Down‐regulated lncRNA SLC25A5‐AS1 facilitates cell growth and inhibits apoptosis via miR‐19a‐3p/PTEN/PI3K/AKT signalling pathway in gastric cancer

Mounting evidence has illustrated the vital roles of long non‐coding RNAs (lncRNAs in gastric cancer (GC). Nevertheless, the majority of their roles and mechanisms in GC are still largely unknown. In this study, we investigate the roles of lncRNA SLC25A5‐AS1 on tumourigenesis and explore its potential mechanisms in GC. The results showed that the expressions of SLC25A5‐AS1 in GC were significantly lower than that of adjacent normal tissues, which were significantly associated with tumour size, TNM stage and lymph node metastasis. Moreover, SLC25A5‐AS1 could inhibit GC cell proliferation, induce G1/G1 cell cycle arrest and cell apoptosis in vitro, as well as GC growth in vivo. Dual‐luciferase reporter assay confirmed the direct interaction between SLC25A5‐AS1 and miR‐19a‐3p, rescue experiment showed that co‐transfection miR‐19a‐3p mimics and pcDNA‐SLC25A5‐AS1 could partially restore the ability of GC cell proliferation and the inhibition of cell apoptosis. The mechanism analyses further found that SLC25A5‐AS1 might act as a competing endogenous RNAs (ceRNA), which was involved in the derepression of PTEN expression, a target gene of miR‐19a‐3p, and regulate malignant phenotype via PI3K/AKT signalling pathway in GC. Taken together, this study indicated that SLC25A5‐AS1 was down‐regulated in GC and functioned as a suppressor in the progression of GC. Moreover, it could act as a ceRNA to regulate cellular behaviours via miR‐19a‐3p/PTEN/PI3K/AKT signalling pathway. Thus, SLC25A5‐AS1 might be served as a potential target for cancer therapeutics in GC.     

Long non‐coding RNA NR2F1‐AS1 promoted proliferation and migration yet suppressed apoptosis of thyroid cancer cells through regulating miRNA‐338‐3p/CCND1 axis

Thyroid cancer (TC) is a prevalent endocrine malignant cancer whose pathogenic mechanism remains unclear. The aim of the study was to investigate the roles of long non‐coding RNA (lncRNA) NR2F1‐AS1/miRNA‐338‐3P/CCND1 axis in TC progression. Differentially expressed lncRNAs and mRNAs in TC tissues were screened out and visualized by R program. Relative expression of NR2F1‐AS1, miRNA‐338‐3p and cyclin D1 (CCND1) was determined by quantitative real time polymerase chain reaction. In addition, Western blot analysis was adopted for evaluation of protein expression of CCND1. Targeted relationships between NR2F1‐AS1 and miRNA‐338‐3p, as well as miRNA‐338‐3p and CCND1 were predicted using bioinformatics analysis and validated by dual‐luciferase reporter gene assay. Besides, tumour xenograft assay was adopted for verification of the role of NR2F1‐AS1 in TC in vivo. NR2F1‐AS1 and CCND1 were overexpressed, whereas miRNA‐338‐3p was down‐regulated in TC tissues and cell lines. Down‐regulation of NR2F1‐AS1 and CCND1 suppressed proliferation and migration of TC cells yet greatly enhanced cell apoptotic rate. Silence of NR2F1‐AS1 significantly suppressed TC tumorigenesis in vivo. NR2F1‐AS1 sponged miRNA‐338‐3p to up‐regulate CCND1 expression to promote TC progression. Our study demonstrated that up‐regulation of NR2F1‐AS1 accelerated TC progression through regulating miRNA‐338‐3P/CCND1 axis.     

LncRNA NR2F1‐AS1 regulates hepatocellular carcinoma oxaliplatin resistance by targeting ABCC1 via miR‐363

Emerging evidence has validated the vital role of long non‐coding RNA (lncRNA) in the chemoresistance of cancer treatment. In the present study, we investigate the function of lncRNA NR2F1‐AS1 on oxaliplatin (OXA) resistance of hepatocellular carcinoma (HCC) and discover the underlying molecular mechanism. Results revealed that lncRNA NR2F1‐AS1 was up‐regulated in oxaliplatin‐resistant HCC tissue and cells using microarray analysis and RT‐PCR. Meanwhile, ABCC1 protein was overexpressed in OXA‐resistant HCC cells (Huh7/OXA and HepG2/OXA). In vitro, NR2F1‐AS1 knockdown reduced the invasion, migration, drug‐resistant gene (MDR1, MRP5, LRP1) and IC50 value in Huh7/OXA and HepG2/OXA cells. In vivo, NR2F1‐AS1 knockdown decreased the tumour weight of HCC cells. Bioinformatics tools and luciferase reporter assay confirmed miR‐363 targeted the 3′‐UTR of NR2F1‐AS1 and ABCC1 mRNA, presenting that NR2F1‐AS1 promoted ABCC1 expression through endogenous sponging miR‐363. In summary, results conclude that NR2F1‐AS1 regulates HCC OXA resistance through targeting miR‐363‐ABCC1 pathway, providing a vital theoretic mechanism and therapeutic target for HCC chemoresistance.     

Loss of PPM1F expression predicts tumour recurrence and is negatively regulated by miR‐590‐3p in gastric cancer

Objectives

MicroRNAs (miRNAs) as small non‐coding RNA molecules act by negatively regulating their target genes. Recent studies have shown that protein phosphatase Mg2+/Mn2+‐dependent 1F (PPM1F) plays a critical role in cancer metastasis. But, the regulation mechanisms of PPM1F by miRNAs in gastric cancer (GC) remain undefined.

Methods

The correlation of PPM1F or miR‐590‐3p (miR‐590) expression with clinicopathological features and prognosis of the patients with GC was analysed by TCGA RNA‐sequencing data. The miRNAs that target PPM1F gene were identified by bioinformatics and Spearman correlation analysis, and the binding site between miR‐590 and PPM1F 3′UTR was confirmed by dual luciferase assay. MTT and Transwell assays were conducted to evaluate the effects of miR‐590 or (and) PPM1F on cell proliferation and invasion.

Results

We found that PPM1F expression was downregulated in GC tissues and cell lines and was correlated with tumour recurrence in patients with GC. The decreased expression of PPM1F was attributed to the dysregulation of miR‐590 expression rather than its genetic or epigenetic alterations. Overexpression of miR‐590 promoted cell proliferation and invasion capability of GC cells, while knockdown of miR‐590 reversed these effects. Moreover, PPM1F was validated as a direct target of miR‐590 and counteracted the tumour‐promoting effects caused by miR‐590. The expression of miR‐590 presented the negative correlation with PPM1F expression and acted as an independent prognostic factor for tumour recurrence in patients with GC.

Conclusion

PPM1F may function as a suppressive factor and is negatively regulated by miR‐590 in GC.

     

Long non‐coding RNA AFAP1‐AS1/miR‐320a/RBPJ axis regulates laryngeal carcinoma cell stemness and chemoresistance

AFAP1‐AS1 is a long non‐coding RNA that is associated with tumorigenesis and poor prognosis in a variety of cancers. We have been suggested that AFAP1‐AS1 increases tumorigenesis in laryngeal carcinoma specifically by enhancing stemness and chemoresistance. We assessed AFAP1‐AS1 expression in human laryngeal specimens, paired adjacent normal tissues and human HEp‐2 cells. Indeed, we found not only that AFAP1‐AS1 was up‐regulated in laryngeal carcinoma specimens and cells, but also that stemness‐associated genes were overexpressed. Silencing of AFAP1‐AS1 promoted HEp‐2 cell chemoresistance under cisplatin treatment. Expression of AFAP1‐AS1 was increased in drug‐resistant Hep‐2 cells. We then probed the mechanism of AFAP1‐AS1 activity and determined that miR‐320a was a potential molecular target of AFAP1‐AS1. Luciferase reporter and qRT‐PCR assays of AFAP1‐AS1 and miR‐320a levels in human specimens and cell cultures indicated that AFAP1‐AS1 negatively regulates miR‐320a. To discover the molecular mechanism of miR‐320a, we again used the DIANA Tools algorithm to predict its genetic target, RBPJ. After cloning the 3′‐untranslated regions (3′‐UTR) of RBPJ into a luciferase reporter, we determined that miR‐320a did in fact reduce RBPJ mRNA and protein levels. Ultimately, we determined that AFAP1‐AS1 increases RBPJ expression by negatively regulating miR‐320a and RBPJ overexpression rescues stemness and chemoresistance inhibited by AFAP1‐AS1 silencing. Taken together, these results suggest that AFAP1‐AS1 can serve as a prognostic biomarker in laryngeal carcinoma and that miR‐320a has the potential to improve standard therapeutic approaches to the disease, especially for cases in which cancer cell stemness and drug resistance present significant barriers to effective treatment.     

Epigenetic silenced miR‐125a‐5p could be self‐activated through targeting Suv39H1 in gastric cancer

Emerging evidence suggests that microRNAs (miRNAs) serve an important role in tumorigenesis and development. Although the low expression of miR‐125a‐5p in gastric cancer has been reported, the underlying mechanism remains unknown. In the current study, the low expression of miR‐125a‐5p in gastric cancer was verified in paired cancer tissues and adjacent non‐tumour tissues. Furthermore, the GC islands in the miR‐125a‐5p region were hypermethylated in the tumour tissues. And the hypermethylation was negatively correlated with the miR‐125a‐5p expression. Target gene screening showed that the histone methyltransferase Suv39H1 was one of the potential target genes. In vitro studies showed that miR‐125a‐5p could directly suppress the Suv39H1 expression and decrease the H3K9me3 levels. On the other hand, the Suv39H1 could induce demethylation of miR‐125a‐5p, resulting in re‐activation of miR‐125a‐5p. What is more, overexpessing miR‐125a‐5p could also self‐activate the silenced miR‐125a‐5p in gastric cancer cells, which suppressed cell migration, invasion and proliferation in vitro and inhibited cancer progression in vivo. Thus, we uncovered here that the epigenetic silenced miR‐125a‐5p could be self‐activated through targeting Suv39H1 in gastric cancer, suggesting that miR‐125a‐5p might be not only the potential prognostic value as a tumour biomarker but also potential therapeutic targets in gastric cancer.     

miR‐124 interacts with the Notch1 signalling pathway and has therapeutic potential against gastric cancer

Aberrant Notch signalling plays an important role in cancer progression. However, little is known about the interaction between miRNA and the Notch signalling pathway and its role in gastric cancer (GC). In this study, we found that miR‐124 was down‐regulated in GC compared with adjacent normal tissue. Forced expression of miR‐124 inhibited GC cell growth, migration and invasion, and induced cell cycle arrest. miR‐124 negatively regulated Notch1 signalling by targeting JAG1. miR‐124 levels were also shown to be inversely correlated with JAG1 expression in GC. Furthermore, we found that the overexpression of the intracellular domain of Notch1 repressed miR‐124 expression, promoted GC cell growth, migration and invasion. Conversely, blocking Notch1 using a γ‐secretase inhibitor up‐regulated miR‐124 expression, inhibited GC cell growth, migration and invasion. In conclusion, our data demonstrates a regulatory feedback loop between miR‐124 and Notch1 signalling in GC cells, suggesting that the miR‐124/Notch axis may be a potential therapeutic target against GC.     

Long non‐coding RNA FEZF1‐AS1 promotes breast cancer stemness and tumorigenesis via targeting miR‐30a/Nanog axis

Long non‐coding RNAs (lncRNAs) have been verified to modulate the tumorigenesis of breast cancer at multiple levels. In present study, we aim to investigate the role of lncRNA FEZF1‐AS1 on breast cancer‐stem like cells (BCSC) and the potential regulatory mechanism. In breast cancer tissue, lncRNA FEZF1‐AS1 was up‐regulated compared with controls and indicated poor prognosis of breast cancer patients. In vitro experiments, FEZF1‐AS1 was significantly over‐expressed in breast cancer cells, especially in sphere subpopulation compared with parental subpopulation. Loss‐of‐functional indicated that, in BCSC cells (MDA‐MB‐231 CSC, MCF‐7 CSC), FEZF1‐AS1 knockdown reduced the CD44⁺/CD24^? rate, the mammosphere‐forming ability, stem factors (Nanog, Oct4, SOX2), and inhibited the proliferation, migration and invasion. In vivo, FEZF1‐AS1 knockdown inhibited the breast cancer cells growth. Bioinformatics analysis tools and series of validation experiments confirmed that FEZF1‐AS1 modulated BCSC and Nanog expression through sponging miR‐30a, suggesting the regulation of FEZF1‐AS1/miR‐30a/Nanog. In summary, our study validate the important role of FEZF1‐AS1/miR‐30a/Nanog in breast cancer stemness and tumorigenesis, providing a novel insight and treatment strategy for breast cancer.     

LINC01303 functions as a competing endogenous RNA to regulate EZH2 expression by sponging miR‐101‐3p in gastric cancer

Long non‐coding RNA (lncRNA) is one of the important regulators of many malignancies. However, the biological function and clinical significance of a large number of lncRNAs in gastric cancer remain unclear. Therefore, we analysed the TCGA data to find that LINC01303 is significantly up‐regulated in gastric cancer tissues. However, the biological function of LINC01303 in GC remains unknown. In our study, we found that the expression of LINC01303 was significantly higher in GC tissues than in adjacent tissues by real‐time quantitative PCR. We can significantly inhibit the malignant proliferation, migration and invasion of GC cells by silencing LINC01303 expression. In addition, LINC01303 knockdown can also inhibit GC growth in vivo. After the bioinformatics analysis, we found that LINC01303 can be used as a miR‐101‐3p sponge to competitively adsorb miR‐101‐3p with EZH2. Therefore, our results indicate that LINC01303 promotes the expression of EZH2 by inhibiting miR‐101‐3p activity and promotes GC progression. In summary, in this study, we demonstrated for the first time that the LINC01303/miR‐101‐3p/EZH2 axis promotes GC progression.     

Down‐regulated lncRNA SBF2‐AS1 in M2 macrophage‐derived exosomes elevates miR‐122‐5p to restrict XIAP,thereby limiting pancreatic cancer development

Evidence has indicated that M2 macrophages promote the progression of cancers, but few focus on the ability of M2 macrophage‐derived exosomes in pancreatic cancer (PC). This study aims to explore how M2 macrophages affect malignant phenotypes of PC through regulating long non‐coding RNA SET‐binding factor 2 antisense RNA 1 (lncRNA SBF2‐AS1)/microRNA‐122‐5p (miR‐122‐5p)/X‐linked inhibitor of apoptosis protein (XIAP) axis. THP‐1 cells were transformed into M1 macrophages by lipopolysaccharide and interferon‐γ treatment, and into M2 macrophages after interleukin‐4 treatment. The PANC‐1 PC cell line with the largest lncRNA SBF2‐AS1 expression was selected, and M2 macrophage‐derived exosomes were isolated and identified. A number of assays were applied for the examination of lncRNA SBF2‐AS1 expression, PC cell biological functions and subcellular localization of lncRNA SBF2‐AS1. XIAP expression was detected, along with the interaction among lncRNA SBF2‐AS1, miR‐122‐5p and XIAP. M2 macrophage exosomal lncRNA SBF2‐AS1 expression's effects on the tumorigenic ability of PANC‐1 cells in nude mice were also investigated. M2 macrophage‐derived exosomes promoted progression of PC cells. Overexpressed lncRNA SBF2‐AS1 promoted progression of PC cells. LncRNA SBF2‐AS1 was found to act as a competing endogenous RNA to repress miR‐122‐5p and up‐regulate XIAP. Constrained lncRNA SBF2‐AS1 in M2 macrophage‐derived exosomes contributed to restraining tumorigenic ability of PC cells. Collectively, our study reveals that constrained lncRNA SBF2‐AS1 in M2 macrophage‐derived exosomes increases miR‐122‐5p expression to restrain XIAP expression, which further inhibits PC progression.     

lncRNA NR2F1‐AS1 promotes breast cancer angiogenesis through activating IGF‐1/IGF‐1R/ERK pathway

Long non‐coding RNAs (lncRNAs) take various effects in cancer mostly through sponging with microRNAs (miRNAs). lncRNA NR2F1‐AS1 is found to promote tumour progression in hepatocellular carcinoma, endometrial cancer and thyroid cancer. However, the role of lncRNA NR2F1‐AS1 in breast cancer angiogenesis remains unknown. In this study, we found lncRNA NR2F1‐AS1 was positively related with CD31 and CD34 in breast cancer through Pearson's correlation analysis, while lncRNA NR2F1‐AS1 transfection promoted human umbilical vascular endothelial cell (HUVEC) tube formation. In breast cancer cells, lncRNA NR2F1‐AS1 enhanced the HUVEC proliferation, tube formation and migration ability through tumour‐conditioned medium (TCM). In zebrafish model, lncRNA NR2F1‐AS1 increased the breast cancer cell‐related neo‐vasculature and subsequently promoted the breast cancer cell metastasis. In mouse model, lncRNA NR2F1‐AS1 promoted the tumour vessel formation, increased the micro vessel density (MVD) and then induced the growth of primary tumour. Mechanically, lncRNA NR2F1‐AS1 increased insulin‐like growth factor‐1 (IGF‐1) expression through sponging miRNA‐338‐3p in breast cancer cells and then activated the receptor of IGF‐1 (IGF‐1R) and extracellular signal‐regulated kinase (ERK) pathway in HUVECs. These results indicated that lncRNA NR2F1‐AS1 could promote breast cancer angiogenesis through IGF‐1/IGF‐1R/ERK pathway.     

LncRNA HOXA11‐AS regulates calcium oxalate crystal–induced renal inflammation via miR‐124‐3p/MCP‐1

Long noncoding RNA (lncRNA) has been suggested to play an important role in a variety of diseases over the past decade. In a previous study, we identified a novel lncRNA, termed HOXA11‐AS, which was significantly up‐regulated in calcium oxalate (CaOx) nephrolithiasis. However, the biological function of HOXA11‐AS in CaOx nephrolithiasis remains poorly defined. Here, we demonstrated that HOXA11‐AS was significantly up‐regulated in CaOx nephrolithiasis both in vivo and in vitro. Gain‐/loss‐of‐function studies revealed that HOXA11‐AS inhibited proliferation, promoted apoptosis and aggravated cellular damage in HK‐2 cells exposed to calcium oxalate monohydrate (COM). Further investigations showed that HOXA11‐AS regulated monocyte chemotactic protein 1 (MCP‐1) expression in HK‐2 cell model of CaOx nephrolithiasis. In addition, online bioinformatics analysis and dual‐luciferase reporter assay results showed that miR‐124‐3p directly bound to HOXA11‐AS and the 3'UTR of MCP‐1. Furthermore, rescue experiment results revealed that HOXA11‐AS functioned as a competing endogenous RNA to regulate MCP‐1 expression through sponging miR‐124‐3p and that overexpression of miR‐124‐3p restored the inhibitory effect of proliferation, promotion effects of apoptosis and cell damage induced by HOXA11‐AS overexpression. Taken together, HOXA11‐AS mediated CaOx crystal–induced renal inflammation via the miR‐124‐3p/MCP‐1 axis, and this outcome may provide a good potential therapeutic target for nephrolithiasis.     

TP73‐AS1 promotes breast cancer cell proliferation through miR‐200a‐mediated TFAM inhibition

P73 antisense RNA 1T (TP73‐AS1 or PDAM) is a long non‐coding RNA, which can regulate apoptosis through regulation of p53 signaling‐related anti‐apoptotic genes. An abnormal change of TP73‐AS1 expression was noticed in cancers. The effects of TP73‐AS1 in breast cancer (BC) growth and the underlying mechanism remain unclear so far. In the present study, the effect of TP73‐AS1 in BC cell lines and clinical tumor samples was detected so as to reveal its role and function. In the present study, TP73‐AS1 was specifically upregulated in BC tissues and BC cell lines and was correlated to a poorer prognosis in patients with BC. TP73‐AS1 knocking down suppressed human BC cell proliferation in vitro through regulation of TFAM. In our previous study, we demonstrated that miR‐200a inhibits BC cell proliferation through targeting TFAM; here we revealed that TP73‐AS1 could regulate miR‐200a through direct targeting. Moreover, TP73‐AS1 might compete with TFAM for miR‐200a binding thus to promote TFAM expression. Data from the present study revealed that TP73‐AS1 promoted BC cell proliferation through acting as a competing endogenous RNA (ceRNA) by sponging miR‐200a. In conclusion, we regarded TP73‐AS1 as an oncogenic lncRNA promoting BC cell proliferation and a potential target for human BC treatment.     

Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p

Long non‐coding RNAs (lncRNAs) could regulate growth and metastasis of hepatocellular carcinoma (HCC). In this study, we aimed to investigate the mechanism of lncRNA F11‐AS1 in hepatitis B virus (HBV)–related HCC. The relation of lncRNA F11‐AS1 expression in HBV‐related HCC tissues to prognosis was analysed in silico. Stably HBV‐expressing HepG2.2.15 cells were established to explore the regulation of lncRNA F11‐AS1 by HBx protein, as well as to study the effects of overexpressed lncRNA F11‐AS1 on proliferation, migration, invasion and apoptosis in vitro. Subsequently, the underlying interactions and roles of lncRNA F11‐AS1/miR‐211‐5p/NR1I3 axis in HBV‐related HCC were investigated. Additionally, the influence of lncRNA F11‐AS1 and miR‐211‐5p on tumour growth and metastasis capacity of HepG2.2.15 cells were studied on tumour‐bearing nude mice. Poor expression of lncRNA F11‐AS1 was correlated with poor prognosis in patients with HBV‐related HCC, and its down‐regulation was caused by the HBx protein. lncRNA F11‐AS1 was proved to up‐regulate the NR1I3 expression by binding to miR‐211‐5p. Overexpression of lncRNA F11‐AS1 reduced the proliferation, migration and invasion, yet induced apoptosis of HepG2.2.15 cells in vitro, which could be abolished by overexpression of miR‐211‐5p. Additionally, either lncRNA F11‐AS1 overexpression or miR‐211‐5p inhibition attenuated the tumour growth and metastasis capacity of HepG2.2.15 cells in vivo. Collectively, lncRNA F11‐AS1 acted as a modulator of miR‐211‐5p to positively regulate the expression of NR1I3, and the lncRNA F11‐AS1/miR‐211‐5p/NR1I3 axis participated in HBV‐related HCC progression via interference with the cellular physiology of HCC.     

circEPSTI1 regulates ovarian cancer progression via decoying miR‐942

Increasing studies show that circular RNAs (circRNAs) play vital roles in tumour progression. But, how circRNAs function in ovarian cancer is mostly unclear. Here, we detected the expression of circEPSTI1 in ovarian cancer and explored the function of circEPSTI1 in ovarian cancer via a series of experiments. Then, we performed luciferase assay and RNA immunoprecipitation (RIP) assay to explore the competing endogenous RNA (ceRNA) function of circEPSTI1 in ovarian cancer. qRT‐PCR verified that circEPSTI1 was overexpressed in ovarian cancer. Inhibition of circEPSTI1 suppressed ovarian cancer cell proliferation, invasion but promoted cell apoptosis. Luciferase assays and RIP assay showed that circEPSTI1 and EPSTI1 (epithelial stromal interaction 1) could directly bind to miR‐942. And circEPSTI1 could regulate EPSTI1 expression via sponging miR‐942. In summary, circEPSTI1 regulated EPSTI1 expression and ovarian cancer progression by sponging miR‐942. circEPSTI1 could be used as a biomarker and therapeutic target in ovarian cancer.     

LncRNA NR2F2‐AS1 promotes tumourigenesis through modulating BMI1 expression by targeting miR‐320b in non‐small cell lung cancer

Recently, long noncoding RNAs (lncRNAs) are attracting wide attention in the field of cancer research because of its important role in cancer diagnosis and prognosis. But studies on the biological effects and relevant mechanisms of lncRNAs in non‐small cell lung cancer (NSCLC) remain few and need to be enriched. Our study discussed the expression and biological effects of LncRNA NR2F2‐AS1, and further explored its possible molecular mechanisms. As a result, elevated expression of NR2F2‐AS1 was detected in NSCLC tissues and cells and was remarkably associated with the tumor, node, metastasis (TNM) stage and the status of lymphatic metastasis of patients. Down‐regulated NR2F2‐AS1 contributed to the promotion of cell apoptosis and the inhibition of cell proliferation and invasion in A549 and SPC‐A‐1 cells in vivo and vitro. Through bioinformatics analysis, NR2F2‐AS1 functions as a ceRNA directly binding to miR‐320b, BMI1 was a direct target of miR‐320b. Combined with the following cellular experiments, the data showed that NR2F2‐AS1 may influence the NSCLC cell proliferation, invasion and apoptosis through regulating miR‐320b targeting BMI1.