A novel lncRNA NR4A1AS up-regulates orphan nuclear receptor NR4A1 expression by blocking UPF1-mediated mRNA destabilization in colorectal cancer.

Long non-coding RNAs (lncRNAs) play important roles in tumorigenesis and cancer progression. The orphan nuclear receptor subfamily 4 group A member 1 (NR4A1) acts as an oncogene, and is involved in colorectal cancer (CRC) development. However, the mechanism through which lncRNA regulates NR4A1 expression remains unknown. We aimed to identify lncRNAs that regulate NR4A1 and assess their underlying mechanisms in CRC. We first identified an antisense lncRNA of NR4A1 that was up-regulated in CRC tissues and cells with rapid amplification of cDNA ends (RACE), and designated it as NR4A1AS. Spearman correlation analysis showed that NR4A1AS was positively correlated with NR4A1 mRNA levels in 37 CRC tissues. Mechanistically, NR4A1AS stabilized NR4A1 mRNA by forming RNA-RNA complexes via partial base-pairing and up-regulated NR4A1 expression in CRC cells. RNA immunoprecipitation (RIP) assays revealed that knockdown of NR4A1AS expression by siRNA enhanced up-frameshift 1 (UPF1) recruitment to NR4A1 mRNA, thereby decreasing NR4A1 mRNA stability. Moreover, depletion of NR4A1AS was found to mimic the effect of NR4A1 knockdown, specifically by suppressing cell proliferation, migration and invasion, and inducing apoptosis and cell cycle arrest. Accordingly, restoring NR4A1 expression ameliorated the effects of NR4A1AS knockdown on tumor growth and metastasis of CRC cells in vitro and in vivo Thus, we conclude that NR4A1AS up-regulates NR4A1 expression by forming RNA-RNA complexes and blocking UPF1-mediated mRNA destabilization, and it functions in tumor growth and metastasis of CRC cells at least partly through regulating NR4A1, suggesting that NR4A1AS might be as a potential target for RNA-based anti-CRC drug studies.

Homocysteine accelerates senescence of endothelial cells via DNA hypomethylation of human telomerase reverse transcriptase.

OBJECTIVE: Homocysteine can accelerate the senescence of endothelial progenitor cells or endothelial cells (ECs) via telomerase inactivation and length shortening. However, the underlying mechanism is unclear. Here, we investigated whether homocysteine promotes endothelial senescence by reducing the expression and activity of human telomerase reverse transcriptase (hTERT) by DNA methylation to reduce ECs telomerase activity. APPROACH AND RESULTS: When compared with primary human umbilical vein endothelial cells grown under standard conditions, ECs with chronic homocysteine treatment showed accelerated upregulation of p16, p21, and p53, markers of cellular senescence, during 6 to 10 passages. Interestingly, homocysteine-stimulated but not angiotensin II-stimulated ECs senescence could be reversed by hypermethylation induced by folic acid or s-adenosylmethionine supplementation. Meanwhile, homocysteine promoted the shortening of telomere length specifically related to restoration of hTERT transcriptional expression and CCCTC-binding factor binding sites with hTERT promoter hypomethylation, as detected by quantitative real-time polymerase chain reaction, Western blot, methylation-specific polymerase chain reaction, and bisulfite sequencing assay. Electrophoretic mobility shift assay and chromatin immunoprecipitation results showed that homocysteine-reduced telomere activity and homocysteine-induced EC senescence might contribute to hTERT promoter demethylation by increasing CCCTC-binding factor repression and interfering in the SP1 binding to the demethylated hTERT promoter, which might relate with reduced of DNA methyltransferase 1. Furthermore, the CCCTC-binding factor-dependent mechanism of homocysteine-reduced hTERT expression via DNA demethylation was confirmed in aortic endothelia of mice with hyperhomocysteine levels. CONCLUSIONS: CCCTC-binding factor and SP1 cross talk may contribute to homocysteine-reduced hTERT DNA methylation and expression in endothelial senescence.

Histone acetylation regulates orphan nuclear receptor NR4A1 expression in hypercholesterolaemia.

Hypercholesterolaemia and inflammation are correlated with atherogenesis. Orphan nuclear receptor NR4A1, as a key regulator of inflammation, is closely associated with lipid levels in vivo. However, the mechanism by which lipids regulate NR4A1 expression remains unknown. We aimed to elucidate the underlying mechanism of NR4A1 expression in monocytes during hypercholesterolaemia, and reveal the potential role of NR4A1 in hypercholesterolaemia-induced circulating inflammation. Circulating leucocytes were collected from blood samples of 139 patients with hypercholesterolaemia and 139 sex- and age-matched healthy subjects. We found that there was a low-grade inflammatory state and higher expression of NR4A1 in patients. Both total cholesterol and low-density lipoprotein cholesterol levels in plasma were positively correlated with NR4A1 mRNA level. ChIP revealed that acetylation of histone H3 was enriched in the NR4A1 promoter region in patients. Human mononuclear cell lines THP-1 and U937 were treated with cholesterol. Supporting our clinical observations, cholesterol enhanced p300 acetyltransferase and decreased HDAC7 (histone deacetylase 7) recruitment to the NR4A1 promoter region, resulting in histone H3 hyperacetylation and further contributing to NR4A1 up-regulation in monocytes. Moreover, cytosporone B, an NR4A1 agonist, completely reversed cholesterol-induced IL-6 (interleukin 6) and MCP-1 (monocyte chemoattractant protein 1) expression to below basal levels, and knockdown of NR4A1 expression by siRNA not only mimicked, but also exaggerated the effects of cholesterol on inflammatory biomarker up-regulation. Thus we conclude that histone acetylation contributes to the regulation of NR4A1 expression in hypercholesterolaemia, and that NR4A1 expression reduces hypercholesterolaemia-induced inflammation.

Homocysteine upregulates soluble epoxide hydrolase in vascular endothelium in vitro and in vivo.

RATIONALE: Hyperhomocysteinemia is a risk factor of atherogenesis. Soluble epoxide hydrolase (sEH) is a major enzyme that hydrolyzes epoxyeicosatrienoic acids and attenuates their cardiovascular protective effects. Whether homocysteine (Hcy) regulates sEH and the underlying mechanism remains elusive. OBJECTIVE: To elucidate the mechanism by which Hcy regulates sEH expression and endothelial activation in vitro and in vivo. METHODS AND RESULTS: Hcy treatment in cultured human endothelial cells dose-dependently and time-dependently upregulated sEH mRNA and protein. Hcy increased the expression of adhesion molecules, which was markedly reversed by inhibiting sEH activity. Hcy-induced sEH upregulation is associated with activation of activating transcription factor-6 (ATF6). Bioinformatics analysis revealed a putative ATF6-binding motif in the promoter region of the sEH gene, which was found at a methylation site. Site-directed mutagenesis and chromatin immunoprecipitation assays demonstrated that Hcy treatment or ATF6 overexpression promoted ATF6 binding to the promoter of sEH and increased its activity. Results of methylation-specific polymerase chain reaction revealed that the ATF6 binding site on the sEH promoter was partially methylated and was demethylated with Hcy. SiRNA knockdown of ATF6α or SP1 blocked and ATF6 overexpression and DNA methyltransferase inhibitor mimicked the effect of homocysteine on sEH upregulation. In vivo, immunofluorescence assay revealed elevated expression of sEH and adhesion molecules in the aortic intima of mice with mild hyperhomocysteinemia, which was attenuated by sEH deletion or inhibition. CONCLUSION: ATF6 activation and DNA demethylation may coordinately contribute to Hcy-induced sEH expression and endothelial activation. Inhibition of sEH may be a therapeutic approach for treating Hcy-induced cardiovascular diseases.

TMEM52B Isoforms P18 and P20 Differentially Promote the Oncogenesis and Metastasis of Nasopharyngeal Carcinoma.

Transmembrane protein 52B (TMEM52B), a newly identified tumor-related gene, has been reported to regulate various tumors, yet its role in nasopharyngeal carcinoma (NPC) remains unclear. Transcriptomic analysis of NPC cell lines reveals frequent overexpression of TMEM52B, and immunohistochemical results show that TMEM52B is associated with advanced tumor stage, recurrence, and decreased survival time. Depleting TMEM52B inhibits the proliferation, migration, invasion, and oncogenesis of NPC cells in vivo. TMEM52B encodes two isoforms, TMEM52B-P18 and TMEM52B-P20, differing in their N-terminals. While both isoforms exhibit similar pro-oncogenic roles and contribute to drug resistance in NPC, TMEM52B-P20 differentially promotes metastasis. This functional discrepancy may be attributed to their distinct subcellular localization; TMEM52B-P18 is confined to the cytoplasm, while TMEM52B-P20 is found both at the cell membrane and in the cytoplasm. Mechanistically, cytoplasmic TMEM52B enhances AKT phosphorylation by interacting with phosphoglycerate kinase 1 (PGK1), fostering NPC growth and metastasis. Meanwhile, membrane-localized TMEM52B-P20 promotes E-cadherin ubiquitination and degradation by facilitating its interaction with the E3 ubiquitin ligase NEDD4, further driving NPC metastasis. In conclusion, the TMEM52B-P18 and TMEM52B-P20 isoforms promote the metastasis of NPC cells through different mechanisms. Drugs targeting these TMEM52B isoforms may offer therapeutic benefits to cancer patients with varying degrees of metastasis.

TLR8 agonist Motolimod-induced inflammatory death for treatment of acute myeloid leukemia.

The clinical treatment of AML is dominated by "7 + 3" therapy, but it often shows great toxicity and limited therapeutic efficacy in application. Therefore, it is urgent to develop novel therapeutic strategies to achieve safe and efficient treatment of AML. Small-molecule inhibitors have the characteristics of high specificity, low off-target toxicity and remarkable therapeutic effect, and are receiving more and more attention in tumor therapy. In this study, we screened a library of 1972 FDA-approved small molecular compounds for those that induced the inflammatory death of AML cells, among which the TLR8 agonist Motolimod (MTL) showed stronger anti-AML activity in the animal model but slight affection on normal lymphocytes in control mice. In terms of mechanism, cellular experiments in AML cell lines proved that TLR8 and LKB1/AMPK are the key distinct mechanisms for MTL triggered caspase-3-dependent cell death and the expression of a large number of inflammatory factors. In conclusion, our findings identified the immunoactivator MTL as a single agent exerting significant anti-AML activity in vitro and in vivo, with strong potential for clinical translation.

Homocysteine activates vascular smooth muscle cells by DNA demethylation of platelet-derived growth factor in endothelial cells.

Hyperhomocysteinemia (HHcy), as an independent risk factor of atherosclerosis, facilitates endothelial dysfunction and activation of vascular smooth muscle cells (VSMCs). However, little is known about the crosstalk between endothelial cells (ECs) and VSMCs under HHcy. We investigated whether homocysteine (Hcy) activates VSMCs by aberrant secretion of mitogen platelet-derived growth factors (PDGFs) from ECs in human and in mice. In this study, we found that increased Hcy level did not affect VSMC activity in 24 hrs until the concentration reached 500 µM. In contrast, Hcy at 100 µM significantly promoted proliferation and migration of VSMCs co-cultured with human ECs. This effect was partially reversed by pretreatment with a PDGF receptor inhibitor. Hcy concentration-dependently upregulated the mRNA level of PDGF-A, -C and -D but not PDGF-B in ECs. Hcy reduced the expression and activity of DNA methyltransferase 1, demethylation of PDGF-A, -C and -D promoters and enhanced the binding activity of transcriptional factor SP-1 to the promoter. Hcy upregulation of PDGF was confirmed in the aortic intima of mice with HHcy. Multivariate regression analysis revealed HHcy was a predictor of increased serum PDGF level in patients. Thus, Hcy upregulates PDGF level via DNA demethylation in ECs, affects cross-talk between ECs and VSMCs and leads to VSMC activation.

NUF2 Drives Clear Cell Renal Cell Carcinoma by Activating HMGA2 Transcription through KDM2A-mediated H3K36me2 Demethylation.

The poor sensitivity of clear cell renal cell carcinoma (ccRCC) to conventional chemotherapy and radiotherapy makes its treatment challenging. The Ndc80 kinetochore complex component (NUF2) is involved in the development and progression of several cancers. However, its role in ccRCC remains unclear. In this study, we investigated the biological functions and underlying mechanism of NUF2 in ccRCC. We found that NUF2 expression was increased in ccRCC and associated with poor prognosis. Altering NUF2 level affected cell proliferation, migration, and invasion. Moreover, NUF2 acted as a potential oncogene to promote the progression of ccRCC through epigenetic activation of high-mobility group AT-hook 2 (HMGA2) transcription by suppressing lysine demethylase 2A expression and affecting its occupancy on the HMGA2 promoter region to regulate histone H3 lysine 36 di-methylation modification. In addition, Kaplan-Meier and multivariate analysis revealed that patients whose NUF2 and HMGA2 were both elevated showed the shortest survival; and the number of upregulated markers acted as an independent predictor to evaluate survival probability. Thus, our results demonstrate that NUF2 promotes ccRCC progression, at least partly by epigenetically regulating HMGA2 transcription, and that the NUF2-HMGA2 axis could be an ideal therapeutic target and a promising prognostic indicator for ccRCC.

YBX3 Mediates the Metastasis of Nasopharyngeal Carcinoma via PI3K/AKT Signaling.

The metastasis of nasopharyngeal carcinoma (NPC) is a complex process associated with oncogenic dysfunction, the deciphering of which remains a challenge and requires more in-depth studies. Y-box protein 3 (YBX3) is a DNA/RNA binding protein associated with gene transcription, DNA repair, and the progression of various diseases. However, whether and how YBX3 affects the metastasis of NPC remains unknown. Thus, in this study, we aimed to investigate the role of YBX3 in the metastasis of NPC and determine its underlying mechanism. Interestingly, it was found that the expression of YBX3, which was associated with NPC metastasis, was upregulated in the clinical NPC tissues and cell lines. Moreover, we found that knockdown of YBX3 expression by lentivirus shRNA significantly suppressed NPC cells migration in vitro and metastasis in vivo. Mechanistically, RNA sequencing results suggested that the genes regulated by YBX3 were significantly enriched in cell adhesion molecules, cAMP signaling pathway, calcium signaling pathway, focal adhesion, PI3K/AKT signaling pathway, Ras signaling pathway, Rap1 signaling pathway, NF-κB signaling pathway, and Chemokine signaling pathway. Of these, PI3K/AKT signaling pathway contained the most genes. Accordingly, YBX3 knockdown decreased the activation of PI3K/AKT signaling pathway, thereby inhibit epithelial-to-mesenchymal transition (EMT) and MMP1. These results have demonstrated that YBX3 are involved in the metastasis of NPC through regulating PI3K/AKT signaling pathway, and serve as a potential therapeutic target for patients with NPC.

LncRNA CDKN2B-AS1 stabilized by IGF2BP3 drives the malignancy of renal clear cell carcinoma through epigenetically activating NUF2 transcription.

Because of the lack of sensitivity to radiotherapy and chemotherapy, therapeutic options for renal clear cell carcinoma (KIRC) are scarce. Long noncoding RNAs (lncRNAs) play crucial roles in the progression of cancer. However, their functional roles and upstream mechanisms in KIRC remain largely unknown. Exploring the functions of potential essential lncRNAs may lead to the discovery of novel targets for the diagnosis and treatment of KIRC. Here, according to the integrated analysis of RNA sequencing and survival data in TCGA-KIRC datasets, cyclin-dependent kinase inhibitor 2B antisense lncRNA (CDKN2B-AS1) was discovered to be the most upregulated among the 14 lncRNAs that were significantly overexpressed in KIRC and related to shorter survival. Functionally, CDKN2B-AS1 depletion suppressed cell proliferation, migration, and invasion both in vitro and in vivo. Mechanistically, CDKN2B-AS1 exerted its oncogenic activity by recruiting the CREB-binding protein and SET and MYND domain-containing 3 epigenetic-modifying complex to the promoter region of Ndc80 kinetochore complex component (NUF2), where it epigenetically activated NUF2 transcription by augmenting local H3K27ac and H3K4me3 modifications. Moreover, we also showed that CDKN2B-AS1 interacted with and was stabilized by insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), an oncofetal protein showing increased levels in KIRC. The Kaplan-Meier method and receiver operating curve analysis revealed that patients whose IGF2BP3, CDKN2B-AS1 and NUF2 are all elevated showed the shortest survival time, and the combined panel (containing IGF2BP3, CDKN2B-AS1, and NUF2) possessed the highest accuracy in discriminating high-risk from low-risk KIRC patients. Thus, we conclude that the stabilization of CDKN2B-AS1 by IGF2BP3 drives the malignancy of KIRC through epigenetically activating NUF2 transcription and that the IGF2BP3/CDKN2B-AS1/NUF2 axis may be an ideal prognostic and diagnostic biomarker and therapeutic target for KIRC.

Small interfering RNA-mediated knockdown of KRT80 suppresses colorectal cancer proliferation.

Colorectal cancer (CRC) is the third most common cancer in the world and its development is associated with oncogenic dysfunction. Therefore, the present study aimed to identify differentially expressed genes (DEGs) in CRC tissues and to determine the role of keratin 80 (KRT80) in CRC cell proliferation. DEGs were initially screened in 32 paired CRC tissues and matched adjacent normal tissues from RNA-Seq datasets in The Cancer Genome Atlas database using the limma package in R software. In total, 2,114 DEGs were identified, of which KRT80 was discovered to be the most upregulated in CRC tissues. Moreover, increased KRT80 expression levels were confirmed in tissues collected from 50 patients with CRC using reverse transcription-quantitative PCR, and its increased expression levels were significantly associated with increased lymph node and distant metastasis and a higher pathological stage. Furthermore, KRT80 knockdown using siRNA decreased the viability and proliferation of CRC cells. Finally, pathway analysis revealed that the proteins co-expressed with KRT80 in CRC were enriched in the cell cycle, DNA replication, immune system, metabolism of protein and RNA, signal transduction and other cellular processes. Among them, the cell cycle and DNA replication pathways contained the highest number of the proteins identified. In conclusion, the findings of the present study suggested that KRT80 may be overexpressed in CRC tissues. Furthermore, KRT80 may be involved in the proliferation of CRC cells, which is likely through its ability to regulate the cell cycle and DNA replication pathways, thus it may serve as a potential therapeutic target for patients with CRC.

FGFR3S249C mutation promotes chemoresistance by activating Akt signaling in bladder cancer cells.

Fibroblast growth factor receptor 3 (FGFR3) is a high frequency mutant gene in bladder cancer (BCa) and has become a promising therapeutic target due to its involvement in cell proliferation and migration. However, whether and how FGFR3 mutations affects BCa cell chemosensitivity is unknown. The current study aimed to elucidate the role of the FGFR3S249C mutation in the development of chemoresistance in BCa cells. The results revealed that 97-7 (FGFR3S249C) cells had decreased sensitivity to cisplatin compared with 5637 (FGFR3WT) and T24 (FGFR3WT) cells. The ratio of phosphorylated-Akt/total-Akt was higher in 97-7 (FGFR3S249C) cells, which was reversed by knockdown of FGFR3. Furthermore, inhibition of Akt signaling by GDC0068 or LY294002 increased the cisplatin sensitivity of 97-7 (FGFR3S249C) cells. GDC0068 or LY294002 was also revealed to augment the effects of cisplatin on 97-7 (FGFR3S249C) cell proliferation and apoptosis. The results of the present study demonstrated that the FGFR3S249C mutation promotes chemoresistance in BCa cells by activating the Akt signaling pathway. The FGFR3S249C mutation may therefore be used as a predictor of chemosensitivity in patients with BCa.

MiR-506 Suppresses Colorectal Cancer Development by Inhibiting Orphan Nuclear Receptor NR4A1 Expression.

NR4A1 acts as an oncogene and plays an important role in colorectal cancer development and progression, but little is known about the regulatory mechanism of NR4A1 expression. MicroRNA (miRNA) is involved in the progression of various tumors, affecting proliferation, apoptosis or migration. We aimed to elucidate whether miRNA regulates NR4A1 expression and determine its underlying significance in colorectal cancer. By using the TargetScan database, we identified a miR-506 binding site in the NR4A1 3'-UTR. Examination of colorectal cancer tissues and cells revealed that NR4A1 mRNA and protein were up-regulated, while miR-506 expression was down-regulated. Spearman correlation analysis revealed that expression of NR4A1 mRNA was negatively correlated with miR-506 levels in colorectal cancer tissue. Further studies indicated that miR-506 decreased NR4A1 expression through directly targeting the NR4A1 mRNA 3'-UTR. Functional experiments showed that rescue of NR4A1 expression in cells reversed the inhibitory effects of miR-506 on proliferation, migration and invasion of colorectal cancer cells. In conclusion, miR-506 acts as a tumor suppressor and inhibits proliferation, migration and invasion in colorectal cancer cells partly through decreasing NR4A1 expression.

Orphan nuclear receptor NR4A1 suppresses hyperhomocysteinemia-induced hepatic steatosis in vitro and in vivo.

Homocysteine (Hcy) is associated with nonalcoholic fatty liver disease (NAFLD). orphan nuclear receptor subfamily 4 group A member 1 (NR4A1) is involved in hepatic lipid metabolism. However, the potential role of NR4A1 in Hcy-associated NAFLD remains elusive. We aimed to elucidate the regulation of NR4A1 and its significance in Hcy-induced NAFLD. Hcy induced steatosis and elevated the expression of CD36 and FATP2 in HepG2 cells. Furthermore, Hcy enhanced p300 and decreased HDAC7 recruitment to the NR4A1 promoter, resulting in histone H3K27 hyperacetylation and NR4A1 upregulation. Moreover, NR4A1 depletion not only mimicked but also exaggerated the effects of Hcy on steatosis, whereas NR4A1 agonist Cytosporone B (CsnB) blocked Hcy-induced steatosis. In hyperhomocysteinemia (HHcy) mice, CsnB attenuated HHcy-induced hepatic steatosis. Thus, Hcy transiently and rapidly induces NR4A1 expression to reduce Hcy-induced steatosis.

Upregulation of miR-200b Inhibits Hepatocellular Carcinoma Cell Proliferation and Migration by Targeting HMGB3 Protein.

HMGB3 belongs to the high-mobility group box subfamily and has been found to be overexpressed in gastric cancer. However, the expression and the role of HMGB3 in human hepatocellular carcinoma remain unknown. Here, we report that HMGB3, which is suppressed by miR-200b, contributes to cell proliferation and migration in human hepatocellular carcinoma. After analyzing The Cancer Genome Atlas data of 371 patients with hepatocellular carcinoma, we identified HMGB3 to be upregulated in human hepatocellular carcinoma tissue. Knockdown of HMGB3 in the hepatocellular carcinoma cell line suppressed cell proliferation and migration. TargetScan analysis showed miR-200b to be a possible regulator for HMGB3. Subsequent luciferase assays indicated that HMGB3 was a direct target of miR-200b. In addition, upregulation of miR-200b inhibited hepatocellular carcinoma cell growth and migration. HMGB3 overexpression or miR-200b downregulation was associated with poor prognosis. Our findings suggest HMGB3 may serve as an important oncoprotein whose expression is negatively regulated by miR-200b in hepatocellular carcinoma.