Nicotinamide Nucleotide Transhydrogenase-antisense RNA1 Research Articles

LncRNA NNT-AS1/hsa-miR-485–5p/HSP90 axis in-silico and clinical prospect correlated-to histologic grades-based CRC stratification: A step toward ncRNA Precision

The oncogenic effects of long non-coding RNA (lncRNA) Nicotinamide Nucleotide Transhydrogenase-antisense RNA1 (NNT-AS1) role in colorectal cancer (CRC) hasn’t been sufficiently inspected in relation to the Homo sapiens (hsa)-microRNA (miR)− 485–5p/ heat shock protein 90 (HSP90) axis, clinically. qRT-PCR was performed to detect lncRNA NNT-AS1 and hsa-miR-485–5p expression levels in 60 Egyptian patients' sera. HSP90 serum level was quantified using Enzyme-linked immunosorbent assay (ELISA). The relative expression level of the studied non-coding RNAs as well as the HSP90 ELISA concentration were correlated with patients clinicopathological characteristics and correlated to each other. The axis diagnostic utility in comparison with carbohydrate antigen 19–9 (CA19–9) and carcinoembryonic antigen (CEA) tumor markers (TMs) was studied by receiver operating characteristic (ROC) curve analysis. The relative lncRNA NNT-AS1 expression level fold change 56.7 (13.5–112) and HSP90 protein ELISA level 6.68 (5.14–8.77) (ng/mL) were elevated, while, for hsa-miR-485–5p 0.0474 (0.0236–0.135) expression fold change was repressed in CRC Egyptian patients’ cohort sera, being compared to 28 apparently healthy control subjects. LncRNA NNT-AS1 specificity is 96.4% and a sensitivity of 91.7%, hsa-miR-485–5p showed 96.4% specificity, 90% sensitivity, and for HSP90 89.3%, 70% specificity and sensitivity, respectively. Those specificities and sensitivities were superior to the classical CRC TMs. A significant negative correlation was found between hsa-miR-485–5p with lncRNA NNT-AS1 (r = −0.933) expression fold change or with HSP90 protein blood level (r = −0.997), but, significant positive correlation was there between lncRNA NNT-AS1 and HSP90 (r = 0.927). LncRNA NNT-AS1/hsa-miR-485–5p/HSP90 axis could be a prospect for CRC development as well as diagnosis. Being correlated and related to CRC histologic grades 1–3, therefore, lncRNA NNT-AS1/hsa-miR-485–5p/HSP90 axis (not individually) expression approved clinically and in silico, could aid treatment precision.

Insights on the potential oncogenic impact of long non-coding RNA nicotinamide nucleotide transhydrogenase antisense RNA 1 in different cancer types; integrating pathway(s) and clinical outcome(s) association.

Long non-coding RNAs (lncRNAs) are becoming more prevalent in the cancer field arena, with functional roles in both oncogenic and onco-suppressive pathways. Despite their widespread aberrant expression in a range of human malignancies, the biological activities of the ncRNAs majority are unknown. All showed the involvement of the lncRNA nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1). Since NNT-AS1 influences cellular proliferation, invasion, migration, apoptosis, and metastasis, this lncRNA appears to be linked to deregulating the normal cellular processes driving malignancy. This was observed in breast cancer (BC), gastric cancer (GC), colorectal cancer (CRC), epithelial ovarian cancer (EOC), and hepatocellular carcinoma (HCC). The current narrative non-systematic review will discuss "the significance of lncRNAs in cancer", as well as "lncRNAs future potential application(s) as diagnostic or predictive biomarkers", therefore, comprising an opportunity as treatment target(s). The review will have a special emphasis on lncRNA NNT-AS1.

Serum long non‑coding RNA NNT‑AS1 protected by exosome is a potential biomarker and functions as an oncogene via the miR‑496/RAP2C axis in colorectal cancer.

Increasing evidence has indicated that long non-coding RNAs (lncRNAs) serve an essential role in carcinogenesis and cancer development. It has been reported that lncRNA nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1) serves a crucial role in several types of cancer. However, the clinical significance of circulating NNT-AS1 expression in colorectal cancer (CRC) remains to be elucidated. The current study aimed to investigate the potential role of NNT-AS1 and the clinical significance of its serum expression levels in patients with CRC. The expression of NNT-AS1 was measured in 40 pairs of tumor and adjacent normal tissues from patients with CRC via reverse transcription-quantitative PCR. The serum expression levels of NNT-AS1 were assayed in an independent cohort of healthy controls and patients with CRC. The levels of NNT-AS1 were also compared between paired preoperative and postoperative serum samples. In addition, the presence of exosomal NNT-AS1 in serum was explored. Furthermore, the biological roles of NNT-AS1 were investigated in CRC cells in vitro. The expression of NNT-AS1 was significantly upregulated in tumor tissues compared with adjacent normal tissues (P<0.05). A higher level of NNT-AS1 was associated with an advanced CRC stage. The serum levels of NNT-AS1 were significantly upregulated in patients with CRC compared with healthy subjects (P<0.05). Furthermore, the NNT-AS1 levels were significantly decreased in postoperative samples compared with preoperative samples (P<0.01). In addition, it was also identified that NNT-AS1 was upregulated in CRC exosomes (P<0.01), whereas no significant difference was observed in NNT-AS1 levels between serum and exosomes. Silencing of NNT-AS1 inhibited the proliferation, migration and invasion of CRC cells. It was also identified that NNT-AS1 exerted its effects via regulation of the microRNA-496/Ras-related protein Rap-2c axis. The present study demonstrated that circulating NNT-AS1, which may be protected by exosomes, could be a novel potential biomarker and therapeutic target in CRC.

The long non-coding RNA NNT-AS1 promotes clear cell renal cell carcinoma progression via regulation of the miR-137/ Y-box binding protein 1 axis

ABSTRACT Long noncoding RNAs (lncRNAs) have been implicated in the progression of malignant tumors, including in clear cell renal cell carcinoma (ccRCC). However, the function and the specific mechanism of lncRNA nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1) in ccRCC remains unknown. Thus, this study explored the role of NNT-AS1 in ccRCC. We evaluated NNT-AS1 expression in ccRCC specimens. Next, CCK-8 and Transwell assays were used to evaluate cell proliferation and metastatic abilities. The interaction between miR-137 and NNT-AS1 or Y-box binding protein 1 (YBX-1) was confirmed using a dual luciferase reporter assay. The results showed that NNT-AS1 was significantly upregulated in ccRCC specimens compared with normal tissues. Inhibition of NNT-AS1 restrained ccRCC proliferation and metastasis. Mechanistically, NNT-AS1 acted as a competitive endogenous RNA to sponge miR-137, which depressed ccRCC cells proliferation and metastasis. Moreover, with the use of bioinformatics analysis, the famous oncogene YBX-1 was selected as the potential target of miR-137. Luciferase assay also confirmed the interaction between miR-137 and YBX-1. Further functional studies demonstrated that the inhibition effect of NNT-AS1 knockdown on ccRCC carcinogenesis could be partially reversed by overexpression of YBX-1, suggesting that NNT-AS1 promotes ccRCC progression through the miR-137/YBX-1 pathway. In summary, these findings indicate that NNT-AS1 promotes ccRCC progression via the miR-137/YBX-1 pathway, which may provide a promising therapeutic target for renal cell carcinoma.

Silencing long non-coding RNAs nicotinamide nucleotide transhydrogenase antisense RNA 1 inhibited papillary thyroid cancer cell proliferation, migration and invasion and promoted apoptosis via targeting miR-199a-5p.

The increasing incidence of papillary thyroid cancer (PTC) has attracted many researchers to investigate the mechanism underlying PTC progression. This study explored the growth and apoptosis of PTC cells based on an lncRNA regulatory mechanism. The expression of nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1) in PTC cell lines and PTC tissues was analyzed by qRT-PCR. The mutual binding site between NNT-AS1 and miR-199a-5p was predicted by starBase and confirmed by dual-luciferase reporter assay. The correlation between NNT-AS1 and miR-199a-5p was shown by Pearson correlation test. The viability, clone formation, migration, invasion and apoptosis of TPC-1 and IHH-4 cells were examined by CCK-8, colony formation, wound-healing, transwell, and flow cytometry assays, respectively. The expressions of Bax, cleaved Caspase-3, Bcl-2, E-Cadherin, N-Cadherin and SNAIL in TPC-1 and IHH-4 cells were determined by Western blot or qRT-PCR. NNT-AS1 expression was upregulated in PTC cells and tissues. In TPC-1 cells, silencing NNT-AS1 inhibited viability, clone formation, migration, and invasion as well as the expressions of N-Cadherin, SNAIL and Bcl-2, but promoted the expressions of E-Cadherin, Bax, and cleaved caspase-3. The effects of NNT-AS1 overexpression on IHH-4 cells were opposite to those of silencing NNT-AS1. In PTC tissues, miR-199a-5p was low-expressed and targeted by NNT-AS1, and it was negatively correlated with NNT-AS1. MiR-199a-5p inhibitor promoted TPC-1 cell progression, but miR-199a-5p mimic inhibited IHH-4 cell progression. NNT-AS1 and miR-199a-5p exerted opposite effects on PTC cells. Silencing NNT-AS1 inhibited PTC cell proliferation, migration and invasion, but promoted apoptosis via upregulation of miR-199a-5p.

Overexpression of NNT-AS1 Activates TGF-β Signaling to Decrease Tumor CD4 Lymphocyte Infiltration in Hepatocellular Carcinoma.

Nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) is a long noncoding RNA (lncRNA) that has been shown to be overexpressed in hepatocellular carcinoma (HCC). However, the molecular mechanism involving NNT-AS1 in HCC remains to be extensively investigated. The activation of TGF-β signaling inhibits tumor-infiltrating lymphocytes (TILs) and results in tumor immune evasion. We thus planned to explore the mechanism by which NNT-AS1 activates the TGF-β signaling pathway and inhibits TILs in HCC. High levels of NNT-AS1 were detected in HCC tissues by both RNAscope and real-time quantitative PCR (RT-qPCR) assays. The levels of proteins involved in TGF-β signaling and those of CD4 T lymphocytes were quantified by immunohistochemistry (IHC). HCC cell lines (HepG2 and Huh7) were used to explore the effects of NNT-AS1 on TGF-β signaling activation. In these analyses, RNAscope detection demonstrated that NNT-AS1 levels were significantly increased in HCC cancer tissues (P = 0.0001). In addition, the elevated NNT-AS1 levels in cancer tissue were further confirmed by RT-qPCR analysis of HCC cancer tissues (n = 64) and normal tissues (n = 26) (P = 0.0003). Importantly, the overall survival time of HCC patients who exhibited higher levels of NNT-AS1 expression was significantly shorter than that of HCC patients who had lower levels of NNT-AS1 expression (P = 0.0402). Further mechanistic investigation indicated that NNT-AS1 inhibition significantly decreased the levels of TGF-β, TGFBR1, and SMAD5 in HCC cells. In HCC tissues, IHC detection showed that relatively high NNT-AS1 levels were associated with a reduction in infiltrated CD4 lymphocyte numbers. In conclusion, this research identifies a novel mechanism by which NNT-AS1 impairs CD4 T cell infiltration via activation of the TGF-β signaling pathway in HCC.

NNT-AS1 modulates prostate cancer cell proliferation, apoptosis and migration through miR-496/DDIT4 axis

BackgroundEmerging studies have disclosed long non-coding RNAs (lncRNAs) as pivotal modulators in the progression of prostate cancer (PCa). Current research planned to figure out the involvement of lncRNA nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1) in PCa.MethodsRNA expression was examined using RT-qPCR in PCa cells. Functional assays assessed the viability, proliferation, apoptosis and migration of PCa cells. RNA pull down and luciferase reporter experiments detected the interplay between miRNA and lncRNA or mRNA.ResultsNNT-AS1 was apparently upregulated in PCa cells. NNT-AS1 deficiency abrogated PCa cell viability, proliferation and migration but promoted apoptosis. Besides, miR-496 could be sequestered by NNT-AS1 to elevate the expression of DNA damage inducible transcript 4 (DDIT4) in PCa. Rescue assays indicated that overexpressed DDIT4 or restrained miR-496 could reverse the influence of NNT-AS1 depletion on malignant processes in PCa cells.ConclusionNNT-AS1 contributes to the malignant phenotypes of PCa cells through targeting miR-496 to boost DDIT4 expression.

Upregulated expression of LncRNA nicotinamide nucleotide transhydrogenase antisense RNA 1 is correlated with unfavorable clinical outcomes in cancers

BackgroundThe nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1) is a long non-coding RNA aberrantly expressed in human malignancies. We aimed to analyze available data to evaluate the correlation between NNT-AS1 expression and cancer prognosis.MethodsLiterature retrieval was performed by systematic searching related databases from inception to April 2, 2020. Studies regarding correlation between NNT-AS1 expression, survival outcomes and clinical characteristics of cancer patients were collected and pooled to calculate the the hazard ratios (HRs) or odds ratios (ORs) with 95% confidence intervals (95% CIs).ResultsTen studies comprising 699 patients were included, all of which were conducted in China according to literature selection criteria. Overexpression of NNT-AS1 had a significant association with unfavorable overall survival (OS) (HR = 2.08, 95% CI: 1.84–2.36, P < 0.001). Stratified analysis showed that tumor type, sample size, follow-up months, and survival analysis approach did not change the predictive value of NNT-AS1 on OS. Furthermore, elevated NNT-AS1 level had significant association with distant metastasis (DM) (OR = 2.45, 95% CI: 1.39–4.30), lymph node metastasis (LNM) (OR = 3.92, 95% CI: 1.35–11.41), TNM stage (OR = 4.25, 95% CI: 1.71–10.56), and vascular invasion (OR = 3.98, 95% CI: 2.06–7.71), but was not associated with age and gender. The TCGA dataset further consistently showed that the NNT-AS1 expression was associated with poor OS and disease-free survival.ConclusionsHigh expression of NNT-AS1 is associated with unfavorable survival outcomes and poor clinicopathologic characteristics. However, large-cohort data and geographical studies are still needed to further validate the prognostic value of NNT-AS1 in cancers.

LncRNA NNT-AS1 contributes to the cisplatin resistance of cervical cancer through NNT-AS1/miR-186/HMGB1 axis

BackgroundCisplatin (DDP) is a major chemotherapeutic drug which was widely used for cervical cancer (CC) patients with advanced or recurrent although its limitation in the development of resistance. LncRNA nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) has been reported to be involved in the DDP resistance. However, the role of NNT-AS1 in DDP resistance in CC remain unknown.MethodsThe mRNA expression of NNT-AS1, microRNA-186 (miR-186) and HMGB1 was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation and apoptosis abilities were measured via MTT assay or flow cytometry, respectively. Western blot was used to measure the expression level of HMGB1, Bax, Bcl-2, Cleaved-caspase 3, N-cadherin, Vimentin and E-cadherin. Cell migration and invasion abilities were analyzed using Transwell assay. The interaction among NNT-AS1, miR-186 and HMGB1 was confirmed by luciferase reporter assay and RNA pull-down assay. Murine xenograft model was established using stably transfected SiHa/DDP cells.ResultsNNT-AS1 level was significantly elevated in CC tissues and cells, especially in DDP-resistant tumors and cell lines. Subsequently, loss-of function assays indicated that NNT-AS1 silence could attenuate DDP resistance by inhibiting proliferation, metastasis and EMT but inducing apoptosis in DDP-resistant CC cells. Besides that, knockdown of NNT-AS1 also antagonized DDP resistance in vivo. Bioinformatics predication revealed NNT-AS1 directly bound to miR-186 and HMGB1 was a target of miR-186. Additionally, NNT-AS1 could regulate HMGB1 expression via targeting miR-186. Furthermore, restoration experiments showed NNT-AS1 knockdown might improve DDP-sensitivity of CC cells via blocking HMGB1 expression by competitive interaction with miR-186.ConclusionNNT-AS1 improved chemoresistance of DDP-resistant CC cells via modulating miR-186/HMGB1 axis.

Long non‑coding RNA NNT‑AS1 knockdown represses the progression of gastric cancer via modulating the miR‑142‑5p/SOX4/Wnt/β‑catenin signaling pathway.

Patients with advanced gastric cancer (GC) have a poor prognosis with a median overall survival of 10–12 months. Long non-coding RNA nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) and sex-determining region Y-related high mobility group box 4 (SOX4) have been reported to be associated with the progression of various types of cancer; however, the regulatory mechanism between NNT-AS1 and SOX4 in GC is not completely understood. Reverse transcription-quantitative PCR was used to detect the expression levels of NNT-AS1, microRNA (miR)-142-5p and SOX4. Western blotting was performed to assess the protein expression levels of SOX4, β-catenin, c-Myc, Bcl-2 and E-cadherin. The proliferation, apoptosis, migration and invasion of GC cells were determined using MTT, flow cytometry and Transwell assays. The relationship between miR-142-5p and NNT-AS1 or SOX4 was investigated using a dual-luciferase reporter assay. NNT-AS1 and SOX4 were upregulated, whereas miR-142-5p was downregulated in GC tissues and cells compared with normal tissues and cells. Both NNT-AS1 and SOX4 knockdown inhibited GC cell proliferation, migration and invasion, and enhanced GC cell apoptosis. Moreover, the results indicated that NNT-AS1 modulated SOX4 expression by sponging miR-142-5p. In addition, SOX4 overexpression reversed NNT-AS1 knockdown-mediated effects on GC cell proliferation, apoptosis, migration and invasion. NNT-AS1 knockdown blocked the Wnt/β-catenin signaling pathway via the miR-142-5p/SOX4 axis. Collectively, the present study indicated that NNT-AS1 knockdown decreased GC cell proliferation, migration and invasion, and induced GC cell apoptosis by regulating the miR-142-5p/SOX4/Wnt/β-catenin signaling pathway axis.

Long Non-Coding RNA NNT-AS1 Contributes to CisplatinResistance viamiR-1236-3p/ATG7 Axis in Lung Cancer Cells.

PurposeLung cancer is one of the most prevailing human cancers worldwide. Emerging evidence implies that long non-coding RNA nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) is implicated in the tumorigenesis of lung cancer. Herein, we aimed to expose the impact of NNT-AS1 on the drug resistance of lung cancer.MethodsLevels of NNT-AS1, microRNA (miR)-1236-3p and autophagy-related gene 7 (ATG7) were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was implemented to detect cell proliferation and the half maximal inhibitory concentration (IC50) of cisplatin (DDP) in vitro. Moreover, flow cytometry was performed to assess cell apoptosis. Cell migration and invasion were examined utilizing transwell assay in lung cancer cells. Besides, levels of ATG7 and cell behavior-related proteins were determined via Western blot. Dual-luciferase reporter assay was administrated to identify the interaction between miR-1236-3p and NNT-AS1 or ATG7. The biological role of NNT-AS1 in DDP resistance of lung cancer was examined by xenograft tumor model in vivo.ResultsNNT-AS1 and ATG7 were upregulated, whereas miR-1236-3p was curbed in lung cancer tissues and in with or without DDP-resistant cell lines. NNT-AS1 detection significantly constrained cell growth, metastasis, and the IC50 of DDP in A549/DDP and H522/DDP cells. Interestingly, the influence of miR-1236-3p mimic on DDP resistance was overturned via NNT-AS1 upregulation in vitro. Reintroduction of miR-1236-3p inhibitor relieved the effect of ATG7 silencing on DDP sensitivity in A549/DDP and H522/DDP cells. Importantly, NNT-AS1 was a sponge of miR-1236-3p to separate ATG7. Besides, NNT-AS1 silencing enhanced DDP sensitivity of lung cancer in vivo.ConclusionNNT-AS1/miR-1236-3p/ATG7 axis regulated DDP resistance in lung cancer cells and might supply a probable target and prognostic marker in lung cancer treatment.

LncRNA NNT-AS1 promotes non-small cell lung cancer progression through regulating miR-22-3p/YAP1 axis.

BackgroundLung cancer is the leading cause of cancer‐related mortality worldwide. Studies have demonstrated that long noncoding RNA nicotinamide nucleotide transhydrogenase‐antisense RNA1 (NNT‐AS1) functioned as an oncogene in most malignancies, including non‐small cell lung cancer (NSCLC). This study aimed to investigate the underlying mechanisms of NNT‐AS1 in NSCLC progression.MethodsThe levels of NNT‐AS1, miR‐22‐3p and Yes‐associated protein (YAP1) were detected by qRT‐PCR in NSCLC tissues and cells. Kaplan‐Meier analysis was conducted to analyze the correlation between NNT‐AS1 expression and overall survival of NSCLC patients. Cell proliferation was evaluated by MTT assay. Cell migration and invasion were assessed using transwell assay. The protein levels of YAP1 and EMT‐related proteins were detected by western blot. The molecular mechanism was predicted by starBase2.0 and validated by dual‐luciferase reporter assay or RNA pull‐down assay. Xenograft analysis was carried out to analyze tumor growth in vivo.ResultsWe found that the levels of NNT‐AS1 and YAP1 were enhanced, while miR‐22‐3p expression was decreased in NSCLC tissues and cells. High NNT‐AS1 expression was correlated with poor prognosis. NNT‐AS1 knockdown impeded proliferation, migration, invasion and EMT of NSCLC cells. NNT‐AS1 targeted miR‐22‐3p, and YAP1 was a target of miR‐22‐3p in NSCLC cells. Furthermore, NNT‐AS1 facilitated the progression of NSCLC by regulating miR‐22‐3p/YAP1 axis. NNT‐AS1 knockdown repressed tumor growth in vivo.ConclusionNNT‐AS1 facilitated proliferation, migration, invasion and EMT of NSCLC cells by sponging miR‐22‐3p and regulating YAP1 expression, which might provide a potential biomarker and therapeutic target for NSCLC.

LncRNA NNT-AS1 regulates the progression of lung cancer through the NNT-AS1/miR-3666/E2F2 axis.

Lung cancer is the main burden on human health, with high mortality and poor prognosis. The involvement of long non-coding RNAs (lncRNAs) in the development of cancer has attracted wide attention. This study aimed to investigate the role and novel mechanisms of lncRNA nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1) in the progression of lung cancer. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to detect the expression of NNT-AS1, microRNA-3666 (miR-3666), and E2F transcription factor 2 (E2F2). 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to analyze cell proliferation. Flow cytometry was carried out to investigate cell apoptosis. Transwell assay was conducted to observe cell invasion. The interaction between miR-3666 and NNT-AS1 or E2F2 was predicted by bioinformatics tool starBase v2.0 and verified by Dual-Luciferase reporter assay. The protein level of E2F2 was quantified by Western blot. NNT-AS1 and E2F2 were upregulated, but miR-3666 was downregulated in lung cancer tissues and cells. NNT-AS1 knockdown attenuated proliferation and invasion but enhanced apoptosis of lung cancer cells, while miR-3666 inhibition reversed these effects. It was confirmed that miR-3666 was a target of NNT-AS1 and it directly interacted with E2F2. The inhibitory proliferation and invasion, and acceleratory apoptosis of lung cancer cells, caused by miR-3666 enrichment, were overturned by E2F2 overexpression. Furthermore, E2F2 was regulated by NNT-AS1 through miR-3666. NNT-AS1 participated in the progression of lung cancer through NNT-AS1/miR-3666/E2F2 regulatory axis at least in part. Our study supplied a promising strategy for the treatment of lung cancer.

Long noncoding RNA NNT-AS1 enhances the malignant phenotype of bladder cancer by acting as a competing endogenous RNA on microRNA-496 thereby increasing HMGB1 expression.

The long noncoding RNA nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1) is a key malignancy regulator in a variety of human cancers. In this study, we first measured the expression of NNT-AS1 in bladder cancer and examined its role in cancer progression. The mechanisms behind the oncogenic functions of NNT-AS1 in bladder cancer were explored. We found that NNT-AS1 was upregulated in bladder cancer tissues and cell lines. This increased expression demonstrated a significant correlation with advanced clinical stage, lymph node metastasis, and shorter overall survival. NNT-AS1 knockdown suppressed bladder cancer cell proliferation, migration, and invasion and facilitated apoptosis in vitro and hindered tumor growth in vivo. NNT-AS1 functioned as a competing endogenous RNA for microRNA-496 (miR-496), and the suppressive effects of NNT-AS1 knockdown on malignant characteristics were abrogated by miR-496 silencing. HMGB1 was identified as a direct target gene of miR-496 in bladder cancer, and HMGB1 expression was enhanced by NNT-AS1 via sponging of miR-496. In conclusion, the NNT-AS1–miR-496–HMGB1 pathway plays a significant role in the aggressive behavior of bladder cancer and may lead to new NNT-AS1–based diagnostics and therapeutics.

Upregulation of long non-coding RNA NNT-AS1 promotes osteosarcoma progression by inhibiting the tumor suppressive miR-320a

ABSTRACTObjective: To investigate the role and mechanism of action of nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1) in osteosarcoma (OS).Methods: Bioinformatic analysis suggested miR-320a as potential target of NNT-AS1. Influence of NNT-AS1 overexpression or knockdown on OS cell proliferation, colony-formation, apoptosis, migration and invasion capacity was first investigated. Expression levels of NNT-AS1, miR-320a, beta-catenin, RUNX2, IGF-1R, c-Myc, Cyclin D1 and MMP13 were also evaluated by RT-qPCR and western blotting accordingly. Xenograft models using U2OS and OS-732 cells with different NNT-AS1 gene modifications were constructed for tumor formation assay as well as evaluation of miR-320a, beta-catenin and RUNX2 expression in primary lesion. NNT-AS1-overexpressing U2OS cells and NNT-AS1-knockdown OS-732 cells were subject to miR-320a mimic and inhibitor transfection, respectively, to investigate the miR-320a dependency of the osteosarcoma-promoting role of NNT-AS1.Results: NNT-AS1 overexpression significantly increased proliferation, survival and mobility of U2OS cells in vitro as well as its tumor formation ability in vivo, while NNT-AS1 knockdown showed opposite effect on OS-732 cells. In both in vitro and in vivo model, NNT-AS1 expression level significantly correlated with that of beta-catenin, RUNX2, IGF-1R, c-Myc, Cyclin D1 and MMP13 as well as Akt phosphorylation level, and inversely correlated with miR-320a expression. Transfection of miR-320a mimic significantly inhibiter the promoting effect of NNT-AS1 on cell proliferation, survival and mobility of U2OS cells, while miR-320 inhibitor partially rescued that of OS-732 cells.Conclusion: NNT-As1 functions as a cancer-promoting lncRNA by downregulating miR-320a, thus increasing the protein expression level of beta-catenin, RUNX2 and IGF-1R as well as activation of Akt in osteosarcoma.

Long noncoding RNA NNT-AS1 promotes gastric cancer proliferation and invasion by regulating microRNA-363 expression.

Increasing studies showed that long noncoding RNAs (lncRNAs) had crucial regulatory roles in various tumors, including gastric cancer (GC). Recent studies demonstrated that lncRNA nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) played an important role in several tumors. However, the role and expression of NNT-AS1 in GC progression remain unknown. In our study, we indicated that NNT-AS1 expression was upregulated in GC samples compared with the nontumor tissues. We also showed that NNT-AS1 expression was upregulated in the GC cell lines. Ectopic expression of NNT-AS1 promoted GC cell line HGC-27 cell proliferation, cell cycle progression, and invasion. In addition, we showed that NNT-AS1 acted as a sponge competing endogenous RNA for microRNA-363 (miR-363), which was downregulated in the GC samples and cell lines. miR-363 expression was negatively related with NNT-AS1 expression in GC samples. Upregulated expression of miR-363 suppressed GC cell growth, cycle, and invasion. Furthermore, we reported that elevated expression of NNT-AS1 promoted GC cell proliferation, cycle, and invasion partly by suppressing miR-363 expression. These results indicated that lncRNA NNT-AS1 acted as an oncogene in the development of GC partly by inhibiting miR-363 expression.

LncRNA NNT-AS1 is a major mediator of cisplatin chemoresistance in non-small cell lung cancer through MAPK/Slug pathway.

To investigate the role of long non-coding ribonucleic acid (lncRNA) nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) in cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC) and its molecular mechanism. Fluorescence quantitative Real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of lncRNA NNT-AS1 in NSCLC cell lines (A549 and SPCA-1) and DDP-resistant cell lines (A549/DDP and SPCA-1/DDP). Corresponding plasmids of si-NTT-AS1 and si-NC were conducted. Then, methyl thiazolyl tetrazolium (MTT) assay was applied to detect the changes in half inhibition concentration (IC50) values of DDP in A549/DDP and SPCA-1/DDP cells after interference in lncRNA NNT-AS1 expression. Clone formation assay and flow cytometry were employed to detect the changes in proliferation, cycle and apoptosis of A549/DDP and SPCA-1/DDP cells caused by si-NNT-AS1. Protein expressions of molecular markers in mitogen-activated protein kinase (MAPK)/Slug signaling pathway after interference in lncRNA NNT-AS1 expression was detected by Western blotting. The differential expressions of lncRNA NNT-AS1 in 10 pairs of drug-resistant and non-resistant tissues were detected by qRT-PCR. QRT-PCR results showed that the expression of lncRNA NNT-AS1 in drug-resistant cells was higher than that in parental cells. The IC50 value of DDP in drug-resistant cells was increased by interfering with the expression of lncRNA NNT-AS1. Clone formation assay and flow cytometry revealed that interfering in the expression of lncRNA NNT-AS1 could inhibit the proliferation, promote the apoptosis and cell cycle arrest of drug-resistant cells. Western blotting assay found that after interfering the expression of lncRNA NNT-AS1, the expressions of molecular markers in MAPK/Slug signaling pathway were downregulated. qRT-PCR assay indicated that there were 9 pairs of drug-resistant tissues with up-regulated lncRNA NNT-AS1 expression in a total of 10 pairs of drug-resistant tissues. LncRNA NNT-AS1 is highly expressed in drug-resistant NSCLC tissues and cells, promoting the DDP resistance of NSCLC cells through the MAPK/Slug signaling pathway.

Overexpression of Long Non-Coding RNA NNT-AS1 Correlates with Tumor Progression and Poor Prognosis in Osteosarcoma

Background/Aims: Increasing evidence demonstrates that long non-coding RNAs (lncRNAs) play critical regulatory roles in cancers, including osteosarcoma. A previous study showed that Nicotinamide Nucleotide Transhydrogenase-antisense RNA1 (NNT-AS1) was aberrantly expressed in several types of cancer. However, the potential biological roles and regulatory mechanisms of NNT-AS1 in osteosarcoma progression remain unknown. Methods: Quantitative RT-PCR was performed to examine the expression of NNT-AS1 in human tissues and cells. The biological functions of NNT-AS1 were determined by CCK-8, colony formation, Flow cytometry and Transwell assays in vitro. A mouse xenograft model was performed to investigate the effect of NNT-AS1 on tumor growth in vivo. Results: In this study, we found the expression of NNT-AS1 was significantly increased in tumor tissues compared to adjacent normal tissues. Furthermore, upregulated NNT-AS1 expression predicted poor prognosis and was an independent and significant risk factor for osteosarcoma patient survival. Further experiments revealed that NNT-AS1 knockdown significantly inhibited cell proliferation by inducing cell cycle arrest and promoting apoptosis in osteosarcoma cells. Moreover, NNT-AS1 silencing suppressed cell migration and invasion in vitro. In a tumor xenograft model, knockdown of NNT-AS1 suppressed tumor growth of OS-732 cells in vivo. Conclusions: Taken together, these findings indicate that NNT-AS1 functions as an oncogene in osteosarcoma and could be a novel diagnostic and therapeutic target for osteosarcoma.

Long noncoding RNA NNT-AS1 promotes hepatocellular carcinoma progression and metastasis through miR-363/CDK6 axis.

Long non-coding RNAs (lncRNAs) have been tested to act as important regulator in liver cancer genesis and progression. LncRNA Nicotinamide Nucleotide Transhydrogenase-antisense RNA1 (NNT-AS1) has been reported to participate in the tumorigenesis. However, the exact molecular mechanism of NNT-AS1 in hepatocellular carcinoma (HCC) is still unknown. In present study, our team identified the up-regulated expression of NNT-AS1 in HCC tissue and cell lines compared with adjacent noncancerous tissue and normal cells. Moreover, HCC patients with high NNT-AS1 levels had poor prognosis than that with low NNT-AS1 level (p=0.0089). In vitro, gain- and loss-of-function experiments revealed that enhanced NNT-AS1 expression promoted the proliferation ability and alleviated the cycle arrest and apoptosis, while NNT-AS1 knockdown suppressed the proliferation and induced G0/G1 phase arrest and apoptosis. In vivo, NNT-AS1 knockdown inhibited the HCC neoplastic tumor volume and weight. Bioinformatics analysis and luciferase reporter assay validated that miR-363 targeted NNT-AS1 and CDK6 3’-UTR. MiR-363 was down-regulated in HCC tissue and cells. NNT-AS1 competed with CDK6 for miR-363 binding and could increase CDK6 expression. In summary, our results suggest the oncogenic role of NNT-AS1 in HCC tumorigenesis through miR-363/CDK6 axis, providing a novel therapeutic target for human HCC.

Upregulated NNT-AS1, a long noncoding RNA, contributes to proliferation and migration of colorectal cancer cells in vitro and in vivo.

The expression patterns of the long non-coding RNA Nicotinamide Nucleotide Transhydrogenase-antisense RNA1 (NNT-AS1) have not been investigated in the context of cancer. In this study, we aim to investigate the NNT-AS1 expression level in colorectal cancer (CRC) patients and its potential roles in tumor biology. We measured the expression of NNT-AS1 in 70 paired tumor tissues and adjacent normal tissues. NNT-AS1 was expressed higher in tumor tissues than that in adjacent noncancer tissues, and higher expression of NNT-AS1 was significantly correlated with lymph node metastasis (Yes vs. No, P=0.004), TNM stage (I/II vs. III/IV, P=0.004), vessel invasion (Yes vs. No, P=0.002) and differentiation (well and moderate vs. poor, P=0.008). Multivariate analyses revealed that NNT-AS1 expression was an independent predictor of overall survival (P=0.0174) and progression free survival (P=0.0132) for CRC. Knockdown of NNT-AS1 using small interfering RNA (siRNA) significantly impaired CRC cell proliferation, migration and invasion in vitro and silencing NNT-AS1 also suppressed tumor growth and metastasis in nude mice. The western blot experiments revealed that silencing NNT-AS1 inhibited epithelial-mesenchymal transition (EMT) and inactivated MAPK/Erk signaling pathway in CRC cell lines. In conclusion, our studies implied that NNT-AS1 may involve in the development and progression of CRC via its regulation of cell proliferation, migration, and invasion by NNT-AS1-mediated activating of MAPK/Erk signaling pathway and EMT. NNT-AS1 may be a useful diagnostic and prognostic biomarker and a potential therapeutic target in CRC patients.