Expression Of NORAD Research Articles

LncRNA NORAD promotes bone marrow stem cell differentiation and proliferation by targeting miR-26a-5p in steroid-induced osteonecrosis of the femoral head

BackgroundSteroid-induced osteonecrosis of the femoral head (SONFH) is a devastating orthopedic disease, which seriously affects the quality of life of patients. The study aims to investigate the effects of LncRNA NORAD on SONFH.MethodsHuman bone marrow-derived mesenchymal stem cells (hBMSCs) were isolated from the proximal femur of patients during routine orthopedic surgery and then cultured with dexamethasone (Dex) and transfected with NORAD overexpression vector, siRNA-NORAD and miR-26a-5p mimics. The mRNA expression of NORAD, miR-26a-5p, OPG, RANK, and RANKL was detected by RT-qPCR. Cell proliferation and apoptosis was measured by CCK-8 assay and flow cytometry, respectively. The protein expression of RUNX2, OPG, RANK, and RANKL was detected by western blot. The dual-luciferase reporter gene assay was performed to confirm the binding between NORAD and miR-26a-5p.ResultsNORAD expression was downregulated in SONFH tissues, while miR-26a-5p expression was upregulated. Overexpression of NORAD improved DEX-induced inhibition of proliferation and differentiation, and promotion of apoptosis in hBMSCs, while knockdown of NORAD led to the opposite results. Moreover, NORAD improved DEX-induced inhibition of proliferation and differentiation, and promotion of apoptosis by regulation of miR-26a-5p in hBMSCs.ConclusionsNORAD expression was downregulated in SONFH tissues, while miR-26a-5p expression was upregulated. NORAD improved DEX-induced inhibition of proliferation and differentiation, and promotion of apoptosis by regulation of miR-26a-5p in hBMSCs.

So alike yet so different. Differential expression of the long non-coding RNAs NORAD and HCG11 in breast cancer subtypes.

Breast cancer (BC) is a heterogeneous disease, and it is the leading cause of death among women. NORAD and HCG11 are highly similar lncRNAs that present binding sites for PUMILIO proteins. PUMILIO acts on hundreds of mRNA targets, contributing to the modulation of gene expression. We analyzed the expression levels of NORAD and HCG11 in the BC subtypes luminal A (LA) and basal-like (BL), and the regulatory networks associated with these lncRNAs. In the analysis of TCGA cohort (n=329) and Brazilian BC samples (n=44), NORAD was up-regulated in LA while HCG11 was up-regulated in BL subtype. An increased expression of NORAD is associated with reduced disease-free survival in basal-like patients (p = 0.002), which suggests that its prognostic value could be different in specific subtypes. The biological pathways observed for the HCG11 network are linked to the epithelial-to-mesenchymal transition; while NORAD associated pathways appear to be related to luminal epithelial cell transformation. NORAD and HCG11 regulons respectively present 36% and 21.5% of PUMILIO targets, which suggests that these lncRNAs act as a decoy for PUMILIO. These lncRNAs seem to work as players in the differentiation process that drives breast cells to acquire distinct phenotypes related to a specific BC subtype.

Oxidative stress activates NORAD expression by H3K27ac and promotes oxaliplatin resistance in gastric cancer by enhancing autophagy flux via targeting the miR-433-3p

Oxaliplatin resistance undermines its curative effects on cancer and usually leads to local recurrence. The oxidative stress induced DNA damage repair response is an important mechanism for inducing oxaliplatin resistance by activating autophagy. ELISA is used to detect target genes expression. TMT-based quantitative proteomic analysis was used to investigate the potential mechanisms involved in NORAD interactions based on GO analysis. Transwell assays and apoptosis flow cytometry were used for biological function analysis. CCK-8 was used to calculate IC50 and resistance index (RI) values. Dual-luciferase reporter gene assay, RIP and ChIP assays, and RNA pull-down were used to detect the interaction. Autophagy flux was evaluated using electron microscope and western blotting. Oxidative stress was enhanced by oxaliplatin; and oxaliplatin resistance gastric cancer cell showed lower oxidative stress. TMT labeling showed that NORAD may regulate autophagy flux. NORAD was highly expressed in oxaliplatin-resistant tissues. In vitro experiments indicate that NORAD knockdown decreases the RI (Resistance Index). Oxaliplatin induces oxidative stress and upregulates the expression of NORAD. SGC-7901 shows enhanced oxidative stress than oxaliplatin-resistant cells (SGC-7901-R). NORAD, activated by H3K27ac and CREBBP, enhanced the autophagy flux in SGC-7901-R to suppress the oxidative stress. NORAD binds to miR-433-3p and thereby stabilize the ATG5- ATG12 complex. Our findings illustrate that NORAD, activated by the oxidative stress, can positively regulate ATG5 and ATG12 and enhance the autophagy flux by sponging miR-433-3p. NORAD may be a potential biomarker for predicting oxaliplatin resistance and mediating oxidative stress, and provides therapeutic targets for reversing oxaliplatin resistance.

LncRNA NORAD promotes proliferation, migration and angiogenesis of hepatocellular carcinoma cells through targeting miR-211-5p/FOXD1/VEGF-A axis

Introduction and objectivesHepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death around the world. Despite improvement in the prevention and treatment of HCC, the clinical prognosis is still poor with increasing mortality. Non-coding RNAs play pivotal roles in HCC oncogenesis, but the detailed mechanism is poorly known. Therefore, the functions and interaction of lncRNA NORAD and miR-211-5p in HCC was investigated in this study. MethodsQuantitative real-time PCR method was used to analyze the expression of NORAD and miR-211-5p in clinical HCC tissues and cultured cell lines. Knockdown of NORAD and overexpression of miR-211-5p were then carried in HCC cells. Moreover, bioinformatics analysis and luciferase report assays were further employed to analyze the interaction between miR-211-5p and NORAD or FOXD1. ResultsIncreased lncRNA NORAD and decreased miR-211-5p expression were first detected in HCC compared with the peritumorial area. Further studies showed that knockdown of NORAD or overexpression of miR-211-5p impaired the proliferation, migration and angiogenesis of HCC cells. Mechanistically, we found that NORAD functions as a sponge for miR-211-5p. Moreover, it was revealed that decreased miR-211-5p induced the expression of FOXD1 as well as its downstream target VEGF-A, thereby contributes to enhanced angiogenesis of HCC. ConclusionElevated NORAD works as a sponge for miR-211-5p in HCC, thus release the inhibition effect of the latter on its downstream target FOXD1 and VEGF-A, which finally promotes angiogenesis. These results provide new insights into the interaction between NORAD and miR-211-5p in HCC and their potential usage as targets for the development of novel therapeutics against HCC.

LncRNA NORAD targets miR-410-3p to regulate drug resistance sensitivity of osteosarcoma

The current experiment was performed to investigate the effect of LncRNA NORAD on the sensitivity of miR-410-3p to drug resistance of osteosarcoma cells. The cisplatin-resistant cell line HOS/DDP was induced; si-NC, si-NORAD, miR-NC, miR-410-3p, pcDNA-NC, and pcDNA-NORAD were transfected into HOS/DDP cells, respectively; record as a si-NC group, si-NORAD group, miR-NC group, miR-410-3p group, pcDNA-NC group, pcDNA-NORAD group; si-NORAD was co-transfected into HOS/DDP cells with anti-miR-NC, anti-miR-410-3p, recorded as an anti-miR-NC+si-NORAD group and anti-miR-410-3p+si-NORAD group. Real-time quantitative PCR (RT-qPCR) was used to detect LncRNA NORAD, miR-410-3p and multidrug resistance protein 1 (MRP1) mRNA expression levels; Western blot was used to detect cyclin D1 (CyclinD1), MRP1, phosphorylated (p-p65), phosphorylated IкBα (p-IкBα) protein expression; cell counting kit 8 (CCK-8) was used to detect cell viability; dual luciferase report assay to detect targeting relationship between LncRNA NORAD and miR-410-3p. Compared with osteosarcoma cells HOS, the expression levels of LncRNA NORAD, MRP1 mRNA and protein in osteosarcoma resistant cells HOS/DDP were significantly increased, and miR-410-3p expression levels were significantly reduced (P<0.05). Low expression of NORAD or high expression of miR-410-3p, CyclinD1, MRP1 expression levels were significantly reduced, the cell survival rate was significantly reduced, and the half inhibitory concentration of cisplatin was significantly reduced (P<0.05). LncRNA NORAD targets and regulates miR-410-3p, and low expression of miR-410-3p can reverse the effects of low NORAD expression on HOS/DDP cell proliferation and cisplatin resistance. Low expressions of NORAD, p-p65, p-IкBα protein expression levels were significantly reduced; low expression of miR-410-3p could reverse the inhibitory effect of low NORAD expression on p-p65, p-IкBα protein expression. Inhibition of LncRNA NORAD expression can inhibit the proliferation of osteosarcoma HOS/DDP cells through targeted regulation of miR-410-3p, increasing its sensitivity to cisplatin, and it may be related to the NF-κB signaling pathway.

In silico identification of MAPK14-related lncRNAs and assessment of their expression in breast cancer samples

Mitogen-activated protein kinase (MAP kinase) pathways participate in regulation of several cellular processes involved in breast carcinogenesis. A number of non-coding RNAs including both microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) regulate or being regulated by MAPKs. We performed an in-silico method for identification of MAPKs with high number of interactions with miRNAs and lncRNAs. Bioinformatics approaches revealed that MAPK14 ranked first among MAPKs. Subsequently, we identified miRNAs and lncRNAs that were predicted to be associated with MAPK14. Finally, we selected four lncRNAs with higher predicted scores (NORAD, HCG11, ZNRD1ASP and TTN-AS1) and assessed their expression in 80 breast cancer tissues and their adjacent non-cancerous tissues (ANCTs). Expressions of HCG11 and ZNRD1ASP were lower in tumoral tissues compared with ANCTs (P values < 0.0001). However, expression levels of MAPK14 and NORAD were not significantly different between breast cancer tissues and ANCTs. A significant association was detected between expression of HCG11 and estrogen receptor (ER) status in a way that tumors with up-regulation of this lncRNA were mostly ER negative (P value = 0.04). Expressions of ZNRD1ASP and HCG11 were associated with menopause age and breast feeding duration respectively (P values = 0.02 and 0.04 respectively). There was a trend towards association between ZNRD1ASP expression and patients’ age of cancer diagnosis. Finally, we detected a trend toward association between expression of NORAD and history of hormone replacement therapy (P value = 0.06). Expression of MAPK14 was significantly higher in grade 1 tumors compared with grade 2 tumors (P value = 0.02). Consequently, the current study provides evidences for association between lncRNA expressions and reproductive factors or tumor features.

Regulatory mechanism of lncRNA NORAD on proliferation and invasion of ovarian cancer cells through miR-199a-3p.

To explore the regulatory mechanism of lncRNA NORAD on proliferation and invasion of ovarian cancer cells through miR-199a-3p. Eighty-six ovarian cancer tissues and 86 tissues adjacent to cancer, human ovarian cancer cell lines SKOV3, HO-8910, A2780, OVCAR-3, and human normal ovarian epithelial cell line IOSE80 were collected. MiR-199a-3p-mimics, miR-199a-3p-inhibitor, miR-NC, si-NORAD, Sh-NORAD, and NC were transfected into HO-8910 and A2780 cells, the expression levels of lncRNA NORAD and miR-199a-3p in ovarian cancer tissues and cells were detected by qRT-PCR, and the expression levels of N-cadherin, E-cadherin, and vimentin in cells were detected by WB. Cell Counting Kit-8 (CCK-8), transwell, and cell scratch tests were used to detect proliferation, invasion, and migration of cells, and the relationship between lncRNA NORAD and miR-199a-3p was confirmed by the Dual-Luciferase reporter assay. LncRNA NORAD was highly expressed and miR-199a-3p was lowly expressed in ovarian cancer, and the expression levels of LNCRNARAD and miR-199a-3p were negatively correlated. Cell experiments showed that inhibiting the expression of lncRNA NORAD or up-regulating the expression of miR-199a-3p could inhibit the proliferation, invasion, migration, and EMT of ovarian cancer cells, while up-regulating the expression of lncRNA NORAD or inhibiting the expression of miR-199a-3p could promote their proliferation, invasion, migration, and EMT. Dual-Luciferase reporter assay confirmed that there was a regulatory relationship between lncRNA NORAD and miR-199a-3p. LncRNA NORAD was highly expressed in ovarian cancer tissues, while silencing lncRNA NORAD expression could inhibit the proliferation, invasion, migration, and EMT of ovarian cancer cells by regulating miR-199a-3p, which might be a new target for the diagnosis and treatment of ovarian cancer.

The lncRNA NORAD/miR-520a-3p Facilitates Malignancy in Non-Small Cell Lung Cancer via PI3k/Akt/mTOR Signaling Pathway.

Background/AimsThe effects of lncRNA-NORAD/mir-520a-3p on proliferation and invasion of non-small cell lung cancer (NSCLC) were studied, and its potential molecular mechanism was discussed.MethodsqRT-PCR was used to detect the expression of lncRNA NORAD and miR-520a-3p in non-small cell lung cancer tissues and cell lines. CCK-8 method and Transwell test were used to identify the effects of lncRNA NORAD on the proliferation and invasion in NSCLC. Target gene prediction and screening and luciferase reporter assay was used to verify downstream target genes of lncRNA NORAD. The expressions of PI3K, AKT, and mTOR proteins were detected by Western blot.ResultsCompared with normal tissues and cells, the expressions of lncRNA NORAD in cancer tissues and cells were significantly higher. Compared with normal cells, the expression of miR-520a-3p in cells was considerably lower. LncRNA NORAD could accelerate the growth and metastasis of NSCLC in vitro and in vivo. Luciferase reporter assay results indicated that miR-520a-3p was a downstream target gene of lncRNA NORAD. Further findings showed that lncRNA NORAD might bind to miR-520a-3p, thereby affecting the PI3k/Akt/mTOR signaling pathway.ConclusionLncRNA NORAD can regulate the proliferation of NSCLC by regulating miR-520a-3p/PI3k/Akt/mTOR signaling pathway, thus promoting the occurrence and development of NSCLC.

Silencing the long noncoding RNA NORAD inhibits gastric cancer cell proliferation and invasion by the RhoA/ROCK1 pathway.

The current study aimed to examine the role and mechanism of a conserved long noncoding RNA termed NORAD (noncoding RNA activated by DNA damage, also named LINC00657) in gastric cancer (GC) progression. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to determine the expression level of relevant genes in GC cell lines. Cell proliferation was examined by cell counting kit-8 (CCK-8) assays. Cell migration and invasion were detected by transwell migration and invasion assays. Protein levels of the indicated genes were detected by Western blotting. Cell apoptosis was examined by flow cytometry. Results showed that NORAD knockdown decreased cell proliferation, migration and invasion but increased cell apoptosis. NORAD knockdown affected the expression of genes related to apoptosis and Epithelial-Mesenchymal Transition (EMT). In addition, NORAD's depletion resulted in reduced Ras Homolog Family Member A (RhoA) and Rho-associated coiled-coil containing protein kinase 1 (ROCK1) expression. Furthermore, NORAD's expression was positively correlated with RhoA and ROCK1 expressions in GC based on The Cancer Genome Atlas (TCGA) database. Our results demonstrate the oncogenic role of NORAD in gastric cancer progression.

LncRNA NORAD is repressed by the YAP pathway and suppresses lung and breast cancer metastasis by sequestering S100P.

Metastasis is responsible for most cancer mortality, but its molecular mechanism has not been completely understood. In addition to coding genes and miRNAs, the contribution of long noncoding RNAs (lncRNAs) to tumor metastatic dissemination and the mechanisms controlling their expression are areas of intensive investigation. Here, we show that lncRNA NORAD is downregulated in lung and breast cancers, and that NORAD low expression in these cancer types is associated with lymph node metastasis and poor prognosis. NORAD is transcriptionally repressed by the Hippo pathway transducer YAP/TAZ-TEAD complex in conjunction with the action of NuRD complex. Functionally, NORAD elicits potent inhibitory effects on migration and invasion of multiple lung and breast cancer cell lines, and repression of NORAD expression participates in the migration- and invasion-stimulatory effects of the YAP pathway. Mechanistically, NORAD exploits its multiple repeated sequences to function as a multivalent platform for binding and sequestering S100P, thereby suppressing S100P-elicited pro-metastatic signaling network. Using cell and mouse models, we show that the S100P decoy function of NORAD suppresses lung and breast cancer migration, invasion, and metastasis. Together, our study identifies NORAD as a novel metastasis suppressor, elucidates its regulatory and functional mechanisms, and highlights its prognostic value.

High long non-coding RNA NORAD expression predicts poor prognosis and promotes breast cancer progression by regulating TGF-\u03b2 pathway

BackgroundTo investigate the expression and significance of long noncoding RNA NORAD (lncRNA-NORAD) in breast cancer.MethodsQ-PCR was adopted to detect the mRNA expression of lncRNA-NORAD in breast cancer and adjacent tissues, survival analysis to compare the low-expression groups with the Kaplan–Meier method. Knockout of lncRNA-NORAD was adopted to observe the effects on the cell proliferation, migration and invasion of breast cancer in vitro and in vivo. The TGF-β/RUNX2 signaling pathway was observed by Western blot after the knockout of lncRNA-NORAD.ResultsIncreased expression of lncRNA-NORAD in breast cancer tissues promotes proliferation, invasion and migration of breast cancer cells and correlated with worse prognosis. LncRNA-NORAD activated TGF-β/RUNX2 signaling pathway in breast cancer cells.ConclusionsThese results strongly suggested that lncRNA-NORAD might play an important role in breast cancer progression and potentially be a new therapeutic target.

Overexpression of long non-coding RNA NORAD promotes invasion and migration in malignant melanoma via regulating the MIR-205-EGLN2 pathway.

Growing evidence suggests that long non‐coding RNAs NORAD and miR‐205 play a significant role in regulating cancer progression and metastasis. In this study, high expression of NORAD was firstly observed in melanoma tissues and human malignant melanoma cell lines, our aim was to study the interaction of them in the process of invasion and migration of malignant melanoma cells. NORAD, miR‐205, and EGLN2 mRNA level in MM cells was detected by qRT‐PCR. In situ hybridization (ISH) was performed to detect NORAD expression in MM tissues specimens. Effects of NORAD and miR‐205 on Prolyl hydroxylase 2 (EGLN2) expression was explored by western blot in MM cells line. Dual‐luciferase reporter assay was performed to verify the interaction relationship between NORAD and miR‐205, as well as, miR‐205 and EGLN2. Transwell assay was conducted to explore the effects of NORAD and miR‐205 in vitro. Xenografts in nude mice experiment were used to confirm the role of NORAD and miR‐205 in vivo. In vitro, NORAD knockdown significantly inhibited migration and invasion of malignant melanoma cells and elevated the expression of miR‐205, there was an interaction between miR‐205 and NORAD in the RNA‐induced silencing complex. Upregulation of miR‐205 induced significant inhibition of migratory and invasive ability compared with the scrambled control. However, downregulating NORAD largely reversed this effect. Furthermore, the regulatory effects of miR‐205 on EGLN2 levels and the induction of endoplasmic reticulum stress were reversed by NORAD. In vivo, deletion of miR‐205 induced tumor growth in nude mice. NORAD may play critical roles in tumorigenesis and progression of malignant melanoma by regulating of the miR‐205‐EGLN2 pathway, and may serve as a new therapeutic target.

The long noncoding RNA NORAD enhances the TGF-β pathway to promote hepatocellular carcinoma progression by targeting miR-202-5p.

Hepatocellular carcinoma (HCC) is one of the most fatal cancers with common features of invasion and metastasis. Recent evidence indicate that the long noncoding RNA NORAD is a potential oncogene and is significantly upregulated in several cancers. However, the general biological role and clinical value of NORAD in HCC remains unknown. Here, NORAD expression was measured in 29 paired tumor and paratumor tissues via quantitative real‐time polymerase chain reaction (qPCR). The effects of NORAD on HCC cell malignant potential were investigated via NORAD overexpression and knockdown both in vitro and in vivo. The mechanism of competitive endogenous RNAs (ceRNAs) was acquired and identified by bioinformatics analyses and luciferase assays. Moreover, the impact of NORAD level on the transforming growth factor β (TGF‐β) pathway was further determined by qPCR. We found that HCC tissues had a high level of NORAD compared with the paratumor tissues, and NORAD upregulation was associated with the shorter overall survival of patients with HCC. Furthermore, NORAD overexpression was demonstrated to promote HCC cell migration and invasion. Mechanically, NORAD might function as a ceRNA to regulate miR‐202‐5p, which served as a tumor‐suppressing microRNA via the TGF‐β pathway. We address that NORAD has a tumor‐promoting effect in HCC and describes a novel mechanism whereby NORAD regulates the TGF‐β pathway as a ceRNA of Homo sapiens (hsa)‐miR‐202‐5p.

LncRNA NORAD Contributes to Colorectal Cancer Progression by Inhibition of miR-202-5p.

Previous study indicates that long noncoding RNA NORAD could serve as a competing endogenous RNA to pancreatic cancer metastasis. However, its role in colorectal cancer (CRC) needs to be investigated. In the present study, we found that the expression of NORAD was significantly upregulated in CRC tissues. Furthermore, the expression of NORAD was positively related with CRC metastasis and patients’ poor prognosis. Knockdown of NORAD markedly inhibited CRC cell proliferation, migration, and invasion but induced cell apoptosis in vitro. In vivo experiments also indicated an inhibitory effect of NORAD on tumor growth. Mechanistically, we found that NORAD served as a competing endogenous RNA for miR-202-5p. We found that there was an inverse relationship between the expression of NORAD and miR-202-5p in CRC tissues. Moreover, overexpression of miR-202-5p in SW480 and HCT116 cells significantly inhibited cellular proliferation, migration, and invasion. Taken together, our study demonstrated that the NORAD/miR-202-5p axis plays a pivotal function on CRC progression.

Long non-coding RNA NORAD upregulate SIP1 expression to promote cell proliferation and invasion in cervical cancer

Long non-coding RNAs (lncRNAs) play important roles in tumor progression. Recently, increasing evidence showed that lncRNA NORAD could be used as an important regulator in tumor progression. However, the roles and underlying mechanism of NORAD in cervical cancer (CC) remain unclear. In the present study, we found that NORAD expression was significantly up-regulated in CC tissues and cell lines. High NORAD expression was correlated with advanced FIGO stage, lymph nodes metastasis, vascular invasion, and poor overall survival of CC patients. In vitro function assays, we showed that NORAD suppression reduced CC cells proliferation, invasion, and epithelial-mesenchymal transition (EMT) processes. Furthermore, the underlying mechanism studies showed that lncRNA NORAD could sponge miR-590-3p to promote the proliferation and invasion of CC cells via upregulating SIP1 expression. In addition, we showed that NORAD inhibition could reduce CC cells growth in vivo. Taken together, these results suggested that NORAD could serve as a ceRNA in CC progression by modulating miR-590-3p/SIP1 axis and act as a therapeutic target for the treatment of CC.

High expression of long noncoding RNA NORAD indicates a poor prognosis and promotes clinical progression and metastasis in bladder cancer

PurposeTo explore the function of NORAD in bladder cancer (BC), and to verify whether NORAD could be used as a biomarker to determine preoperative presence of progression and lymph node metastasis. To our knowledge, it is the first study investigating NORAD and its implications in BC. MethodsBC specimens of 90 patients underwent bladder cystectomy or transurethral resection between January 2012 to December 2016 were tested by fluorescence in situ hybridization. The association between NORAD expression and clinicopathological features and prognosis of the patients was analyzed using Kaplan-Meier survival analysis and Cox regression analysis. Quantitative real-time polymerase chain reaction was performed in 4 BC cell lines and 10 fresh tumor sample together with adjacent tissues. MTT, colony formation assay, and Annexin-V apoptosis detection were performed after knockdown of NORAD using shRNA in TSSCUP cells. Western blot was performed to related proteins extracted from these cells. ResultsFluorescence in situ hybridization indicated that high NORAD expression was associated with more advanced histological grade and clinical stage for patients with BC. Higher NORAD expression resulted in lower overall survival, and was an independent prognostic indicator. Real-time polymerase chain reaction showed that the expression of NORAD in BC tissues was higher than those measured in adjacent normal tissues. MTT and colony formation assay demonstrated that knockdown of NORAD results in lower proliferation in TSSCUP cells, whereas PUM2 expression was upregulated and E2F3 downregulated. ConclusionsHigh NORAD expression could serve as an independent prognostic factor for overall survival of patients with transitional BC. NORAD could be considered as a promising candidate for novel biomarker and therapeutic target for human BC.

Long noncoding RNA NORAD, a novel competing endogenous RNA, enhances the hypoxia-induced epithelial-mesenchymal transition to promote metastasis in pancreatic cancer

BackgroundPancreatic cancer, one of the top two most fatal cancers, is characterized by a desmoplastic reaction that creates a dense microenvironment, promoting hypoxia and inducing the epithelial-to-mesenchymal transition (EMT) to facilitate invasion and metastasis. Recent evidence indicates that the long noncoding RNA NORAD may be a potential oncogenic gene and that this lncRNA is significantly upregulated during hypoxia. However, the overall biological role and clinical significance of NORAD remains largely unknown.MethodsNORAD expression was measured in 33 paired cancerous and noncancerous tissue samples by real-time PCR. The effects of NORAD on pancreatic cancer cells were studied by overexpression and knockdown in vitro. Insights into the mechanism of competitive endogenous RNAs (ceRNAs) were gained from bioinformatics analyses and luciferase assays. In vivo, metastatic potential was identified using an orthotopic model of PDAC and quantified using bioluminescent signals. Alterations in RhoA expression and EMT levels were identified and verified by immunohistochemistry and Western blotting.ResultsNORAD is highly expressed in pancreatic cancer tissues and upregulated in hypoxic conditions. NORAD upregulation is correlated with shorter overall survival in pancreatic cancer patients. Furthermore, NORAD overexpression promoted the migration and invasion of pancreatic carcinoma cells, while NORAD depletion inhibited EMT and metastasis in vitro and in vivo. In particular, NORAD may function as a ceRNA to regulate the expression of the small GTP binding protein RhoA through competition for hsa-miR-125a-3p, thereby promoting EMT.ConclusionsElevated expression of NORAD in pancreatic cancer tissues is linked to poor prognosis and may confer a malignant phenotype upon tumor cells. NORAD may function as a ceRNA to regulate the expression of the small GTP binding protein RhoA through competition for hsa-miR-125a-3p. This finding may contribute to a better understanding of the role played by lncRNAs in hypoxia-induced EMT and provide a potential novel diagnostic and therapeutic target for pancreatic cancer.

Subtyping muscle-invasive bladder cancer to assess clinical response to trimodality therapy.

287 Background: Trimodality therapy with TURBT followed by chemoradiation is an acceptable alternative to cystectomy for muscle invasive bladder cancer (MIBC). Recently, genomic profiling has demonstrated MIBC can be divided into three or more subtypes with differing responses to chemotherapy, suggesting genomic subtype may impact therapeutic response. Here, we explore the utility of genomic information to better select patients for bladder-sparing trimodality therapy. Methods: Transcriptome-wide gene expression profiles were generated for 189 MIBC TURBT samples from patients undergoing trimodality therapy at the Massachusetts General Hospital. Of these, 108 passed microarray QC and 100 had complete clinical information. The patient tumors were classified as basal, basal claudin-low, infilrated luminal or luminal subtype. The subtype and the expression of a number of bladder cancer genes were assessed for their association with need for salvage cystectomy and for overall survival. Finally, transcriptome-wide differential expression analysis was used to explore gene set enrichment in trimodality therapy response groups. Results: Our chemoradiation cohort (n = 108) was classified into the four subtypes: basal (n = 45), basal claudin low (n = 13), infiltrated luminal (n = 17) and luminal tumors (n = 33). Survival analysis (n = 100) showed that patients of the luminal subtype trended to better overall survival, but did not reach significance (HR = 0.63, p = 0.1). Fewer patients with infiltrated luminal tumors (12%) required a salvage cystectomy compared to all other subtypes (34+/-1.5%, p = 0.08). We found high expression of BLACAT1 and NORAD (a lncRNA with a role in genome stability) correlated with worse (p = 0.01) and better prognosis (p = 0.008), respectively. Likewise, patients with high levels of the luminal-associated PPARG showed a significant increase in overall survival (p = 0.0002). Gene set enrichment revealed differential regulation of immune pathways in the trimodality therapy responders relative to the non-responders (p &lt; 0.05). Conclusions: Preliminary data exploring MIBC subtyping suggests the possibility of using genomics to predict response to trimodality bladder-sparing therapy.

Long non-coding RNA C5orf66-AS1 is downregulated in pituitary null cell adenomas and is associated with their invasiveness.

Pituitary null cell adenoma is a challenging clinical condition, and its pathogenesis remains to be elucidated. We performed this study to determine the roles of C5orf66-AS1, NORAD, and TINCR in the pathogenesis and invasion of pituitary null cell adenomas. Expression of the three long non-coding RNAs in pituitary null cell adenoma tissues of 11 patients and normal pituitary tissues from four donors was examined by performing quantitative reverse transcription-polymerase chain reaction. We found that C5orf66-AS1 expression was lower in pituitary null cell adenoma tissues than in normal pituitary tissues. Moreover, C5orf66-AS1 expression level was significantly lower in invasive pituitary null cell adenomas than in non-invasive ones. After transfection of C5orf66-AS1 into pituitary adenoma cells, assessment of cell viability and invasion suggested that overexpressed C5orf66-AS1 inhibited cell viability and cell invasion. In silico algorithms predicted several cis- and trans-acting target genes of C5orf66-AS1, including PITX1 and SCGB3A1. In addition, expression of some of the predicted target genes was determined using microarray data of another cohort with pituitary null cell adenomas. It showed that some of these target genes were differentially expressed between pituitary null cell adenoma tissues and normal pituitary tissues as well as between invasive and non-invasive tumors. Co-expression analysis in RNA sequencing data showed that PAQR7 was the most correlated gene of C5orf66-AS1 and that several predicted trans-acting target genes, including SCGB3A1, were highly correlated with C5orf66-AS1. NORAD and TINCR expression was not statistically significant in the complete cohort; however, a negative correlation was observed between NORAD expression and maximum tumor diameter in some subgroups. These results indicate that C5orf66-AS1 suppresses the development and invasion of pituitary null cell adenomas. However, our results do not provide enough statistical evidence to support the roles of NORAD and TINCR in the development and invasion of pituitary null cell adenomas.