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.     

Nidogen‐1 is a common target of microRNAs MiR‐192/215 in the pathogenesis of Hirschsprung's disease

Recent studies have emphasized the important role of microRNA (miRNA) clusters and common target genes in disease progression. Despite the known involvement of the miR‐192/215 family in many human diseases, its biological role in Hirschsprung disease (HSCR) remains undefined. In this study, we explored the role of the miR‐192/215 family in the pathogenesis of HSCR. Quantitative real‐time PCR and western blotting measured relative expression levels of miRNAs, mRNAs, and proteins in 80 HSCR patients and 77 normal colon tissues. Targets were evaluated by dual‐luciferase reporter assays, and the functional effects of miR‐192/215 on human 293T and SH‐SY5Y cells were detected by the Transwell assay, CCK8 assay and flow cytometry. MiR‐192/215 was significantly down‐regulated in HSCR tissue samples, and their knockdown inhibited cell migration and proliferation in the human 293T and SH‐SY5Y cell lines. Nidogen 1 (NID1) was confirmed as a common target gene of miR‐192/215 by dual‐luciferase reporter gene assay and its expression was inversely correlated with that of miR‐192/215 in tissue samples and cell lines. Silencing of NID1 could rescue the extent of the suppressing effects by miR‐192/215 inhibitor. The down‐regulation of miR‐192/215 may contribute to HSCR development by targeting NID1.

     

IGF2‐derived miR‐483‐3p associated with Hirschsprung's disease by targeting FHL1

HSCR (Hirschsprung's disease) is a serious congenital defect, and the aetiology of it remains unclear. Many studies have highlighted the significant roles of intronic miRNAs and their host genes in various disease, few was mentioned in HSCR although. In this study, miR‐483‐3p along with its host gene IGF2 (Insulin‐like growth factor 2) was found down‐regulated in 60 HSCR aganglionic colon tissues compared with 60 normal controls. FHL1 (Four and a half LIM domains 1) was determined as a target gene of miR‐483‐3p via dual‐luciferase reporter assay, and its expression was at a higher level in HSCR tissues. Here, we study cell migration and proliferation in human 293T and SH‐SY5Y cell lines by performing Transwell and CCK8 assays. In conclusion, the knockdown of miR‐483‐3p and IGF2 both suppressed cell migration and proliferation, while the loss of FHL1 leads to opposite outcome. Furthermore, miR‐483‐3p mimics could rescue the negative effects on cell proliferation and migration caused by silencing IGF2, while the FHL1 siRNA may inverse the function of miR‐483‐3p inhibitor. This study revealed that miR‐483‐3p derived from IGF2 was associated with Hirschsprung's disease by targeting FHL1 and may provide a new pathway to understand the aetiology of HSCR.     

Long non‐coding RNA FAL1 functions as a ceRNA to antagonize the effect of miR‐637 on the down‐regulation of AKT1 in Hirschsprung's disease

Objectives

Emerged evidence demonstrates that long non‐coding RNAs (lncRNAs) may play quintessential regulatory roles in the cellular processes, tumourigenesis and the development of disease. Though focally amplified lncRNA on chromosome 1 (FAL1) has been identified to have crucial functions in many diseases, its biological mechanism in the development of Hirschsprung's disease (HSCR) still remains unknown.

Materials and methods

The expression levels of FAL1 in HSCR aganglionic tissues and matched normal specimens were detected by quantitative real‐time PCR (qRT‐PCR). Cell proliferation and migration were detected by Cell Counting Kit‐8 (CCK‐8) assay, Ethynyl‐deoxyuridine (EdU) assay and transwell assay relatively. Cell cycle and apoptosis were assessed using flow cytometer analysis. Moreover, the novel targets of FAL1 were confirmed with the help of bioinformatics analysis and dual‐luciferase reporter assay. Western blot assay as well as RNA immunoprecipitation (RIP) assay was conducted to investigate the potential mechanism.

Results

FAL1 expression was markedly down‐regulated in HSCR aganglionic tissues and decreased FAL1 expression was associated with the diagnosis of HSCR. Cell functional analyses indicated that FAL1 overexpressing notably promoted cell proliferation and migration, while down‐regulation of FAL1 suppressed cell proliferation and migration. Additionally, Flow cytometry assay demonstrated that knockdown of FAL1 induced markedly cell cycle stalled in the G0/G1 phase. Furthermore, FAL1 could positively regulate AKT1 expression by competitively binding to miR‐637.

Conclusions

These results illuminated that FAL1 may work as a ceRNA to modulate AKT1 expression via competitively binding to miR‐637 in HSCR, suggesting that it may be clinically valuable as a biomarker of HSCR.

     

CircRNA circPDSS1 promotes the gastric cancer progression by sponging miR-186-5p and modulating NEK2

The aim of this study is to investigate the regulatory mechanism of circPDSS1/miR-186-5p/NEK2 axis on the viability and proliferation in gastric cancer (GC) cell line. Differentially expressed circRNAs, miRNAs, and mRNAs in GC tissues and paracarcinoma tissues were analyzed using gene chips GSE83521, GSE89143, and GSE93415. Then, the expression of circPDSS1, miR-186-5p, and NEK2 was analyzed via quantitative real-time polymerase chain reaction (qRT-PCR). Survival analysis was adopted to explore the association between the circPDSS1 expression and the prognosis of GC. The effect of circPDSS1 on GC cell cycle and apoptosis was verified with the flow cytometry. Targeting relationships among circPDSS1, miR-186-5p, and NEK2 were predicted via bioinformatics analysis and demonstrated by the dual-luciferase reporter assay. Our results showed that circPDSS1 and NEK2 were high-expressed whereas miR-186-5p was low-expressed in GC tissues and cells. CircPDSS1 promoted GC cell cycle and inhibited apoptosis by sponging miR-186-5p, while miR-186-5p inhibited cell cycle and promoted apoptosis by targeting NEK2. Thus, circPDSS1 acts as a tumor promoter by regulating miR-186-5p and NEK2, which could be a potential biomarker and therapeutic target for the management of GC.     

长非编码 RNA ILF3-AS1 通过调控 miR-130a-3p/PTEN轴抑制宫颈癌细胞的增殖

ILF3反义 RNA 1(ILF3 antisense RNA 1,ILF3-AS1)是一条定位于染色体 19p13.2的lncRNA,它是白介素增强子结合因子3(interleukin enhancer binding factor 3,ILF3)的反义 RNA.ILF3-AS1在多种肿瘤发生发展中发挥关键作用,但其...     

LINC00839 Promotes Neuroblastoma Progression by Sponging miR-454-3p to Up-Regulate NEUROD1

Neuroblastoma (NB) is the most common extracranial solid malignancy in children. Increasing long non-coding RNAs (lncRNAs) are reported to be associated with NB tumorigenesis and aggressiveness. Here, we attempted to investigate the biological functions of LINC00839 in NB progression as well as its possible pathogenic mechanisms. Public microarray datasets were applied to unearth the abnormally expressed lncRNAs in NB. RT-qPCR analysis was used to measure the expression of LINC00839, miR-454-3p, and neuronal differentiation 1 (NEUROD1) mRNA. The protein level was determined by a western blot assay. CCK-8, plate clone formation, EdU, wound-healing scratch, and transwell assays were employed to evaluate cell proliferation, migration, and invasion. Xenografts were developed in nude mice to determine the effects of LINC00839 on NB tumor growth. Dual-luciferase reporter and RNA immunoprecipitation (RIP) experiments were performed to identify the interaction between miR-454-3p and LINC00839 or NEUROD1. According to GSE datasets (GSE16237 and GSE16476), LINC00839 was found as a potential driver of NB progression. LINC00839 expression was higher in NB tumor tissues and cells. Also, LINC00839 expression was positively correlated with MYCN amplification, advanced INSS stages, and worse prognosis. Silencing of LINC00839 suppressed cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro. Mechanistically, LINC00839 could act as a sponge of miR-454-3p to facilitate the expression of its target NEUROD1. Moreover, miR-454-3p was demonstrated to exert an anti-cancer activity in NB. More importantly, the tumor-suppressive properties mediated by LINC00839 knockdown were significantly counteracted by the inhibition of miR-454-3p or overexpression of NEUROD1. Our study demonstrates that LINC00839 exerts an oncogenic role in NB through sponging miR-454-3p to up-regulate NEUROD1 expression, deepening our comprehension of lncRNA involved in NB and providing access to the possibility of LINC00839 as a therapeutic target for NB.

     

Long noncoding RNA FOXD2-AS1 promotes glioma cell cycle progression and proliferation through the FOXD2-AS1/miR-31/CDK1 pathway

Long noncoding RNAs (lncRNAs) are vital mediators involved in cancer progression. Previous studies confirmed that FOXD2 adjacent opposite strand RNA 1 (FOXD2-AS1) is upregulated in tumor diseases. The potential influence of FOXD2-AS1 in glioma progression, however, remains unknown. In this paper, FOXD2-AS1 was found to be upregulated in glioma tissues. Its level was linked with glioma stage. Moreover, glioma patients expressing high level of FOXD2-AS1 suffered worse prognosis. Biological functions of FOXD2-AS1 in glioma cells were analyzed through integrative bioinformatics and TCGA RNA sequencing data analysis. Pathway enrichment analysis uncovered that FOXD2-AS1 was mainly linked with cell cycle regulation in both low-grade glioma and glioblastoma. Further experiments demonstrated that silence of FOXD2-AS1 inhibited proliferation, arrested cell cycle and downregulated cyclin-dependent kinase 1 (CDK1) in human glioma cells. Dual-luciferase reporter assay confirmed that FOXD2-AS1 upregulated CDK1 by sponging miR-31. Rescue assays were performed and confirmed the regulatory loop FOXD2-AS1/miR-31/CDK1 in glioma. Collectively, our results indicated that the FOXD2-AS1/miR-31/CDK1 axis influenced glioma progression, providing a potential new target for glioma patients.     

MiR‐128‐1‐5p regulates tight junction induced by selenium deficiency via targeting cell adhesion molecule 1 in broilers vein endothelial cells

Vein endothelial cells (VECs) constitute an important barrier for macromolecules and circulating cells from the blood to the tissues, stabilizing the colloid osmotic pressure of the blood, regulating the vascular tone, and rapidly changing the intercellular connection, and maintaining normal physiological function. Tight junction has been discovered as an important structural basis of intercellular connection and may play a key role in intercellular connection injuries or vascular diseases and selenium (Se) deficiency symptoms. Hence, we replicated the Se‐deficient broilers model and detected the specific microRNA in response to Se‐deficient vein by using quantitative real time‐PCR (qRT‐PCR) analysis. Also, we selected miR‐128‐1‐5p based on differential expression in vein tissue and confirmed its target gene cell adhesion molecule 1 (CADM1) by the dual luciferase reporter assay and qRT‐PCR in VECs. We made the ectopic miR‐128‐1‐5p expression for the purpose of validating its function on tight junction. The result showed that miR‐128‐1‐5p and CADM1 were involved in the ZO‐1‐mediated tight junction, increased paracellular permeability, and arrested cell cycle. We presumed that miR‐128‐1‐5p and Se deficiency might trigger tight junction. Interestingly, miR‐128‐1‐5p inhibitor and fasudil in part hinder the destruction of the intercellular structure caused by Se deficiency. The miR‐128‐1‐5p/CADM1/tight junction axis provides a new avenue toward understanding the mechanism of Se deficiency, revealing a novel regulation model of tight junction injury in vascular diseases.     

CircRNA WHSC1 targets the miR‐646/NPM1 pathway to promote the development of endometrial cancer

Circular RNAs (circRNAs) play important roles in human cancer progression. Their high stability and tissue specificity make circRNAs important molecular targets for clinical diagnosis, treatment and prognosis. However, the functions and molecular mechanisms of circRNA WHSC1 in endometrial cancer are unknown. CircWHSC1 expression in normal endometrial and endometrial cancer tissues was detected using PCR. Overexpression or knockdown of circWHSC1 in endometrial cancer cell lines HEC‐1B or Ishikawa, respectively, cell function experiments were used to detect the impact of circWHSC1 on endometrial cancer cells. A nude mouse xenograft model was used to detect changes in tumorigenesis of HEC‐1B cells after circWHSC1 overexpression. Bioinformatics and dual luciferase reporter gene technology were used to predict and validate the sponging ability of circWHSC1 on microRNAs. Gene expression changes were detected by using Western blotting. CircWHSC1 expression was increased in endometrial cancer tissues. CircWHSC1 overexpression promoted the proliferation, migration and invasion of endometrial cancer cells and decreased apoptosis. CircWHSC1 knockdown had the opposite effect. CircWHSC1 overexpressed nude mice showed increased tumorigenicity. Bioinformatics predicted that circWHSC1 binds to miR‐646, which was confirmed using luciferase reporter gene assays. High expression of miR‐646 could reverse the effect of circWHSC1 on endometrial cancer cells. Western blotting showed increased or decreased levels of nucleophosmin 1 (NPM1), an miR‐646 downstream target, after circWHSC1 overexpression or knockdown, respectively. CircWHSC1 promotes endometrial cancer development through sponging miR‐646 and targeting NPM1.     

Aberrant expression of lncRNAs SNHG6, TRPM2-AS1, MIR4435-2HG,and hypomethylation of TRPM2-AS1 promoter in colorectal cancer

Accumulating evidence has indicated that deregulation of lncRNAs plays essential roles in colorectal cancer (CRC) carcinogenesis. The goal of this study was to analyze the expression of lncRNAs in colorectal cancer and their association with clinicopathological variables. Bioinformatics analysis of published CRC microarray data was performed to identify the important lncRNAs. The expression levels of candidate genes were assessed in the human colon cancer/normal cell lines, CRC, adenomatous colorectal polyps, and their marginal tissues by qRT-PCR. Moreover, the methylation status of the TRPM2-AS1 promoter was studied using qMSP assay. Furthermore, we investigated the molecular mechanisms of these lncRNAs in CRC progression using in silico analysis. Microarray analysis revealed that lncRNAs SNHG6, MIR4435-2HG, and TRPM2-AS1 were upregulated in CRC. These results were validated in colon cell lines. Moreover, qRT-PCR showed that the expression levels of SNHG6 and TRPM2-AS1 were upregulated in the colorectal tumor tissues compared with their paired tissues. Nonetheless, there was no significant increase in MIR4435-2HG expression in CRC samples. Furthermore, we observed a significant hypomethylation of TRPM2-AS1 promoter and its activation in CRC tissues. By in silico analysis, we found that the lncRNAs upregulation could promote proliferation and drug resistance of colorectal cancer cells via miRNAs sponging and modulation of their targets expression. In conclusion, based on our results upregulation of SNHG6 and TRPM2-AS1, and hypomethylation of TRPM2-AS1 promoter might be considered as potential diagnostic biomarkers for CRC initiation and development.     

LINC00963 targeting miR-128-3p promotes acute kidney injury process by activating JAK2/STAT1 pathway

The role of long non-coding RNAs (lncRNAs) in kidney diseases has been gradually discovered in recent years. LINC00963, as an lncRNA, was found to be involved in chronic renal failure. However, the role and molecular mechanisms of LINC00963 engaged in acute kidney injury (AKI) were still unclear. In this study, we established rat AKI models by ischaemia and reperfusion (I/R) treatment. Urea and creatinine levels were determined, and histological features of kidney tissues were examined following HE staining. CCK8 assay was chosen to assess the viability of hypoxia-induced HK-2 cells. Dual-luciferase reporter gene assays were performed to verify the target relationship between LINC00963 and microRNA. The mRNA and protein levels were assayed by RT-qPCR and Western blot, respectively. Annexin V-FITC/PI and TUNEL staining were used to evaluate apoptosis. LINC00963 was highly expressed in the cell and rat models, and miR-128-3p was predicted and then verified as a target gene of LINC00963. Knockdown of LINC00963 reduced acute renal injury both in vitro and in vivo. LINC00963 activated the JAK2/STAT1 pathway to aggravate renal I/R injury. LINC00963 could target miR-128-3p to reduce G1 arrest and apoptosis through JAK2/STAT1 pathway to promote the progression of AKI.     

LncRNA SNHG6 knockdown inhibits cisplatin resistance and progression of gastric cancer through miR-1297/BCL-2 axis

Cisplatin (DDP) resistance is a huge obstacle to gastric cancer (GC) treatment. Long non-coding RNAs (lncRNAs) have been manifested to exert pivotal functions in GC development. Herein, we aimed to explore the functional impact of lncRNA small nucleolar RNA host gene 6 (SNHG6) on DDP resistance and progression of GC. Quantitative real-time PCR (qRT-PCR) assay or Western blotting was performed to detect the expression of SNHG6, microRNA(miR)-1297, and epithelial–mesenchymal transition (EMT)-related factors and B-Cell Lymphoma 2 (Bcl-2) in DDP-resistant GC cells. Half inhibition concentration (IC50) to DDP, clonogenicity, apoptosis and invasion were examined via CCK-8 assay, colony formation assay, flow cytometry and Transwell assay, respectively. Target association between miR-1297 and SNHG6 or BCL-2 was demonstrated via dual-luciferase reporter assay or RIP assay. Xenograft models in nude mice were formed to investigate role of SNHG6 in vivo. We found that SNHG6 and BCL-2 were up-regulated, while miR-1297 expression was declined in GC tissues and DDP-resistant cells. Moreover, depletion of SNHG6 or gain of miR-1297 could repress DDP resistance, proliferation and metastasis of DDP-resistant cells, which was weakened by miR-1297 inhibition or BCL-2 overexpression. Besides, SNHG6 positively regulated BCL-2 expression by sponging miR-1297. Furthermore, SNHG6 knockdown repressed GC tumor growth in vivo. In a word, lncRNA SNHG6 knockdown had inhibitory effects on DDP resistance and progression of GC by sponging miR-1297, highlighting its potential in GC treatment.