Anti-RAF1 monoclonal antibody, clone 5H5

The long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) was reported to be upregulated and be involved in oncogenic growth and drug resistance in nasopharyngeal carcinoma (NPC). However, the exact roles of NEAT1 and its underlying mechanisms in the drug resistance of NPC remain largely unclear. In this study, the expressions of NEAT1, let-72-5p and Rsf-1 mRNA were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The effects of NEAT1 and let-72-5p on cell proliferation and cisplatin resistance of NPC cells were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and 5-ethynyl-20-deoxyuridine (EdU) assay. Western blot analysis was performed to detect the protein levels of Rsf-1, Ras, p-Raf1, Raf1, p-MEK1, MEK1, p-ERK1/2 and ERK1/2. Xenograft tumor assay was done to elucidate the role of NEAT1 involved in NPC tumor growth in vivo. We found that NEAT1 was upregulated and let-7a-5p was downregulated in NPC tissues, as well as NPC cell lines. Inhibition of NEAT1 markedly repressed the cisplatin resistance of NPC cells. NEAT1 was demonstrated to interact with let-7a-5p. Besides, a negative correlation between NEAT1 and let-7a-5p expression was observed in NPC tissues. Rsf-1 was confirmed as a target of let-7a-5p. NEAT1 remarkably reversed the inhibitory effect of let-7q-5p on the cisplatin resistance of NPC cells in vitro. Additionally, NEAT1 knockdown inhibited the Ras-MAPK pathway in NPC cells. NEAT1 knockdown suppressed tumor growth in the presence of cisplatin in vivo. Overall, these findings suggest that NEAT1/let-7a-5p axis regulates the cisplatin resistance in NPC by targeting Rsf-1 and modulating the Ras-MAPK signaling pathway.

Long non-coding RNAs (ncRNAs) are major regulators of gene expression and cell fate. TheINK4locus encodes the tumour suppressor proteins p15(INK4b), p16(INK4a)and p14(ARF)required for cell cycle arrest and whose expression increases during senescence.ANRILis a ncRNA antisense to thep15gene. In proliferative cells,ANRILprevents senescence by repressingINK4genes through the recruitment of Polycomb-group proteins. In models of replicative and RASval12 oncogene-induced senescence (OIS), the expression ofANRILand Polycomb proteins decreases, thus allowingINK4derepression. Here, we found in a model of RAF1 OIS thatANRILexpression rather increases, due in particular to an increased stability. This led us to search for circularANRILisoforms, as circular RNAs are rather stable species. We found that the expression of two circularANRILincreases in several OIS models (RAF1, MEK1 and BRAF). In proliferative cells, they repressp15expression, while in RAF1 OIS, they promote full induction ofp15, p16andp14(ARF)expression. Further analysis of one of these circularANRILshows that it interacts with Polycomb proteins and decreases EZH2 Polycomb protein localization and H3K27me3 at thep15andp16promoters, respectively. We propose that changes in the ratio between Polycomb proteins and circularANRILisoforms allow these isoforms to switch from repressors ofp15gene to activators of allINK4genes in RAF1 OIS. Our data reveal that regulation ofANRILexpression depends on the senescence inducer and underline the importance of circularANRILin the regulation ofINK4gene expression and senescence.