Identification of long noncoding RNAs downregulated specifically in ovarian high-grade serous carcinoma.

Purpose: To investigate whether long noncoding RNAs (lncRNAs) are involved in the development or malignant behavior of ovarian high-grade serous carcinoma (HGSC), we attempted to identify lncRNAs specific to HGSC. Methods: Total RNAs were isolated from HGSC, normal ovarian, and fallopian tube tissue samples and were subjected to a PCR array that can analyze 84 cancer-associated lncRNAs. The lncRNAs that were upregulated and downregulated in HGSC in comparison to multiple samples of normal ovary and fallopian tube were validated by real-time RT-PCR. To infer the function, ovarian cancer cell lines that overexpress the identified lncRNAs were established, and the activation of cell proliferation, migration, and invasion was analyzed. Results: Eleven lncRNAs (ACTA2-AS1, ADAMTS9-AS2, CBR3-AS1, HAND2-AS1, IPW, LINC00312, LINC00887, MEG3, NBR2, TSIX, and XIST) were downregulated in HGSC samples. We established the cell lines that overexpress ADAMTS9-AS2, CBR3-AS1, or NBR2. In cell lines overexpressing ADAMTS9-AS2, cell proliferation was suppressed, but migration and invasion were promoted. In cell lines overexpressing CBR3-AS1 or NBR2, cell migration tended to be promoted, although cell proliferation and invasion were unchanged. Conclusion: We identified eleven lncRNAs that were specifically downregulated in HGSC. Of these, CBR3-AS1, NBR2, and ADAMTS9-AS2 had unique functions in the malignant behaviors of HGSC.

Long non-coding RNA NBR2 suppresses the progression of colorectal cancer by downregulating miR-19a to regulate M2 macrophage polarization.

Colorectal cancer (CRC) is a malignant tumor of the gastrointestinal tract that significantly impacts the health of patients and lacks promising methods of diagnosis. Tumor-associated macrophages (TAMs) are involved in CRC progression, and their function is regulated by long non-coding RNAs (lncRNAs). The lncRNA NBR2 was recently reported as an oncogene, whose function in CRC remains uncertain. The present study aimed to investigate the biological function of lncRNA NBR2 in the progression of CRC and its underlying molecular mechanisms. Ten pairs of clinical CRC and para-carcinoma tissues were collected to determine the expression levels of lncRNA NBR2 and miR-19a, and the polarization state of TAMs. Quantitative reverse transcriptase-polymerase chain reaction was used to evaluate the expression of miR-19a, and western blotting was used to determine the expression levels of tumor necrosis factor-α, human leukocyte antigen-DR, arginase-1, CD163, CD206, interleukin-4, AMP-activated protein kinase (AMPK), p-AMPK, hypoxia-inducible factor-1α (HIF-1α), protein kinase B (AKT), p-AKT, mechanistic target of rapamycin (mTOR), and p-mTOR in TAMs. The proliferative ability of HCT-116 cells was detected using the CCK8 assay, and the migratory ability of HCT-116 cells was evaluated using the Transwell assay. The interaction between lncRNA NBR2 and miR-19a was determined using the luciferase assay. The lncRNA NBR2 was downregulated and miR-19a was highly expressed in CRC cells, accompanied by a high M2 polarization. Downregulated miR-19a promoted M1 polarization, activated AMPK, suppressed HIF-1α and AKT/mTOR signaling pathways, and promoted antitumor properties in NBR2-overexpressed TAMs, which were all reversed by the introduction of the miR-19a mimic. LncRNA NBR2 was verified to target miR-19a in macrophages according to the results of the luciferase assay. Collectively, lncRNA NBR2 may suppress the progression of CRC by downregulating miR-19a to regulate M2 macrophage polarization.

Remifentanil reduces the proliferation, migration and invasion of HCC cells via lncRNA NBR2/miR-650/TIMP3 axis.

Cancer cell hyperproliferation and metastasis are major causes of cancer-associated mortality. Although the use of anaesthetics and analgesics may affect cancer cell metastasis, the underlying molecular mechanism remains unclear. This study aimed to explore the mechanisms of action of remifentanil on hepatocellular carcinoma (HCC) progression. Cell viability was measured by the 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-h-tetrazolium bromide assay. Quantitative real-time polymerase chain reaction and Western blotting were performed to assess the expression levels of long non-coding RNA (lncRNA) neighbour of BRCA1 gene 2 (NBR2), microRNA (miR)-650 and tissue inhibitor of metalloproteinase-3 (TIMP3) in HCC cells. Wound healing and transwell assays were employed to evaluate the migration and invasion of HCC cells respectively. The target relationships between miR-650 and NBR2/TIMP3 were confirmed by dual luciferase reporter assay. Remifentanil reduced the viability of HCC cells in a dose-dependent manner. Remifentanil treatment significantly increased the expression of lncRNA NBR2 and TIMP3, and repressed miR-650 expression in HCC cells. Decreased lncRNA NBR2 or increased miR-650 promoted the proliferation, migration and invasion of remifentanil-treated HCC cells. LncRNA NBR2 targeted miR-650, and miR-650 further targeted TIMP3. Moreover, miR-650 down-regulation or TIMP3 up-regulation reversed the effects of lncRNA NBR2 knockdown that caused an enhancement of cell viability, migration and invasiveness in remifentanil-treated HCC cells. Thus remifentanil reduces the proliferation, migration and invasion of HCC cells via the lncRNA NBR2/miR-650/TIMP3 axis in vitro.

LncRNA NBR2 inhibits epithelial-mesenchymal transition by regulating Notch1 signaling in osteosarcoma cells.

Long noncoding RNAs (lncRNAs) have been identified to have increasingly important roles in tumorigenesis, and they may serve as novel biomarkers for cancer therapy. Recent studies have demonstrated that lncRNA NBR2 (neighbor of BRCA1 gene 2), a novel identified lncRNA, is decreased in several cancers; however, the role of NBR2 in the development of osteosarcoma has not been elucidated. In our study, we found that NBR2 expression was downregulated in osteosarcoma tissues, and osteosarcoma cases with lower NBR2 expression exhibited a shorter overall survival time compared with those with higher NBR2 expression. NBR2 overexpression inhibited osteosarcoma cell proliferation, invasion, and migration but did not increase apoptosis. Furthermore, RNA-binding protein immunoprecipitation assays confirmed that NBR2 directly binds to Notch1 protein. Furthermore, overexpression of Notch1 in NBR2-overexpressing osteosarcoma cells reversed the effects of NBR2 on cell proliferation, invasion, migration, and epithelial-mesenchymal transition. The in vivo results showed that NBR2 overexpression inhibited tumor growth in nude mice that were inoculated with osteosarcoma cells. NBR2 overexpression also suppressed the messenger RNA (mRNA) expression of Notch1, N-cadherin, and vimentin and increased the mRNA expression of E-cadherin in the tumor tissues. These data indicated that NBR2 served as a tumor suppressor gene in osteosarcoma and inhibited osteosarcoma cell proliferation, invasion, and migration. The current study provides a novel insight and treatment strategy for osteosarcoma.

MiR-19a Affects Hepatocyte Autophagy via Regulating lncRNA NBR2 and AMPK/PPARα in D-GalN/Lipopolysaccharide-Stimulated Hepatocytes.

This study aims to evaluate the potential involvement and regulatory mechanism of miR-19a in hepatocytes autophagy of acute liver failure (ALF). The in vitro hepatocytes injury model of primary hepatocyte and hepatocytes line HL-7702 was established by D-galactosamine (D-GalN) and lipopolysaccharide (LPS) co-treatment. Relative expression level of miR-19a and NBR2 was determined by qRT-PCR. Protein expression of AMPK/PPARα and autophagy-related gene was determined by Western blot. In hepatic tissue of 20 ALF patients and D-GalN/LPS-stimulated hepatocytes, miR-19a was upregulated and NBR2 was downregulated. D-GalN/LPS stimulation caused the inactivation of AMPK/PPARα signaling and the decrease of autophagy-related LC3-II/LC3-I ratio and beclin-1 expression in hepatocytes. The expression of both AMPK/PPARα and NBR2 were negatively controlled by miR-19a overexpression or knockdown. Moreover, both NBR2 and PPARα were targeted regulated by miR-19a according to luciferase reporter assay. In D-GalN/LPS-stimulated hepatocytes, AMPK activation promoted PPARα expression. AMPK inactivation inhibited the pro-autophagy effect of miR-19a and caused the decrease of LC3-II/LC3-I ratio and beclin-1 expression. PPARα activation abrogated the anti-autophagy effect of miR-19a mimic and caused the increase of LC3-II/LC3-I ratio and beclin-1 expression. NBR2 knockdown reversed the anti-autophagy impact of miR-19a inhibitor and caused the decrease of LC3-II/LC3-I ratio and beclin-1 expression. In summary, our data suggested that miR-19a negatively controlled the autophagy of hepatocytes attenuated in D-GalN/LPS-stimulated hepatocytes via regulating NBR2 and AMPK/PPARα signaling. J. Cell. Biochem. 119: 358-365, 2018. © 2017 Wiley Periodicals, Inc.

Long non-coding RNAs in cancer metabolism.

Altered cellular metabolism is an emerging hallmark of cancer. Accumulating recent evidence links long non-coding RNAs (lncRNAs), a still poorly understood class of non-coding RNAs, to cancer metabolism. Here we review the emerging findings on the functions of lncRNAs in cancer metabolism, with particular emphasis on how lncRNAs regulate glucose and glutamine metabolism in cancer cells, discuss how lncRNAs regulate various aspects of cancer metabolism through their cross-talk with other macromolecules, explore the mechanistic conceptual framework of lncRNAs in reprogramming metabolism in cancers, and highlight the challenges in this field. A more in-depth understanding of lncRNAs in cancer metabolism may enable the development of novel and effective therapeutic strategies targeting cancer metabolism.

Effects of the lncRNA NBR2 on the proliferation and autophagy of breast cancer cells under starvation conditions.

An increasing number of studies indicate that long noncoding RNAs (lncRNAs) play important roles in tumour proliferation, migration and other vital processes and are expected to become novel biomarkers for early cancer screening. The expression of the lncRNA NBR2 (adjacent breast cancer suppressor BRCA1) has been found to decrease in several cancer types. However, it is still unknown whether the lncRNA NBR2 is involved in breast cancer and autophagy. According to the Kaplan-Meier plotter survival curve analysis, the survival rate of the group with high lncRNA-NBR2 expression was higher than that of the group with low lncRNA-NBR2 expression. The suppression of cancer cell proliferation, invasion and migration by the lncRNA NBR2 has been demonstrated, suggesting that this lncRNA is involved in the development and progression of cancer. Our subsequent study revealed that the lncRNA NBR2 inhibited autophagy in breast cancer cells, and that starvation conditions enhanced this inhibitory effect. Moreover, this lncRNA changed the proliferation ability of breast cancer cells by affecting protective autophagy. The aim of this study was to investigate the link between starvation and lncRNAs by evaluating changes in autophagy-related proteins, cell proliferation and other biological processes. Together, these studies provide strategies for the early screening of breast cancer and suggest that starvation therapy can be used as a new approach for the treatment of cancer.

Study of the role of leukocyte telomere length-related lncRNA NBR2 in Alzheimer's disease.

Alzheimer's Syndrome (AD) is a neurodegenerative disease that is prevalent in middle-aged and elderly people. As the disease progresses, patients gradually lose the ability to take care of themselves, which brings a heavy burden to the family. There is a link between leukocyte telomere length (LTL) and cognitive ability. To search for possible pathogenic mechanisms and potential therapeutic agents, we demonstrated a causal link between LTL and AD using Mendelian randomization analysis (MR). The expression of the target gene NBR2 and the downstream mRNA GJA1 and GJA1-related genes, pathway enrichment, and association with immune cells were further explored. Using the gene cluster-drug target interaction network, we obtained potential therapeutic drugs. Our study provides evidence for a causal link between AD and LTL, suggesting medicines that may treat and alleviate AD symptoms.

Multiwalled Carbon Nanotubes-Reprogrammed Macrophages Facilitate Breast Cancer Metastasis via NBR2/TBX1 Axis.

In recent years, carbon nanotubes have emerged as a widely used nanomaterial, but their human exposure has become a significant concern. In our former study, we reported that pulmonary exposure of multiwalled carbon nanotubes (MWCNTs) promoted tumor metastasis of breast cancer; macrophages were key effectors of MWCNTs and contributed to the metastasis-promoting procedure in breast cancer, but the underlying molecular mechanisms remain to be explored. As a follow-up study, we herein demonstrated that MWCNT exposure in breast cancer cells and macrophage coculture systems promoted metastasis of breast cancer cells both in vitro and in vivo; macrophages were skewed into M2 polarization by MWCNT exposure. LncRNA NBR2 was screened out to be significantly decreased in MWCNTs-stimulated macrophages through RNA-seq; depletion of NBR2 led to the acquisition of M2 phenotypes in macrophages by activating multiple M2-related pathways. Specifically, NBR2 was found to positively regulate the downstream gene TBX1 through H3k27ac activation. TBX1 silence rescued NBR2-induced impairment of M2 polarization in IL-4 & IL-13-stimulated macrophages. Moreover, NBR2 overexpression mitigated the enhancing effects of MWCNT-exposed macrophages on breast cancer metastasis. This study uncovered the molecular mechanisms underlying breast cancer metastasis induced by MWCNT exposure.

LncRNA NBR2 Regulates Cancer Cell Stemness and Predicts Survival in Non-small Cell Cancer Patients by Downregulating TGF-ß1.

BACKGROUND: LncRNA NBR2 is a key regulator in cancer metabolism. However, its role in lung cancer is unknown. OBJECTIVE: This study aimed to explore the function of NBR2 in non-small cell lung cancer (NSCLC), which is the most common type of lung cancer. METHODS: Paired NSCLC and non-cancer tissues were collected from 68 patients with NSCLC. The expression of NBR2 and transforming growth factor-ß1 (TGF-ß1) in these samples was analyzed by RT-qPCR. The prognostic value of NBR2 for NSCLC was explored by performing a 5-year follow-up study. The interaction between NBR2 and TGF-ß1 in two NSCLC cell lines was detected by overexpression assay, followed by RT-qPCR and Western blot analysis. Flow cytometry was performed to evaluate the role of NBR2 and TGF-ß1 in regulating NSCLC cell stemness. RESULTS: NBR2 was significantly downregulated in NSCLC tissues than that in non-cancer tissues of NSCLC patients, and low expression levels of NBR2 predicted poor survival. TGF-ß1 was significantly upregulated in NSCLC tissues than that in non-cancer tissues, and was inversely correlated with NBR2. Overexpression of NBR2 downregulated TGF-ß1, while overexpression of TGF-ß1 did not affect the expression of NBR2. Overexpression of NBR2 inhibited, while overexpression of TGF-ß1 promoted NSCLC cell stemness. Overexpression of TGF-ß1 attenuated the effects of overexpression of NBR2. Mechanically, NBR2 interacted with Notch1 protein to inhibit its expression, thereby inhibiting the expression of TGF-ß1 and further affecting the proportion of CD133+ cells. CONCLUSION: LncRNA NBR2 regulates cancer cell stemness and predicts survival in NSCLC possibly by downregulating TGF-ß1 through Notch1.

lncRNA NBR2 attenuates angiotensin II-induced myocardial hypertrophy through repressing ER stress via activating LKB1/AMPK/Sirt1 pathway.

Myocardial hypertrophy leads to heart failure (HF), and emerging researchers have illustrated that long noncoding RNAs (lncRNAs) modulate myocardial hypertrophy. Here, we explored the role and mechanism of a novel lncRNA, NBR2, in modulating angiotensin II (Ang II)-induced myocardial hypertrophy. First, we examined plasma NBR2 levels in 25 patients with HF and myocardial hypertrophy and ten healthy donors and analyzed the correlation between NBR2 profiles and patients' clinical indicators. In addition, the overexpression experiment of NBR2 was carried out to probe the influence of NBR2 on myocardial hypertrophy. lncRNA NBR2 was down-regulated in plasma of patients with HF and myocardial hypertrophy (vs. healthy controls), and its level was negatively correlated with cardiac function (represented by left ventricular end-diastolic diameter and left ventricular ejection fraction) and degree of myocardial hypertrophy. Besides, Ang II treatment intensified the hypertrophy of human myocardial cell lines (HCM and AC16) and curbed the NBR2 expression. Overexpressing lncRNA NBR2 alleviated Angiotension II-induced myocardial hypertrophy and declined the profiles of hypertrophic markers. Moreover, up-regulating lncRNA NBR2 weakened Ang II-mediated endoplasmic reticulum (ER) stress and activated the LKB1/AMPK/Sirt1 pathway. Interfering with the LKB1/AMPK/Sirt1 axis abated the lncRNA NBR2-mediated inhibitory effect on myocardial hypertrophy and ER stress. This study confirmed that lncRNA NBR2 dampened myocardial hypertrophy and ER stress by modulating the LKB1/AMPK/Sirt1 pathway. Our study provides the first evidence that lncRNA NBR2 is positively associated with myocardial hypertrophy.

Corrigendum: LncRNA NBR2 Inhibits the Malignancy of Thyroid Cancer, Associated With Enhancing the AMPK Signaling.

[This corrects the article DOI: 10.3389/fonc.2020.00956.].

LncRNA NBR2 inhibits tumorigenesis by regulating autophagy in hepatocellular carcinoma.

Long noncoding RNAs (lncRNAs) have been identified to play increasingly important roles in tumorigenesis, and they may serve as novel biomarkers for cancer therapy. LncRNA NBR2 (neighbor of BRCA1 gene 2), a novel identified lncRNA, is demonstrated to decrease in several cancers. However, it is still unknown whether lncRNA NBR2 is involved in hepatocellular carcinoma and autophagy. We found that HCC cases with lower NBR2 expression had significantly worse overall survival than those with higher NBR2 expression in advanced patients. And the expression of NBR2 was negatively correlated with the degree of malignancy of HCC cell lines and differentiation of hepatocellular carcinoma. Besides, NBR2 inhibited the proliferation, invasion, and migration of liver cancer cells. We further found that NBR2 repressed cytoprotective autophagy to restrain HCC cell proliferation. Moreover, NBR2 inhibited Beclin 1-dependent autophagy through ERK and JNK pathways. Taken together, NBR2 suppressed autophagy-induced cell proliferation at least partly through ERK and JNK pathways. These data indicated that NBR2 served as a tumor suppressor gene in hepatocellular carcinoma. The current study provides a novel insight and treatment strategy for hepatocellular carcinoma.

LncRNA NBR2 aggravates hepatoblastoma cell malignancy and promotes cell proliferation under glucose starvation through the miR-22/TCF7 axis.

Hepatoblastoma (HB) is the most commonly seen pediatric liver malignancy. With frequent mutations in CTNNB1 gene that encodes ß-catenin, hepatoblastoma has been considered as a Wnt/ß-catenin-activated malignant tumor. Altered glucose metabolism upon nutrient deprivation (glucose starvation) might also be a critical event in hepatoblastoma carcinogenesis. The present study provides a lncRNA NBR2/miR-22/TCF7 axis modulating proliferation, invasion, migration, and apoptosis of hepatoblastoma cells upon glucose starvation through Wnt and downstream TCF7 signaling pathways. The expression of NBR2 is significantly increased within hepatoblastoma tissue samples; moreover, under incubation with 0 mM glucose (glucose starvation), NBR2 expression is significantly upregulated. NBR2 silencing not only inhibited hepatoblastoma cell viability, invasion, and migration under normal culture condition but also promoted the cell apoptosis under glucose starvation. NBR2 silencing in hepatoblastoma cells also decreased TCF7 mRNA expression and TCF7 protein levels, as well as the protein levels of the cell cycle, glucose entrapment, and EMT markers. miR-22 is directly bound to both NBR2 and TCF7; lncRNA NBR2 counteracted miR-22-mediated repression on TCF7 via acting as a ceRNA. The effects of NBR2 silencing on TCF7 expression, hepatoblastoma cell phenotype, and cell cycle, glucose entrapment, and EMT markers were all significantly reversed by miR-22 inhibition. In conclusion, lncRNA NBR2 aggravates hepatoblastoma cell malignancy through competing with TCF7 for miR-22 binding, therefore counteracting miR-22-mediated repression on TCF7. LncRNA NBR2 might be a promising target to inhibit hepatoblastoma cell proliferation under glucose starvation.

Long non-coding RNA NBR2 suppresses the progress of colorectal cancer in vitro and in vivo by regulating the polarization of TAM.

Colorectal cancer (CRC) threatens the health of patients with high mortality, which lacks sensitive biomarkers for diagnosis to improve total survival. The lncRNA NBR2 is reported to be downregulated in CRC and suppresses the proliferation of CRC cells. However, the underlying mechanisms remain unclear. The present study aimed to explore the regulatory function of the lncRNA NBR2 on tumor-associated macrophage (TAM) polarization and its consequent anti-tumor effect. Two CRC cell lines were used in this study. We found that the lncRNA NBR2, TNF-α, and HLA-DR were downregulated, and Arg-1, CD163, CD206, and IL-4 were upregulated in CRC tumors. M1 polarization was activated and M2 polarization was suppressed in NBR2-overexpressed macrophages, accompanied by increased production of inflammatory factors, decreased proliferation, and inhibited migration ability in the co-culture system of HCT-116 cells (SW480 cells) and NBR2-overexpressed macrophages. The promoted proliferation and migration were observed in the co-culture system of HCT-116 cells (SW480 cells) and NBR2-knockdown macrophages. The tumor growth of both HCT-116 cells and SW480 cells in the xenograft model was suppressed by co-planting NBR2-overexpressed macrophages and was facilitated by the co-planting of NBR2-knockdown macrophages. The release of inflammatory factors was induced, M1 polarization was facilitated, and M2 polarization was suppressed in tumor tissues in the NBR2-overexpressed group, which were all reversed in the NBR2-knockdown group. Therefore, the lncRNA NBR2 suppressed the progression of colorectal cancer in vitro and in vivo by regulating TAM polarization.

LncRNA NBR2 Inhibits the Malignancy of Thyroid Cancer, Associated With Enhancing the AMPK Signaling.

Long non-coding RNA NBR2 is a transcript of the neighbor of BRCA1 gene 2 and can regulate tumor development. However, there is little information on the role of NBR2 in the progression of thyroid cancers (TC). Here, we show that NBR2 expression is down-regulated in TC tissues and associated with histologic subtypes of TC. NBR2 expression was variably reduced in different TC cells. While NBR2 silencing significantly enhanced the malignancy of BCPAP cells by increasing cell proliferation, clonogenicity, wound healing, and invasion as well as tumor growth in vivo, and decreasing spontaneous apoptosis, NBR2 over-expression had opposite effects in BHT101 cells. Furthermore, treatment with A-769662 (a specific AMPK activator), like NBR2 over-expression, significantly attenuated the malignancy of BHT101 cells while treatment with Compound C (a specific AMPK inhibitor) significantly rescued that NBR2-reduced malignancy of BHT101 cells. In comparison with non-tumor thyroid epithelial Nthy-ori 3-1 cells, obviously increased GLUT-1 expression, but decreased AMPK and ACC phosphorylation were detected in TC cells. While NBR2 silencing further enhanced GLUT-1 expression and reduced AMPK and ACC phosphorylation as well as the EMT process in BCPAP cells. NBR2 over-expression also had opposite effects in BHT101 cells. Similar patterns of GLUT-1 expression and AMPK and ACC phosphorylation were detected in the different types of xenograft TC tumors in vivo. Therefore, such data indicated that NBR2 acted as a tumor suppressor of thyroid cancers associated with enhancing the AMPK signaling and NBR2 may be a potential biomarker and therapeutic target for thyroid cancers.

LncRNA NBR2 suppresses migration and invasion of colorectal cancer cells by downregulating miRNA-21.

It has been reported that lncRNA NBR2 regulates cancer metabolism. We investigated the role of NBR2 in colorectal cancer. We found that NBR2 was downregulated in colorectal cancer tissues than in adjacent healthy tissues. Decreased expression levels of NBR2 in tumor tissues were observed with the increase of clinical stages. MiRNA-21 was upregulated in colorectal cancer tissues than in adjacent healthy tissues, and was significantly and inversely correlated with NBR2. NBR2 overexpression downregulated miRNA-21 in colorectal cancer cells, while miRNA-21 overexpression failed to significantly affect NBR2 expression. NBR2 overexpression suppressed migration and invasion of colorectal cancer cells. MiRNA-21 overexpression played an opposite role and attenuated the effects of NBR2 overexpression. NBR2 overexpression did not significantly alter cancer cell proliferation. Therefore, lncRNA NBR2 inhibited colorectal cancer cell migration and invasion possibly by downregulating miRNA-21.

Curcumin Regulates the Progression of Colorectal Cancer via LncRNA NBR2/AMPK Pathway.

OBJECTIVE: To identify the effect of curcumin on tumor suppression and the possible molecular pathways involved. METHODS: The expression of long noncoding RNA neighbor of BRCA1 lncRNA 2 (NBR2) was quantified using reverse transcription-polymerase chain reaction on cultured colorectal cancer cells. Next, we used Western blot to measure the activation of adenosine monophosphate-activated protein kinase and mechanistic target of rapamycin kinase (mTOR) signaling molecules. Both cell proliferation and viability were measured via MTT assay, and the cell ratio and S phase were detected by BrdU assay. Colorectal cancer cells were pretreated with curcumin or transfected with shNBR2 or adenosine monophosphate-activated protein kinase inhibitor Compound C to examine the molecular pathway involved. RESULTS: Current data showed that glucose deficiency increased the expression of NBR2 in colorectal cancer cells, and NBR2 knockdown affected the progression of colorectal cancer cells under glucose starvation conditions. When NBR2 was silenced in the treated colorectal cancer cells, the proliferation, the clone formation, and the percentage of S-phase cells suppressed by glucose deprivation were compromised. Furthermore, NBR2 knockdown could suppress glucose deprivation-induced adenosine monophosphate-activated protein kinase activation plus mTOR inactivation. Similarly, when colorectal cancer cells were treated with curcumin, the expression of NBR2 was significantly increased. NBR2 knockdown reversed curcumin-suppressed proliferation, clone formation, and the percentage of S-phase colorectal cancer cells. Furthermore, NBR2 knockdown abolished curcumin-induced activation of adenosine monophosphate-activated protein kinase and inactivation of the mTOR signaling pathway. CONCLUSION: This study revealed a novel mechanism by which long noncoding RNA NBR2 mediates curcumin suppression of colorectal cancer proliferation by activating adenosine monophosphate-activated protein kinase and inactivating the mTOR signaling pathway.

Induction of the long noncoding RNA NBR2 from the bidirectional BRCA1 promoter under hypoxic conditions.

BRCA1 plays an important role in preventing breast cancer and is often silenced or repressed in sporadic cancer. The BRCA1 promoter is bidirectional: it drives transcription of the long non-coding (lnc) NBR2 transcript in the opposite orientation relative to the BRCA1 transcript. Hypoxic conditions repress BRCA1 transcription, but their effect on expression of the NBR2 transcript has not been reported. We used quantitative RT-PCR to measure BRCA1 and NBR2 transcript levels in 0% and 1% oxygen in MCF-7 breast cancer cells and found that NBR2 transcript levels increased as a function of time under hypoxic conditions, whereas BRCA1 mRNA levels were repressed. Hypoxic conditions were ineffective in reducing BRCA1 mRNA in the UACC-3199 breast cancer cell line, which is reported to have an epigenetically silenced BRCA1 promoter, even though appreciable levels of BRCA1 and NBR2 mRNA were detected. Significant recovery back to baseline RNA levels occurred within 48h after the MCF-7 cells were restored to normoxic conditions. We used a construct with the 218bp minimal BRCA1 promoter linked to marker genes to show that this minimal promoter repressed expression bidirectionally under hypoxic conditions, which suggests that the elements necessary for induction of NBR2 are located elsewhere.

NBR2: A former junk gene emerges as a key player in tumor suppression.

NBR2 (neighbor of BRCA1 gene 2) is a non-protein coding gene that resides adjacent to tumor suppressor gene BRCA1, but its role in cancer biology has remained unknown. Our recent study showed that NBR2 encodes a long non-coding RNA and suppresses tumor development through regulation of adenosine monophosphate-activated protein kinase (AMPK) activation.