LncRNA <Emphasis Type="Italic">ANRIL</Emphasis> Expression and <Emphasis Type="Italic">ANRIL</Emphasis> Gene Polymorphisms Contribute to the Risk of Ischemic Stroke in the Chinese Han Population

The aim of the present study was to explore the role of lncRNA ANRIL in the pathogenesis of ischemic stroke (IS) and coronary artery disease (CAD) and to determine the association between ANRIL variants and the genetic susceptibility of IS and CAD in the Chinese Han population. A genetic association study including 550 IS patients, 550 CAD patients, and 550 healthy controls was conducted. The expression levels of lncRNA ANRIL, CDKN2A, and CDKN2B were detected using qRT-PCR. Genotyping was performed by Sequenom MassARRAY on an Agena platform. Our study showed that IS patients had an increased lncRNA ANRIL expression (P?=?0.002) and a decreased CDKN2A expression (P?<?0.001) compared with normal controls. A significant difference with regard to the genotype distribution of rs2383207 was found between male IS patients and controls (P?=?0.011). The minor allele of rs2383207 significantly increased the IS risk under a recessive model (OR?=?1.52, 95% CI?=?1.05–2.21, P?=?0.027). The minor allele of rs1333049 was significantly associated with the risk of IS among the male patients under a recessive model (OR?=?1.56, 95% CI?=?1.04–2.35, P?=?0.031). However, no significant association was found between the ANRIL variants and the risk of CAD (all P?>?0.050). In addition, we found a decreased lncRNA ANRIL expression in IS patients who carried the GG genotype of rs1333049 compared with IS patients who carried the CC or CG genotype (P?=?0.041). In summary, we found that IS patients had an increased lncRNA ANRIL expression and a decreased CDKN2A expression compared with the controls, which might play an impellent role in pathological processes of IS. The ANRIL variants rs2383207 and rs1333049 were significantly associated with the risk of IS among males but not females in the Chinese Han population.     

Expression analysis of lncRNA <Emphasis Type="Italic">AK370814</Emphasis> involved in the barley vitamin B6 salvage pathway under salinity

Long non-coding RNAs (lncRNAs), which are longer than >?200 nt, perform various functions in a variety of important biological processes. The aim of this study is the investigation of relative expression levels of AK372815 putative pyridoxal reductase (PLR) gene and sense lncRNA AK370814 on four barley genotypes (Hasat, Beysehir 99, Konevi 98 and Tarm 92) in response to 150 mM salinity application during 3 days post-germination. Seeds were placed randomly in petri dishes containing (a) only H₂O (control), (b) 150 mM NaCl, for 72 h. RNA isolation was carried out using TriPure® reagent from 150 mM salt-treated root and shoot samples. Relative expression levels of AK372815 PLR and sense lncRNA AK370814 were determined by qPCR. Results demonstrated that salinity affected the expression levels of both AK372815 PLR gene and sense lncRNA AK370814 during germination. Although expression levels of AK372815 PLR tended to be down-regulated under salinity, expression levels of sense lncRNA AK370814 were up-regulated. Another goal of this study is improvement of alternative approach to NGS technologies for determination of relative expression levels of sense lncRNAs under particular circumstances. This is the first report that demonstrates a relationship between lncRNA and vitamin B6 salvage pathway.     

An Immune-Related Six-lncRNA Signature to Improve Prognosis Prediction of Glioblastoma Multiforme

Recent studies have demonstrated the utility and superiority of long non-coding RNAs (lncRNAs) as novel biomarkers for cancer diagnosis, prognosis, and therapy. In the present study, the prognostic value of lncRNAs in glioblastoma multiforme was systematically investigated by performing a genome-wide analysis of lncRNA expression profiles in 419 glioblastoma patients from The Cancer Genome Atlas (TCGA) project. Using survival analysis and Cox regression model, we identified a set of six lncRNAs (AC005013.5, UBE2R2-AS1, ENTPD1-AS1, RP11-89C21.2, AC073115.6, and XLOC_004803) demonstrating an ability to stratify patients into high- and low-risk groups with significantly different survival (median 0.899 vs. 1.611 years, p = 3.87e?09, log-rank test) in the training cohort. The six-lncRNA signature was successfully validated on independent test cohort of 219 patients with glioblastoma, and it revealed superior performance for risk stratification with respect to existing lncRNA-related signatures. Multivariate Cox and stratification analysis indicated that the six-lncRNA signature was an independent prognostic factor after adjusting for other clinical covariates. Further in silico functional analysis suggested that the six-lncRNA signature may be involved in the immune-related biological processes and pathways which are very well known in the context of glioblastoma tumorigenesis. The identified lncRNA signature had important clinical implication for improving outcome prediction and guiding the tailored therapy for glioblastoma patients with further prospective validation.     

Gene Ontology-based function prediction of long non-coding RNAs using bi-random walk

Background

With the development of sequencing technology, more and more long non-coding RNAs (lncRNAs) have been identified. Some lncRNAs have been confirmed that they play an important role in the process of development through the dosage compensation effect, epigenetic regulation, cell differentiation regulation and other aspects. However, the majority of the lncRNAs have not been functionally characterized. Explore the function of lncRNAs and the regulatory network has become a hot research topic currently.

Methods

In the work, a network-based model named BiRWLGO is developed. The ultimate goal is to predict the probable functions for lncRNAs at large scale. The new model starts with building a global network composed of three networks: lncRNA similarity network, lncRNA-protein association network and protein-protein interaction (PPI) network. After that, it utilizes bi-random walk algorithm to explore the similarities between lncRNAs and proteins. Finally, we can annotate an lncRNA with the Gene Ontology (GO) terms according to its neighboring proteins.

Results

We compare the performance of BiRWLGO with the state-of-the-art models on a manually annotated lncRNA benchmark with known GO terms. The experimental results assert that BiRWLGO outperforms other methods in terms of both maximum F-measure (F_max) and coverage.

Conclusions

BiRWLGO is a relatively efficient method to predict the functions of lncRNA. When protein interaction data is integrated, the predictive performance of BiRWLGO gains a great improvement.

     

Long non-coding RNA <Emphasis Type="Italic">HOTAIR</Emphasis> regulates proliferation and invasion via activating Notch signalling pathway in retinoblastoma

Retinoblastoma is the most frequently occurring tumour in the eyes in early childhood. Novel targets that are important for the diagnosis or treatment of retinoblastoma could be valuable in increasing the survival rate of patients affected by this disease. Long non-coding RNAs (lncRNAs) are a recently discovered type of RNAs with no protein-coding function; yet it has become increasingly clear that lncRNAs are responsible for important gene regulatory functions in various diseases. In this study, the expression of lncRNA HOTAIR was measured by qRT-PCR, and HOTAIR expression was found to be significantly upregulated in human retinoblastomas tissues as compared with that in paracancerous tissues. Knockdown of HOTAIR restricted the proliferation and invasion of the more invasive retinoblastoma Y79 cells, and led to G0/G1 arrest, possibly through inhibiting phospho-RB1, RB1 and CCNE. Furthermore, we found that the Notch signalling pathway was activated abnormally in retinoblastoma cell lines, while knockdown of HOTAIR attenuated the endogenous Notch signalling pathway in vitro and in vivo. In addition, knockdown of HOTAIR could inhibit the tumour progression in a xenograft model of retinoblastoma. In summary, our findings indicate that HOTAIR may play important roles in retinoblastoma progression via Notch pathway. HOTAIR has the potential to enhance the development of novel targeted diagnostic and therapeutic approaches for retinoblastoma.     

Novel link prediction for large-scale miRNA-lncRNA interaction network in a bipartite graph

Background

Current knowledge and data on miRNA-lncRNA interactions is still limited and little effort has been made to predict target lncRNAs of miRNAs. Accumulating evidences suggest that the interaction patterns between lncRNAs and miRNAs are closely related to relative expression level, forming a titration mechanism. It could provide an effective approach for characteristic feature extraction. In addition, using the coding non-coding co-expression network and sequence data could also help to measure the similarities among miRNAs and lncRNAs. By mathematically analyzing these types of similarities, we come up with two findings that (i) lncRNAs/miRNAs tend to collaboratively interact with miRNAs/lncRNAs of similar expression profiles, and vice versa, and (ii) those miRNAs interacting with a cluster of common target genes tend to jointly target at the common lncRNAs.

Methods

In this work, we developed a novel group preference Bayesian collaborative filtering model called GBCF for picking up a top-k probability ranking list for an individual miRNA or lncRNA based on the known miRNA-lncRNA interaction network.

Results

To evaluate the effectiveness of GBCF, leave-one-out and k-fold cross validations as well as a series of comparison experiments were carried out. GBCF achieved the values of area under ROC curve of 0.9193, 0.8354+/??0.0079, 0.8615+/??0.0078, and 0.8928+/??0.0082 based on leave-one-out, 2-fold, 5-fold, and 10-fold cross validations respectively, demonstrating its reliability and robustness.

Conclusions

GBCF could be used to select potential lncRNA targets of specific miRNAs and offer great insights for further researches on ceRNA regulation network.

     

Inhibition of breast cancer cell proliferation and tumorigenesis by long non-coding RNA <Emphasis Type="Italic">RPPH1</Emphasis> down-regulation of miR-122 expression

Background

Recent studies showed that long non-coding RNA (lncRNA) plays an important role in many life activities. RPPH1 is one of the lncRNA genes that are expressed differently between breast cancer and normal tissues by the lncRNA gene chip. Our study was conducted to examine the regulation of lncRNA RPPH1 in breast cancer.

Methods

Two cell lines, MCF-7 and MDA-MB-231, were selected to be the research objects in this study; RPPH1 overexpression and knockdown models were established by transforming vectors. Real-time polymerase chain reaction, MTT assay, clone formation and cell flow cytometer assay were used to test the function of RPPH1. Dual-luciferase assay was used to detect a target relationship between RPPH1 and miR-122.

Results

RPPH1 overexpression promoted cell cycle and proliferation and increased colony formation. In the RPPH1 overexpression model, there was a target relationship between RPPH1 and miR-122, and some of the downstream genes of miR-122, including ADAM10, PKM2, NOD2 and IGF1R, were increased. Moreover, we found that lentivirus-mediated interference of lncRNA RPPH1 inhibited tumour growth in nude mice.

Conclusion

Breast cancer progression can be promoted by directly targeting miR-122 through lncRNA RPPH1. This study provided evidence that can serve as the molecular basis for improving treatment options for patients.

     

Microarray analysis reveals a potential role of LncRNAs expression in cardiac cell proliferation

Background

Long non-coding RNAs (LncRNAs) have been identified to play important roles in epigenetic processes that underpin organogenesis. However, the role of LncRNAs in the regulation of transition from fetal to adult life of human heart has not been evaluated.

Methods

Immunofiuorescent staining was used to determine the extent of cardiac cell proliferation. Human LncRNA microarrays were applied to define gene expression signatures of the fetal (13–17 weeks of gestation, n?=?4) and adult hearts (30–40 years old, n?=?4). Pathway analysis was performed to predict the function of differentially expressed mRNAs (DEM). DEM related to cell proliferation were selected to construct a lncRNA-mRNA co-expression network. Eight lncRNAs were confirmed by quantificational real-time polymerase chain reaction (n?=?6).

Results

Cardiac cell proliferation was significant in the fetal heart. Two thousand six hundred six lncRNAs and 3079 mRNAs were found to be differentially expressed. Cell cycle was the most enriched pathway in down-regulated genes in the adult heart. Eight lncRNAs (RP11-119 F7.5, AX747860, HBBP1, LINC00304, TPTE2P6, AC034193.5, XLOC_006934 and AL833346) were predicted to play a central role in cardiac cell proliferation.

Conclusions

We discovered a profile of lncRNAs differentially expressed between the human fetal and adult heart. Several meaningful lncRNAs involved in cardiac cell proliferation were disclosed.

     

Expression profile of long non-coding RNAs during the differentiation of human umbilical cord derived mesenchymal stem cells into cardiomyocyte-like cells

We aimed to investigate the differentially expressed long non-coding RNAs (lncRNAs) during the differentiation of human umbilical cord derived mesenchymal stem cells (hUCMSCs) into cardiomyocyte-like cells induced by 5-aza. hUCMSCs were isolated and purified from umbilical cords. After treated with 10 μmol/L 5-Aza for 24 h, hUCMSCs wereas continued to be cultured for 14 days. Comparison of cardiac specific genes and the expression profile of lncRNAs on hUCMSCs between day 14 and day 0 was performed using immunofluorescence staining, immunohistochemistry, Western blot assay, RT-PCR and lncRNA microarray. Results show that well-organized sarcomeric structure and more cTnI and MLC2a staining were seen in hUCMSCs of day 14 after 5-aza-induced compared to those in day 0. Expression of Desmin, Nkx2.5, cTnI and MLC2a of hUCMSCs was much higher on day 14 compared with day 0 (P?<?0.01). 41 differentially expressed lncRNAs were found on day 14 hUCMSCs compared those of day 0 were identified. Among them, 25 upregulated and 16 downregulated. Four out of the five upregulated lncRNAs (P?=?0.00035, 0.014, 0.016 and 0.005 for uc010vei.1, X72487, BC064139, AK092074) and four out of the five downregulated lncRNAs (P?=?0.038, 0.0014, 0.00026 and 0.004 for X85157, uc007keu.1, AK309872, NR_029399) showed significantly different expressions in further validation using RT-PCR. Our results illustrated that there was a dysregulation of the lncRNA profile during the differentiation of hUCMSCs into cardiomyocyte-like cells, which will provide the foundation for further study of the biological functions and mechanism of lncRNAs in the differentiation of hUCMSCs into cardiomyocyte-like cells.     

Subchronic nandrolone administration reduces cardiac oxidative markers during restraint stress by modulating protein expression patterns

lncRNAs are an emerging class of regulators involved in multiple biological processes. MEG3, an lncRNA, acts as a tumor suppressor, has been reported to be linked with osteogenic differentiation of MSCs. However, limited knowledge is available concerning the roles of MEG3 in the multilineage differentiation of hASCs. The current study demonstrated that MEG3 was downregulated during adipogenesis and upregulated during osteogenesis of hASCs. Further functional analysis showed that knockdown of MEG3 promoted adipogenic differentiation, whereas inhibited osteogenic differentiation of hASCs. Mechanically, MEG3 may execute its role via regulating miR-140-5p. Moreover, miR-140-5p was upregulated during adipogenesis and downregulated during osteogenesis in hASCs, which was negatively correlated with MEG3. In conclusion, MEG3 participated in the balance of adipogenic and osteogenic differentiation of hASCs, and the mechanism may be through regulating miR-140-5p.     

Identification and association of novel lncRNA <Emphasis Type="BoldItalic">pouMU1</Emphasis> gene mutations with chicken performance traits

The biological functions of long noncoding RNAs (lncRNAs), which play an important role in regulating development and gene expression, may be affected by variations in lncRNA gene loci or associated genomic sequences. However, the functions of many lncRNAs remain unknown. To analyse correlations between mutations in pouMU1 with chicken growth and carcass traits, 860 chickens from a Gushi \(\times \) Anka F2 resource population and 96 Lushi, Xichuan, Changshun and recessive white chickens were used to evaluate the genetic effect of the pouMU1 gene. We performed quantitative real-time polymerase chain reaction (qRT-PCR) to analyse the relative expression levels of pouMU1 in nine different tissues and stages of development. pouMU1 expression was highest in pectoralis and leg muscles, whereas no expression was observed in the heart, liver and abdominal fat. Using direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods, two novel sequence mutations (g.1198A>G and g.1238-1239del/insGA) were detected in the pouMU1 gene. SPSS software was used for statistical analysis in association studies. Based on the association data, the presence of both variants was significantly associated with leg muscle fibre width and leg muscle fibre roundness (\(P < 0.05\)) and highly associated with leg muscle fibre girth and body weight at 0 week of age (\(P < 0.01\)). These data suggest that pouMU1 might participate in regulating chicken muscle development and growth, and the findings offer new insight into the functions of sequence mutations in lncRNAs.     

Suppression of long non-coding RNA CCAT2 improves tamoxifen-resistant breast cancer cells’ response to tamoxifen

Breast cancer is one of the most common cancers in women worldwide. Tamoxifen (TAM) provided a successful treatment for ER-positive (ER+) breast cancer for many years. However, ER+ patients with metastatic diseases respond poorly to TAM therapy and many initial responders eventually relapse. Emerging evidence indicates that long non-coding RNAs (lncRNAs) may have a critical role in the regulation of cellular processes such as cancer progression and metastasis, though the function of lncRNAs in TAM-resistance is still unclear. To investigate the role of CCAT2 in the development of resistance to TAM treatment of breast cancer, we established TAM-resistant models in MCF-7 and T47D cells. The present study demonstrates that TAM-resistant cells show a higher level of CCAT2 expression compared with TAM-sensitive cells. Biologically, CCAT2 knockdown could inhibit proliferation and induce apoptosis in TAM-resistant cells exposed to TAM. Furthermore, knockdown of CCAT2 improves the sensitivity to TAM in TAM-resistant cells. Overall, the study results provide a novel therapeutic approach for TAM-resistant patients through depleting CCAT2 expression.