Circular RNA differential expression profiles and bioinformatics analysis of hsa_circRNA_079422 in human endometrial carcinoma

Abstract The aim of our study was to explore circular RNA (circRNA) expression profiles associated with human endometrial carcinoma (EC) and to analyse the molecular mechanisms involved in cancer development and their potential clinical importance. Differential expression profiles were revealed by Arraystar human circRNA microarray analysis. The results of the circRNA microarray were confirmed by quantitative real-time PCR. Interactions between circRNAs and microRNAs (miRNAs) were predicted using Arraystar’s miRNA target prediction software. The functions of the circRNA-miRNA coexpression network were identified by KEGG pathway analysis and GO analysis. Compared with para-tumorous tissues, 14 genes were significantly upregulated and 12 genes were significantly downregulated in EC tissues (P < 0.05). The quantitative real-time PCR data demonstrated consistency with the results of the microarray profile analysis. We generated a circRNA-miRNA coexpression network. Hsa_circRNA_079422 expression was significantly lower and miR-136-5p expression was higher in EC tissues than in normal endometrial tissues. KEGG pathway analysis and GO analysis indicated that hsa_circRNA_079422 might play roles in different signalling pathways and biological functions. We confirmed the presence of different circRNA expression profiles and predicted the circRNA-miRNA coexpression network in human EC tissues. Hsa_circRNA_079422 might be involved in the pathogenesis and biological process of EC via interactions with miRNAs. IMPACT STATEMENT What is already known on this subject? EC is a common malignancy of the female reproductive system. CircRNAs were demonstrated to exert critical roles in cancers, including EC. What do the results of this study add? The results of this study add circRNAs expression profiles, the circRNA-miRNA coexpression network and cancer-related circRNA-miRNA target genes in EC. It was first found that hsa_circRNA_079422 was downregulated, while miR-136-5p was upregulated in EC tissues. What are the implications of these findings for clinical practice and/or further research? In clinical practice, early EC diagnosis lacks specific biomarkers, so most EC patients are diagnosed at an advanced stage. In the management of EC patients, we also lack personalised adjuvant treatment that combines the clinical pathological characteristics. For the existing literature, we identified a new EC differential expression biomarker, hsa_circ_079422. It can be used to verify the correlation with EC clinical severity or poor prognosis. Its targeting can also be used to stratify EC patients with different molecular types, including to guide adjuvant therapy. In addition, we can verify and analyse regulatory pathways associated with it for the design of regulating engineering circRNA.


Introduction
Endometrial carcinoma (EC) is the third most common cancer in females.In recent years, there has been a distinct growth in the incidence of EC.According to the American Cancer Society, there will be approximately 66,570 new confirmed cases of EC in women in the United States and approximately 12,940 deaths due to EC in 2021 (Siegel et al., 2021).EC develops in approximately 142,000 women worldwide, and an estimated 42,000 women die from EC each year (Amant et al., 2005).High body mass index (BMI), hypertension, diabetes mellitus, obesity and genetic history have all been reported as potential risk factors for EC (Gunter et al., 2008, Raglan et al., 2019).However, the aetiology and mechanism of EC remain unclear and are worth exploring.
CircRNAs are closed circular structures with covalently linked 3 0 and 5 0 ends, which distinguish them from other types of RNA (Sanger et al., 1976).As a new class of noncoding RNAs (ncRNAs), circRNAs have been considered secondary byproducts of linear mRNA splicing events (Chen, 2016).Due to a lack of free ends, circRNAs are more stable and resistant to RNases than linear RNAs (Suzuki et al., 2006, Enuka et al., 2016).They are widely expressed in exosomes (Li et al., 2015b).An increasing number of scholars are focussing on the role of miRNA sponges by studying miRNA response elements (MREs); for example, circRNAs can bind to miRNAs and competitively inhibit the activity of miRNAs.In this way, circRNA-miRNA interactions might regulate gene expression (Panda, 2018).
Growing evidence reveals that circRNAs may play crucial roles in the incidence and development of diseases by the mechanisms mentioned above, particularly in carcinoma.In cancers such as colorectal, bladder, oesophageal, and lung cancer (Huang et al., 2015, Li et al., 2015a, Wan et al., 2016, Yang et al., 2018), circRNAs act as oncogenes by sponging miRNAs and affect the expression of target genes or proteins.Then, the relevant signalling pathways that regulate tumour progression, such as the Wnt/b-catenin pathway (Yu et al., 2016), are activated.These pathways influence the proliferation and invasion of tumour cells.Therefore, we believe that circRNA should be highly utilised to leverage its characteristics of stability and high abundance.CircRNAs could be a potential diagnostic and prognostic biomarker for diseases, especially carcinomas.
circRNAs play regulatory roles in EC that are gradually being revealed by high-throughput technologies.To analyse the expression profiles of circRNAs in EC tissues and paratumorous tissues, we performed a study that combined the use of the Arraystar Human circRNA array and quantitative real-time PCR.We identified 13,617 circRNAs in EC tissue, of which the changes in 5 upregulated and 3 downregulated circRNAs were validated.We further used miRNA target gene prediction software to predict circRNA and miRNA interactions.The expression levels of hsa_circRNA_079422 in EC were significantly lower than those in para-tumorous tissue.Next, we utilised Gene Ontology (GO) function and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis to explore the role of hsa_circRNA_079422.Consequently, we discovered the importance of hsa_ circRNA_079422 in EC for the first time.It has been suggested from a new perspective that circRNA is a potential biomarker for the diagnosis and prognosis of EC.

Clinical sample collection
Four groups of EC tissues (group-Ca) and para-tumorous tissues (group-Pa) were obtained from patients who were diagnosed with endometrioid carcinoma by diagnostic curettage and then received a hysterectomy at the First Affiliated Hospital of Harbin Medical University and the Cancer Hospital of Harbin Medical University, Harbin, China.The para-tumorous tissues were confirmed to lack tumour cells by immunohistochemistry.The 8 samples were sent to Kangcheng (Shanghai, China) for Arraystar circRNA microarray analysis.Moreover, we collected clinical samples from the First Affiliated Hospital of Harbin Medical University, comprising 7 endometrial cancer tissues and 7 normal endometrial tissues as controls, for further validation by qRT-PCR.Endometrial cancer tissues came from patients with a preoperative pathological diagnosis of endometrioid cancer and indications for radical surgery.Postoperative pathological analysis, which represents the standard of reference, confirmed a diagnosis of endometrioid cancer.Normal endometrial tissue came from patients with hysteromyoma who require a total hysterectomy.Postoperative pathology of patients with hysteromyoma did not have complicated endometrial hyperplasia, endometrial intraepithelial neoplasia (EIN), adenomyosis, etc.All enrolled patients underwent surgical treatment that met clinical guidelines.None of the patients had other malignant tumours or received any previous anticancer treatment, such as radiotherapy, chemotherapy and hormonal therapy.After being obtained, the tissues were immediately placed into liquid nitrogen and preserved at À 80 � C for further use.Enrolled patients signed informed consent for all the procedures used for research purposes, and this study was approved by the institutional ethics board of the First Affiliated Hospital of Harbin Medical University.All research complied with the principles of medical ethics.

RNA extraction and detection
Total RNA was extracted from frozen tissues using TRIzol (Invitrogen Life Technologies) according to the manufacturer's instructions.We used a NanoDrop ND-1000 spectrophotometer to evaluate the quality of RNA samples.The integrity of RNA was measured by ultraviolet-visible absorbance at 260 nm and 280 nm (A260/280/230) and verified by gel electrophoresis.

Screening and identification of EC-associated circRNAs
Microarray detection was performed by KangCheng Biotech (Shanghai, China) under the guidance of an experimental workflow.Total RNA was digested with RNase R to exclude linear RNA and enrich circRNAs.The circRNAs were transcribed and amplified with fluorescent complementary RNA using the Arraystar Super RNA Labelling kit, and the labelled cRNAs were purified using the RNeasy Mini kit (Qiagen).The concentration and specific activity of the labelled cRNAs were measured by a NanoDrop ND-1000.Fluorescent cRNAs were hybridised with Arraystar Human circRNA Arrays V2.0 (8 � 15, Arraystar), followed by incubation in an Agilent hybridisation oven (Agilent).Hybridised arrays were washed, fixed and scanned using the Agilent Wash Kit (Agilent).

Differentially expressed gene analysis
We used the Axon GenePix Pro 6.0 software graph chip to read the values and to obtain the original data.R software was used for quantile normalisation and subsequent processing of the original data.The significant differences between the Ca group and Pa group were evaluated using a T test.The circRNAs with fold changes >1.5 and P values <0.05 were selected as significantly differentially expressed genes.

Quantitative real-time PCR (qRT-PCR)
Total RNA isolated from all samples was reverse transcribed into cDNA.The cDNA samples were amplified in the 2X PCR master mix of the Arraystar assay.Amplifications were performed as follows: 10 minutes at 95 � C, followed by denaturation for 10 seconds at 95 � C and 40 cycles of 60 � C for 60 seconds.All reactions were run in triplicate.Specific primers were designed by Primer 5.0 according to the particular loop structure of circRNAs.b-actin was used as an endogenous reference control.Moreover, the expression levels of hsa_circRNA_079422 and miR-136-5p in 7 EC tissues and paired normal tissues were measured.U6 was used as an internal control to normalise the differences in the levels of the target genes.The primer pairs for circRNAs/b-actin and miR-136-5p/U6 are shown in Table 1.The relative gene expression level was calculated using the 2 À DDCt method.All of these assays were performed in a blinded fashion.

Prediction and analysis of circRNA-miRNA
The connectivity of circRNA-miRNA and target genes was predicted and analysed by Arraystar miRNA target prediction software, which generated a circRNA-miRNA network based on TargetScan (http://www.targetscan.org/vert_72)and miRanda (http://www.microrna.org/microrna/home.do).The graph of the circRNA-miRNA network was drawn by Cytoscape.

GO analysis and KEGG pathway analysis of target genes
We performed GO enrichment analysis and KEGG pathway analysis on circRNAs using DAVID (The Database for Annotation, Visualisation and Integrated Discovery, https:// david.ncifcrf.gov).The log10(P value) represented the significant enrichment score, which suggested the possible functions of circRNAs in different processes and pathways.

Statistical analysis
All statistical analyses were performed in SPSS 17.0 software.The results are presented as the means ± standard deviations (SD), and the differences between two groups were assessed using Student's t test.All statistical tests were two sided, and results with P < 0.05 were considered statistically significant.

Overview of circRNA profiles in EC tissues
Using the Arraystar Human circRNA Array, we detected a total of 13,617 circRNAs in four paired human EC tissues and para-tumorous tissues.Differentially expressed circRNAs with statistical significance (fold changes > 1.5 and P < 0.05) between the Ca and Pa groups were identified by a volcano plot (Figure 1(a)) and scatter plot (Figure 1(b)).Based on microarray analysis, 14 upregulated and 12 downregulated circRNAs were specifically and significantly differentially expressed in EC tissues and were used to generate an expression heatmap (Figure 1(c)).The successful clustering of circRNAs expressed differentially between four paired EC tissues and para-tumorous tissues supported the reliability of these circRNA sequencing data.Among the 14 upregulated circRNAs, 10 were exonic, 3 were intronic and 1 was intergenic.Among the 12 downregulated circRNAs, 10 were exonic, and 1 was intronic.

Validation of circRNA differential expression profiles
Furthermore, we used quantitative real-time PCR (qRT-PCR) to validate the circRNA microarray profiles.Based on our previous study, we verified five upregulated circRNAs (hsa_ circRNA_007192, hsa_circRNA_100297, hsa_circRNA_101474, hsa_circRNA_104434, and hsa_circRNA_407202) and three downregulated circRNAs (hsa_circRNA_010906, hsa_circRNA_ 079422 and hsa_circRNA_100438) based on fold changes and P values.Ultimately, the qRT-PCR data demonstrated consistency with the results of the microarray profile analysis (Figure 1(d)).We selected has_circRNA_079422, which exhibited the most significant difference (P ¼ 0.008), for further study.The expression levels hashsa_circRNA_079422 in EC tissue were significantly lower than those in para-tumorous tissue.

Generation of the circRNA-miRNA coexpression network and prediction of cancer-related circRNA-miRNA target genes
A circRNA-miRNA coexpression network was generated to show the potential associations among the representative

Validation of hsa_circRNA_079422 and miR-136-5p expression in EC tissues
We performed agarose gel electrophoresis of the PCR product, which showed a monospecific amplified band (Figure 3(a)).Additionally, we analysed melting and amplification curves.The melting curve had a single peak, which suggested that the amplified product of hsa_circRNA_079422 was specific.Then,

GO and KEGG pathway analysis
To explore how hsa_circRNA_079422 plays a role in diseases, we investigated the potential mechanisms of circRNAs in EC with bioinformatics techniques.The results of GO analysis suggested that the target genes of hsa_circRNA_079422 were involved in some biological processes, such as enzyme activity, cellular structural composition, ion channel complex and nucleotide biosynthetic process (Figure 3(d)).According to the KEGG pathway analysis of hsa_circRNA_079422, the top 6 predicted pathways included the Notch signalling pathway, metabolism of amino acids and many other biosynthesis pathways (Figure 3(e)).These results suggested that hsa_circRNA_079422 might directly inhibit some signalling pathways leading to cancer.

Discussion
In recent years, due to the development of our country's economy, the incidence of EC has gradually increased.The  gold standard treatment for EC is surgery during the early stages.Assessing the status of lymph nodes by sentinel lymph node biopsy can help us accurately scope surgery and assess patient outcomes (Chiu et al., 2022).In addition, the doctor will personalise the choice of adjuvant therapy according to the risk of recurrence, including radiotherapy, chemotherapy, or a combination of both.But we are facing a significant issue of rapid drug resistance (Neri et al., 2019).
There is an urgent need to integrate molecular classification with clinicopathological features to develop biomarkers for EC early diagnosis, management and prognosis.For example, the predictive score of nodal involvement in EC patients is a useful test to identify patients at low risk of nodal involvement (Capozzi et al., 2022).For early identification of EC in high-risk populations, Benati et al. discovered a possible highly diagnostic blood biomarker, aberrant telomere length in circulating cell-free DNA (Benati et al., 2020).
Based on the type of mutations and somatic copy-number variations, genome and exome sequencing, and microsatellite instability (MSI) assay, the Cancer Gene Atlas (TCGA) Research Network divides EC into four prognostically relevant groups: polymerase epsilon (POLE) ultramutated, MSI hypermutated, copy-number (CN) low, and CN high (Kandoth et al., 2013).
This classification takes into account the histopathologic and clinical features of EC.It goes beyond the limitations of traditional binary classification.However, due to the high cost and complexity of TCGA research in clinical applications, scientists need to develop cheaper, more practical, and more accurate methods (Cuccu et al., 2023).Then, the proposal of the ProMisE (Proactive Molecular Risk Classifier for Endometrial Cancer) model increases the possibility of establishing targeted therapeutic methods based on tumour molecular biology (Kommoss et al., 2018).Radiogenomics, which is more advantageous, can achieve precision medicine by combining molecular genetics and radiology data (Lo Gullo et al., 2020).In EC patients, targeted operative or postoperative treatment may be tailored by combining radiomic and molecular biological features of ultrasound images (Bogani et al., 2022).
Since circRNAs have special structures that enable them to resist the digestion and degradation of RNases, they are stable more than linear RNA.The most important biological function of circRNAs is their regulation of gene expression.An increasing number of studies have revealed that many circRNAs, such as miRNA sponges (Panda, 2018, Misir et al., 2020) or competitive endogenous RNAs (ceRNAs) (Rong et al., 2019, Su et al., 2019, Wang et al., 2020), can regulate the expression of miRNA targets.Another regulatory function of circRNAs is achieved through their ability to interact with RNA-binding proteins (RBPs) (Zang et al., 2020) to enable transcription and alternative splicing.In addition to the noncoding function of circRNAs, scientists have studied circRNA translation (Lei et al., 2020, Shi et al., 2020).In recent years, the important phenotypic roles of circRNAs in cells and tissues have been gradually revealed in the context of many other carcinomas, such as gastric, hepatocellular and laryngeal carcinoma (Zeng et al., 2018, Zhang et al., 2019a, 2019b).In addition to differential expression in malignancies (Chen et al., 2018), circRNAs also fulfil a role in tumour proliferation, invasion, metastasis and drug resistance, and they are associated with clinical features such as tumour size, lymph node metastasis and cancer stage.Therefore, circRNAs have been considered as molecular biomarkers for diagnosis, monitoring, treatment and prognosis of human cancer.Taken together, our data suggest that circRNAs provide new ideas for the mechanisms underlying EC and its diagnosis and treatment.
Based on the characteristics and functions of miRNA sponges, some differentially expressed circRNAs we detected can bind to multiple MREs to function.CircRNAs modulate the activity of target miRNAs through competitive binding, thereby inhibiting the transcription of downstream products.Some evidence has indicated that generating artificial miRNA sponges is a new technology that achieves knockdown of miRNA, which could be a practicable option for the treatment of diseases, especially carcinomas (Tay et al., 2015).For example, miR-135b has been proven to promote growth and metastasis in lung cancer.In a mouse lung cancer in situ model experiment, the expression of miRNA was inhibited, and the progression of tumour growth and metastasis was retarded after transfection of a miR-135b sponge (Lin et al., 2013).There have been few studies on downregulated circRNAs in EC.Jia et al. found that circ_0001776 was significantly downregulated in EC tissues and could inhibit EC progression by the miR-182/LRIG2 axis (Jia et al., 2020).In addition, Yang et al. analysed the role of downregulated circ-ATAD1 in EC, and the results showed that circ-ATAD1 can inhibit EC cell invasion and migration by downregulating miR-10a through methylation (Yang et al., 2021).A review summarised the current regulatory roles of circRNAs involved in EC progression: regulate cell proliferation and motility; participate in angiogenesis; participate in glycolysis; participate in drug and radiation resistance (Gao et al., 2022).The significant differential downregulation of hsa_circRNA_079422 indicated the possibility of circRNAs as clinically detectable markers for the diagnosis, treatment and prognosis of EC.
In this study, bioinformatics software was used to predict that hsa_circRNA_079422 may have cross-binding sites with miR-4659a-3p, miR-4659b-3p, miR-526b-5p, and miR-136-5p.Researchers have confirmed that miR-136-5p can serve as a tumour suppressor factor and prevent the development of tumours via the Bcl-2 signalling pathway and Wnt signalling pathway (Li et al., 2017).MiR-136-5p expression is lower in individuals with hepatocellular carcinoma than in controls and is related to stage, vascular invasion and portal vein thrombosis (Ding et al., 2017).A study by Luhong Li showed that long noncoding RNA (lncRNA) down syndrome cell adhesion molecule antisense 1 (DSCAM-AS1) stimulates EC progression by regulating miR-136-5p expression.The effects of DSCAM-AS1 silencing on EC cell proliferation can be partially abolished by miR-136-5p knockdown (Li et al., 2021).Bioinformatic analysis through databases such as TargetScan, miRanda, and DAVID suggests that candidate target genes for miR-136-5p include Bcl-2, Wnt2, and NF-jB.Hsa_circRNA_ 079422/miRNA binding helps to further reveal the mechanism of EC initiation, invasion, and metastasis.
Moreover, GO analysis and KEGG pathway analysis provided us with a new approach for studying the mechanism of circRNA_079422, which revealed target genes enriched in many important biological processes and certain signalling pathways.Through GO enrichment analysis, it was found that the target genes of circRNA_079422 were enriched in enzymatic activity, the composition of cell membrane and ion channels, transmembrane transport, and nucleotide synthesis.Therefore, circRNA_079422 may be involved in cell composition, signal transduction and some important biological processes.In addition, through KEGG pathway analysis, our study suggested that the target genes of circRNA_079422 were enriched in polysaccharide synthesis, amino acid metabolism and the Notch signalling pathway.This finding suggests that circRNA_079422 may be involved in the synthesis and metabolism of macromolecules and several important pathogenic signalling pathways, such as the Notch signalling pathway.Based on previous research, the Notch signalling pathway not only plays a role in cell differentiation, proliferation and apoptosis (Fi� uza and Arias, 2007) but also affects the development and progression of cancer (Hu et al., 2012).In mammals, the Notch signalling pathway consists of the membrane receptors Notch1, Notch2, Notch3, and Notch4 and the membrane ligands DSL proteins Jag1, Jag2, DLL1, DLL3, and DLL4.Both the DSL ligand-activated pathway and the non-DSL ligand-activated pathway can activate the Notch signalling pathway.When the Notch pathway is activated, a series of signalling pathways are triggered, such as the Wnt signalling pathway and the MAPK signalling pathway.Eventually, the cells proliferate and differentiate into malignant cells.
Notch signals are active in the epithelial cells and stromal cells of human endometrial cells.Four NOTCH receptors are commonly expressed in endometrial cells, while the ligands JAG1 and DLL4 and the targeting bodies HES1 and HEY are mainly expressed in endometrial epithelial cells (Mikhailik et al., 2009).The mRNA levels of Notch receptor, ligand and Notch target gene HES1 in EC tissues are lower than those in normal endometrium (Jonusiene et al., 2013).This finding indicates that Notch signalling is less active in EC, so it is speculated that Notch signalling has a tumour suppressor effect in EC (Lachej et al., 2019).Zong et al. proved that circ_ PUM1 promoted the development of EC by acting as a miR-136 sponge and upregulating its target gene NOTCH3 (Zong et al., 2020).Moreover, the overexpression of miR-136 could reverse this result (Zong et al., 2020).Whether the regulatory network of hsa_circ_079422 can serve as an upstream regulatory mechanism of the Notch pathway remains to be further demonstrated.These future studies may provide new potential markers for the molecular classification and prognosis of EC or therapeutic targets.
We selected a circRNAs chip to perform microarray analysis on collected specimens and established the expression profile of circRNAs in EC tissues, which is a highlight compared to RNA-sequencing (RNA-seq) (Li et al., 2019).Because circRNAs are extremely low in cells, RNA-seq cannot accurately quantify its expression.And increasing sequencing depth requires a large cost.In addition, RNA-seq has limitations on the circRNAs annotation information and depends on the size of computer's data storage.Compared with RNAseq, Arraystar circRNA microarray has high sensitivity, accuracy, and comprehensive annotation.This analytical tool increases the confidence that our screened circRNAs are expressed in EC.For the circRNAs in EC mentioned in the existing literature, we found a new downregulated gene, hsa_circRNA_079422.Our study proposed the first explanation of the relationship between hsa_circRNA_079422 and EC.Our result includes the generation of a circRNA-miRNA coexpression network, prediction of cancer-related circRNA-miRNA target genes, and related functional pathway analysis.
However, there were several limitations in our research.On the one hand, we only selected endometrioid cancer samples.In further experiments, the sample size and type can be increased to verify our findings, and the study can involve analyses of the pathological tissue subtype and stage of EC.On the other hand, uncovering the relationship between the circRNA-miRNA combined network and the possible signalling pathway requires more work.In the future, more detailed verification of the correlation between hsa_ circRNA_079422 and miR-136-5p in EC is needed.
In conclusion, this study confirmed the different expression profiles of circRNAs in EC tissues, which might expand knowledge and provide new avenues for EC diagnosis and therapy.Additionally, the results of bioinformatics analysis showed that hsa_circ_079422, as a downregulated gene, might play a potential role in signalling pathways and the biological processes of EC.

Figure 1 .
Figure 1.CircRNAs differentially expressed in EC tissues.(a) Volcano plot of circRNA expression.The red point represents 1.5-fold up-and downregulation of circRNA expression.(b) Scatter plot of distinguished circRNAs.The points above the top and bottom green lines revealed that the difference in expression was more than 1.5-fold.(c) Heatmap of differentially expressed circRNAs in EC.Each column represents the expression profile of one sample, and each row corresponds to one circRNA.Red represents high expression, and green represents low expression.(d) Validation of circRNA profiles by qRT-PCR.The X-axis represents circRNAs.The Y-axis represents the P value.The bar chart in red represents EC tissues, while blue represents para-tumorous tissues.

Figure 3 .
Figure 3. qRT-PCR validation and functional analysis of hsa_circRNA_079422 in EC tissues.(a) Electropherogram of qRT-PCR products.Up: circRNA_079422, Down: the reference gene b-actin.Ca represents endometrial cancer tissue group, Pa represents para-tumorous tissue group.(b) Relative expression of circRNA_079422 (P ¼ 0.045).(c) Relative expression of miR-136-5p (P ¼ 0.002).(d) GO analysis results of hsa_circRNA_079422 in EC.The Y-axis represents the enrichment score, and the X-axis represents GO terms.(e) KEGG pathway analysis of hsa_circRNA_079422 in EC.The Y-axis represents the KEGG pathway, and the X-axis represents the enrichment score.

Table 1 .
Sequences of primers used for qRT-PCR.