Characterization of circSCL38A1 as a novel oncogene in bladder cancer via targeting ILF3/TGF-ß2 signaling axis.

The regulatory role of circRNAs in cancer metastasis has become a focused issue in recent years. To date, however, the discovery of novel functional circRNAs and their regulatory mechanisms via binding with RBPs in bladder cancer (BC) are still lacking. Here, we screened out circSLC38A1 based on our sequencing data and followed validation with clinical tissue samples and cell lines. Functional assays showed that circSLC38A1 promoted BC cell invasion in vitro and lung metastasis of mice in vivo. By conducting RNA pull-down, mass spectrum, and RIP assays, circSLC38A1 was found to interact with Interleukin enhancer-binding factor 3 (ILF3), and stabilize ILF3 protein via modulating the ubiquitination process. By integrating our CUT&Tag-seq and RNA-seq data, TGF-ß2 was identified as the functional target of the circSLC38A1-ILF3 complex. In addition, m6A methylation was enriched in circSLC38A1 and contributed to its upregulation. Clinically, circSLC38A1 was identified in serum exosomes of BC patients and could distinguish BC patients from healthy individuals with a diagnostic accuracy of 0.878. Thus, our study revealed an essential role and clinical significance of circSLC38A1 in BC via activating the transcription of TGF-ß2 in an ILF3-dependent manner, extending the understanding of the importance of circRNA-mediated transcriptional regulation in BC metastasis.

Evaluation of Serum Exosomal lncRNAs as Diagnostic and Prognostic Biomarkers for Esophageal Squamous Cell Carcinoma.

BACKGROUND: Exosomal long non-coding RNAs (lncRNAs) have been recognised as promising stable biomarkers in cancers. The aim of this study was to identify an exosomal lncRNA panel for diagnosis and prognosis of esophageal squamous cell carcinoma (ESCC). MATERIALS AND METHODS: Exosomes were isolated from serum by ExoQuick Solution. To validate the exosomes, exosomal markers and characterization of nanoparticle were performed. Quantitative real-time PCR was used to measure the levels of lncRNAs in exosomes from ESCC patients and healthy subjects. In the training set, exosomal lncRNA profiles from 404 samples were conducted and established new models by multivariate logistic regression. In the validation set, the diagnostic performance of the panel was further validated in 222 additional individuals with a receiver operating characteristic curve (ROC). Kaplan-Meier and multivariate Cox proportional hazards analysis were applied to assess the correlation between lncRNAs and survival rate of ESCC patients. RESULTS: A 4-lncRNA panel (UCA1, POU3F3, ESCCAL-1 and PEG10) in exosomes for ESCC diagnosis was developed by logistic regression model. The diagnostic accuracy of panel was evaluated with AUC value of 0.844 and 0.853 for training and validation stage, respectively. The corresponding AUCs for patients with TNM stage I-II and III were 0.820 and 0.935, significantly higher than squamous cell carcinoma antigen (P<0.001), which were 0.652 and 0.642, respectively. Kaplan-Meier analysis indicated that patients with higher level of UCA1 and POU3F3 had lower survival rate (P<0.001). Additionally, POU3F3 might be as an independent prognostic factor for ESCC patients (P=0.004). CONCLUSION: These findings suggested that serum exosomal 4-lncRNA panel has considerable value for ESCC diagnosis, and POU3F3 may serve as a novel and independent prognostic predictor in clinical applications.

Circulating long non-coding RNA colon cancer-associated transcript 2 protected by exosome as a potential biomarker for colorectal cancer.

BACKGROUND: Colon cancer-associated transcript 2 (CCAT2) plays a crucial role in several cancers. However, the clinical significance of circulating CCAT2 expression in colorectal cancer (CRC) has not previously been elucidated. In this study, we aimed to elucidate the potential role of CCAT2 and its clinical significance of circulating expression level in CRC. METHODS: We detected the expression of CCAT2 in 75 pairs of tumorous and adjacent non-tumorous tissues derived from CRC patients by quantitative real-time polymerase chain reaction. The serum levels of CCAT2 expression were detected in an independent cohort of healthy controls and CRC patients. We analyzed the relationship between CCAT2 levels in serum and clinicopathological features of CRC patients. We also compared CCAT2 levels in paired pre-operative and post-operative serum samples. Furthermore, the existence of serum CCAT2 in exosomes was investigated. RESULTS: The levels of CCAT2 expression were significantly over-expressed in tumor tissues (p < 0.05) compared to adjacent non-tumorous tissues. Higher CCAT2 expression was associated with advanced CRC patients. Moreover, the serum levels of CCAT2 expression were significantly over-expressed in CRC patients (p < 0.05) compared to those in healthy subjects. In addition, the CCAT2 levels were significantly decreased in post-operative samples than those in pre-operative ones (P = 0.01). We also found that CCAT2 expression was up-regulated in CRC exosomes (P < 0.001) and no significant differences of CCAT2 levels were found between in serum and in exosomes. CONCLUSIONS: Our data indicate that circulating CCAT2 which might protected by exosomes can serve as a novel potential predictor in CRC.

Improving seed germination and oil contents by regulating the GDSL transcriptional level in Brassica napus.

KEY MESSAGE: Seed germination rate and oil content can be regulated at theGDSL transcriptional level by eitherAtGDSL1 orBnGDSL1 inB. napus. Gly-Asp-Ser-Leu (GDSL)-motif lipases represent an important subfamily of lipolytic enzymes, which play important roles in lipid metabolism, seed development, abiotic stress, and pathogen defense. In the present study, two closely related GDSL-motif lipases, Brassica napus GDSL1 and Arabidopsis thaliana GDSL1, were characterized as functioning in regulating germination rate and seed oil content in B. napus. AtGDSL1 and BnGDSL1 overexpression lines showed an increased seed germination rate and improved seedling establishment compared with wild type. Meanwhile, the constitutive overexpression of AtGDSL1 and BnGDSL1 promoted lipid catabolism and decreased the seed oil content. While RNAi-mediated suppression of BnGDSL1 (Bngdsl1) in B. napus improved the seed oil content and decreased seed germination rate. Moreover, the Bngdsl1 transgenic seeds showed changes in the fatty acid (FA) composition, featuring an increase in C18:1 and a decrease in C18:2 and C18:3. The transcriptional levels of six related core enzymes involved in FA mobilization were all elevated in the AtGDSL1 and BnGDSL1 overexpression lines, but strongly suppressed in the Bngdsl1 transgenic line. These results suggest that improving the seed germination and seed oil content in B. napus could be achieved by regulating the GDSL transcriptional level.

Overexpression of long noncoding RNA NORAD in colorectal cancer associates with tumor progression.

PURPOSE: The aim of this study was to elucidate the role and clinical significance of long noncoding RNA-activated by DNA damage (NORAD) in colorectal cancer (CRC). METHODS: Sixty pairs of tumorous and adjacent nontumorous tissues derived from CRC patients were subjected to quantitative real-time polymerase chain reaction to determine the expression level of NORAD. The serum levels of NORAD expression were also measured in an independent cohort of CRC patients as well as patients with benign diseases and healthy controls. Comparative analyses were performed to investigate the relationships between NORAD levels in tissues and clinicopathological features of CRC. Receiver operating characteristic (ROC) curve analysis was used to assess the diagnostic value of NORAD in patients with CRC. Furthermore, the potential functions of NORAD in the development of CRC were explored in vitro, using the HCT116 and SW1116 CRC cell lines. RESULT: NORAD expression was significantly upregulated in the tumorous tissues of CRC patients (P<0.001) compared to the adjacent nontumorous tissues. Higher NORAD expression was associated with advanced CRC. Moreover, serum levels supported that NORAD could distinguish CRC patients from healthy controls and patients with benign diseases, indicating a potential diagnostic role in CRC. The ROC curve analysis showed a diagnostic efficacy with area under the curve of 0.800 (95% CI: 0.737-0.853). Mechanistic investigations indicated that NORAD silencing reduced CRC cell proliferation, migration, and invasion. CONCLUSION: NORAD may serve as a novel predictor in CRC and may be a potential target for future therapy.

Circulating long noncoding RNA act as potential novel biomarkers for diagnosis and prognosis of non-small cell lung cancer.

Lung cancer is the first leading cause of cancer deaths worldwide. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Increasing evidence shows that long noncoding RNA (lncRNA) are capable of modulating tumor initiation, proliferation and metastasis. In the present study, we aimed to evaluate whether circulating lncRNA could be used as biomarkers for diagnosis and prognosis of NSCLC. Expression profiles of 14 lncRNA selected from other studies were validated in 20 pairs of tissues by quantitative real-time PCR, and the dysregulated lncRNA thus identified were further validated in serum samples from two independent cohorts along with three tumor makers (CEA, CYFRA21-1, and SCCA). Receiver-operating characteristic analysis was utilized to estimate the diagnostic efficiency of the candidate lncRNA and tumor markers. Importantly, we observed an association between lncRNA expression and overall survival (OS) rate of NSCLC. The expressions of SOX2 overlapping transcript (SOX2OT) and ANRIL were obviously upregulated in NSCLC tissues and serum samples compared with normal controls (P < 0.01). Based on the data from the training set, we next used a logistic regression model to construct an NSCLC diagnostic panel consisting of two lncRNA and three tumor markers. The area under the curve of this panel was 0.853 (95% confidence interval = 0.804-0.894, sensitivity = 77.1%, specificity = 79.2%), and this was distinctly superior to any biomarker alone (all at P < 0.05). Similar results were observed in the validation set. Intriguingly, Kaplan-Meier analysis demonstrated that low expressions of SOX2OT and ANRIL were both associated with higher OS rate (P = 0.008 and 0.017, respectively), and SOX2OT could be used as an independent prognostic factor (P = 0.036). Taken together, our study demonstrated that the newly developed diagnostic panel consisting of SOX2OT, ANRIL, CEA, CYFRA21-1, and SCCA could be valuable in NSCLC diagnosis. LncRNA SOX2OT and ANRIL might be ideal biomarkers for NSCLC prognosis.

Cell-free lncRNA expression signatures in urine serve as novel non-invasive biomarkers for diagnosis and recurrence prediction of bladder cancer.

Cell-free long non-coding RNAs (lncRNAs) are stably present in urine and can serve as non-invasive biomarkers for cancer. We aimed to identify signatures of lncRNAs in urine for diagnosis and prognosis of bladder cancer (BC). Screening of lncRNAs by microarray analysis was performed using urine samples of 10 BC patients and 10 controls. Expressions of candidate lncRNAs were evaluated in the training and validation set including 230 BC patients and 230 controls by quantitative reverse transcription polymerase chain reaction (qRT-PCR). A two-lncRNA panel (uc004cox.4 and GAS5) was constructed and provided high diagnostic accuracy of BC with an area under the curve (AUC) of 0.885 (95% CI, 0.836-0.924). The AUCs of the lncRNA panel for Ta, T1 and T2-T4 were 0.843, 0.867 and 0.923, respectively, significantly higher than those of urine cytology (all P < .05). Kaplan-Meier analysis revealed that higher level of uc004cox.4 was associated with poor recurrence-free survival (RFS) of non-muscle invasive BC (NMIBC) (P = .008). Additionally, Cox regression analysis indicated that uc004cox.4 was an independent prognostic factor for RFS of NMIBC (P = .018). Taken together, our findings indicated that urinary lncRNA signatures possessed potential clinical value for BC diagnosis. Moreover, uc004cox.4 could provide prognostic information for NMIBC.

Identification of a serum circulating lncRNA panel for the diagnosis and recurrence prediction of bladder cancer.

Accumulating evidence indicates that long non-coding RNAs (lncRNAs) play important roles in tumorigenesis and progression. We aimed to identify a panel of lncRNAs for the diagnosis and recurrence prediction in bladder cancer (BC). The expression of 13 candidate lncRNAs was investigated in 80 BC and matched adjacent normal tissues via quantitative real-time PCR. The differentially expressed lncRNAs were then analyzed in 240 serum samples (training set) and three lncRNAs (MEG3, SNHG16 and MALAT1) showed differential expression. A logistic regression model was constructed using the training set and validated in an independent cohort of 200 serum samples (validation set). The AUC of the three-lncRNA panel was 0.865 for the training and 0.828 for the validation set. The diagnostic performance of the lncRNA panel for Ta, T1, and T2-T4 were 0.778, 0.805, and 0.880, which were significantly higher than those of urine cytology (0.548, 0.604, and 0.682, respectively). Moreover, we determined that low expression of MEG3 was associated with poor recurrence-free survival by Kaplan-Meier analysis (p = 0.028), univariate Cox analysis (p = 0.033) and multivariate Cox analysis (p = 0.046). In conclusion, our results identified a three-lncRNA panel for BC diagnosis and a recurrence-independent prognostic factor, MEG3.

Identification of long noncoding RNAs as potential novel diagnosis and prognosis biomarkers in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is prevalent worldwide, and improvements in timely and effective diagnosis are imperatively needed. We aimed to identify potential long noncoding RNA (lncRNA) biomarkers for CRC diagnosis along with prognosis prediction. METHODS: LncRNA expression profiles were studied by microarray in paired tumor and normal tissues from six patients with CRC. The expression levels of candidate lncRNAs were analyzed in 80 pairs of tissues and two independent cohorts of serum samples. Receiver-operating characteristic (ROC) curves were employed to evaluate the performance of the lncRNAs identified. The correlation between lncRNAs and disease-specific survival rate of CRC patients was assessed to explore prognostic potential. RESULTS: Four lncRNAs (BANCR, NR_026817, NR_029373, and NR_034119) were identified to be significantly dysregulated in both tissue and serum samples with consistent pattern, and a panel was established based on this result. The performance of the 4-lncRNA panel was measured with an area under the ROC curve (AUC) of 0.881. The corresponding AUCs of the panel for patients with TNM stageI, II and III were 0.774, 0.844 and 0.949, respectively, significantly higher than that of CEA. Kaplan-Meier analysis showed that patients with low levels of NR_029373 and NR_034119 had significantly lower disease-specific survival rate (p = 0.013 and 0.044, respectively). Multivariate Cox analysis demonstrated that NR_029373 and NR_034119 were both independently associated with disease-specific survival rate (p = 0.013 and 0.038, respectively). CONCLUSIONS: Our study established a distinctive 4-lncRNA panel with considerable diagnostic value and identified NR_029373 and NR_034119 as potential biomarkers for CRC prognosis prediction.

Characterization and expression of a GDSL-like lipase gene from Brassica napus in Nicotiana benthamiana.

The GDSL esterase and lipase families play important roles in abiotic stress, pathogen defense, seed development and lipid metabolism. Identifying the lipase activity of the putative GDSL lipase is the prerequisite for dissecting its function. According to the sequence similarity and the conserved domains, we cloned the Brassica napus BnGLIP gene, which encodes a GDSL-like protein. We failed to identify the BnGLIP lipase activity in the bacterium and yeast expression systems. In this paper, we expressed the BnGLIP gene by fusing a 6× His tag in Nicotiana benthamiana and purified the recombinant protein. The extraction buffer contained 1 % (v/v) n-caprylic acid and was able to remove most of the protein impurities. About 50 µg of recombinant BnGLIP was obtained from 1 g of N. benthamiana leaves. The lipase activity was tested with the purified BnGLIP and the maximum enzyme activity reached 17.7 mM/mg. In conclusion, this study found that the recombinant protein BnGLIP expressed in tobacco system was effectively purified and was detected as a GDSL lipase.