者螯合miRNA来调节miRNA丰度,发挥lncRNA的“海绵”作用,从而调控一系列的病理生理过程。lncRNAs及miRNA在呼吸系统疾病的发生、发展、治疗和预后起重要作用。本文就lncRNAs及其“海绵”作用对呼吸系统疾病的影响及可能的机制进行综述。     

LINC01116 promotes proliferation and migration of endometrial stromal cells by targeting FOXP1 via sponging miR-9-5p in endometriosis

Endometriosis is a common multi-factorial gynaecological disease. Recent studies have revealed that long non-coding RNAs (lncRNAs) are involved in the pathogenesis of endometriosis. In the present study, the expression profiles of lncRNAs in 6 pairs of endometriosis ectopic endometrium (ecEM) and eutopic endometrium (euEM) tissues were analysed by RNA sequencing. From the profiles, LINC01116 was found to be up-regulated in ecEM tissues compared to euEM tissues and was verified by quantitative real-time PCR (qRT-PCR). Then, functional experiments demonstrated that LINC01116 promoted the proliferation and migration of ectopic primary endometrial stromal cells (ESCs), while miR-9-5p exerted the opposite effects. Dual-luciferase reporter assays verified that LINC01116 directly sponged miR-9-5p and relieved the suppression of its target, Forkhead box protein P1 (FOXP1). Rescue experiments further demonstrated that LINC01116 could promote proliferation and migration of ESCs by targeting FOXP1 via sponging miR-9-5p. Overall, our study illuminates that LINC01116 promotes the progression of endometriosis through the miR-9-5p/FOXP1 axis. This finding provides a novel therapeutic target for patients with endometriosis.     

Combined analysis of microRNome and 3'-UTRome reveals a species-specific regulation of progesterone receptor expression in the endometrium of rhesus monkey

The establishment of endometrial receptivity is a prerequisite for successful pregnancy, which is controlled by a complex mechanism. MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as important regulators of gene expression. However, the contribution of miRNAs in endometrial receptivity is still unknown. Here we used rhesus monkey as an animal model and compared the endometrial miRNA expression profiles during early-secretory (pre-receptive) phase and mid-secretory (receptive) phase by deep sequencing. A set of differentially expressed miRNAs were identified, 8 of which were selected and validated using quantitative RT-PCR. To facilitate the prediction of their target genes, the 3'-UTRome was also determined using tag sequencing of mRNA 3'-termini. Surprisingly, about 50% of the 10,677 genes expressed in the rhesus monkey endometrium exhibited alternative 3'-UTRs. Of special interest, the progesterone receptor (PGR) gene, which is necessary for endometrial receptivity, processes an ultra long 3'-UTR (~10 kb) along with a short variant (~2.5 kb). Evolutionary analysis showed that the 3'-UTR sequences of PGR are poorly conserved between primates and rodents, suggesting a species-biased miRNA binding pattern. We further demonstrated that PGR is a valid target of miR-96 in rhesus monkey and human but not in rodents, whereas the regulation of PGR by miR-375 is rhesus monkey-specific. Additionally, we found that miR-219-5p regulates PGR expression through a primate-specific long non-coding RNA immediately downstream of the PGR locus. Our study provides new insights into the molecular mechanisms underlying endometrial receptivity and presents intriguing species-specific regulatory roles of miRNAs.     

LncRNA SNHG20 contributes to cell proliferation and invasion by upregulating ZFX expression sponging miR-495-3p in gastric cancer

Long noncoding RNAs (lncRNAs) exert key regulators in cancer development and progression. The functional significance of lncRNA small nucleolar RNA host gene 20 (SNHG20) was reported in gastric cancer (GC); however, the underlying molecular mechanism in GC development is largely unknown. Here, our results showed that the lncRNA SNHG20 expression was significantly higher in GC tissues compared with adjacent normal tissues by quantitative real-time PCR (qRT-PCR) analysis. Higher lncRNA SNHG20 expression was highly associated with tumor size and lymphatic metastasis of patients. Patients with higher lncRNA SNHG20 expression predicted a short disease-free survival (DFS) and overall survival (OS). Furthermore, lncRNA SNHG20 expression negatively associated with miR-495-3p expression and regulated miR-495-3p expression. Function assays confirmed that lncRNA SNHG20 knockdown using RNA interference suppressed cell proliferation and invasion of GC by negatively regulating miR-495-3p expression. Moreover, we demonstrated that lncRNA SNHG20 inhibited zinc finger protein X-linked (ZFX) expression by negatively miR-495-3p expression in GC cells. In vivo, the current study also indicated that lncRNA SNHG20 knockdown reduced the tumor growth by downregulating ZFX expression. Thus, our results implied that inhibition of SNHG20/miR-495-3p/ZFX axis may provide valuable target for GC treatment.     

Administration of dexamethasone disrupts endometrial receptivity by alteration of expression of miRNA 223, 200a,LIF, Muc1, SGK1, and ENaC via the ERK1/2-mTOR pathway

Successful implantation of embryos requires endometrial receptivity. Glucocorticoids are one of the factors influencing the implantation window. In this study, 40 female BALB/c mice were used to study the impacts of dexamethasone administration on endometrial receptivity markers during implantation window. The mice mated and were randomly divided into four groups: control (vehicle), dexamethasone (100 μg/kg, IP), PP242 (30 mg/kg, IP), and dexamethasone + PP242 (Dex + PP242). On the Day 4th and 5th of gestation, mice received their respective treatments and were killed on the 5th day. To assess the expression of Muc1, leukemia inflammatory inhibitor (LIF), serum/glucocorticoid-inducible kinase 1 (SGK1), epithelial Na+ channel (ENaC), miRNA 200a, and miRNA 223-3p in the endometrium real-time polymerase chain reaction was performed. Furthermore, using Western blot analysis protein expressions of extracellular signal-regulated kinase 1/2 (ERK1/2), mammalian target of rapamycin (mTOR), and euk皓aryotic translation initiation factor 4E-binding protein 1 (4E-BP1) were evaluated. Periodic Acid-Schiff staining was used to examine the histomorphological changes of the uterus. According to the results dexamethasone declined the expression of LIF, whereas upregulated expression of Muc1, SGK1, ENaC mRNA, miRNA 200a, and miRNA 223-3p in the endometrium. In addition, PP242, an mTOR inhibitor, induced mRNA expression of Muc1, miRNA200a, and miRNa223-3p whereas it declined the expression of LIF. Moreover, activity of the ERK1/2-mTOR pathway in the endometrial cells was deterred by dexamethasone and PP242. Nonstop epithelium proliferation and elevated surface glycoproteins layer on epithelium of dexamethasone and/or PP242-received groups were divulged through histochemical analysis. According to the above mentioned results, uterine receptivity during implantation period was declined by dexamethasone, at least in part, through modulation of involved genes in endometrial receptivity and inhibition of the ERK1/2-mTOR pathway.