Fig. 3: LUCAT1 regulates MYC expression by binding to NCL in the nucleus. | Cell Death & Disease

Fig. 3: LUCAT1 regulates MYC expression by binding to NCL in the nucleus.

From: The long noncoding RNA LUCAT1 promotes colorectal cancer cell proliferation by antagonizing Nucleolin to regulate MYC expression

Fig. 3

(A) The amounts of LUCAT1 in the nuclear and cytoplasmic fractions of HCT116 cells were quantified by qRT-PCR. GAPDH was used as a control for cytoplasmic transcripts. MALAT1 was used as a nuclear marker. A nuclear/cytoplasmic ratio >1 proved that LUCAT1 was a nuclear-enriched lncRNA. (B) ChIRP assay using LUCAT1 probes followed by PRM analysis, which detected and quantified the amounts of NCL. Production pattern and chromatograph of two labeled NCL peptides (GYAFIEFASFEDAK and GFGFVDFNSEEDAK). Different colors represent different fragment ions of the same polypeptide. Each peptide was quantified using six fragment ions. U1 probes were used as a positive control. (C) Immunofluorescence staining using anti-NCL antibodies (green) in HCT116 cells. Scale bars, 10 µm. DAPI, 4′,6-diamidino-2-phenylindole. (D) Colocalization analysis: RNA FISH assay of LUCAT1 (red) combined with the immunofluorescence detection of NCL (green) in HCT116 cells. Scale bars, 20 µm. (E) A RIP assay was performed in HCT116 and SW620 cells using anti-NCL and anti-IgG antibodies (control). The amount of LUCAT1 mRNA was quantified by qRT-PCR. The results are presented as the mean±s.d. and are representative of at least three independent experiments. (F–H) Expression changes in four G4-associated genes, MYC, HIF-1α, VEGF, and KRAS, were determined in the NC and sgRNA HCT116 and SW620 cells with qRT-PCR (G) and Western blot analyses (F, H). (I) ChIP analysis of the interaction between the NCL protein and the MYC promoter in HCT116 cells. ChIP-qPCR of NCL at the MYC promoter when LUCAT1 or the vector was overexpressed in HCT116 cells. (J) NCL binding to the MYC promoter was detected by a luciferase assay. The relative luciferase activity of the reporter containing the MYC NHE III1 promoter/mutant was cotransfected with the indicated constructs in HCT116 cells. LUCAT1 interfered with NCL binding to the MYC promoter via its potential G4-forming sequence. (K) Relative luciferase activity of the reporter containing the MYC NHE III1 promoter/mutant that was cotransfected with the indicated constructs into HCT116 cells. The results are presented as the mean±s.d. and are representative of at least three independent experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001, ns p > 0.05.

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