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. 2022 Apr 24;12(7):3518–3533. doi: 10.7150/thno.72581

Figure 3.

Figure 3

mTORC1 acts at NEAT1 promoter and negatively regulates NEAT1 transcription. (A-B) Rapamycin stimulates NEAT1 promoter activity. Activity of NEAT1 promoter-driven luciferase reporter was measured in SNU423 (A) and Huh1 (B) cells treated with or without 100 nM rapamycin for 24 h. Mean ± SEM (n = 3), Unpaired two-tail t test; (C) NEAT1 promoter activity is regulated by glucose and growth factors. SNU423 cells transfected with NEAT1 promoter luciferase reporter were starved from fetal bovine serum (FBS) or glucose. Data (mean ± SEM, n = 3) were analyzed by unpaired two-tail t test. (D) Shown are peaks of mTOR-binding and histone H3K4me3 across a region of Chromosome 19 in mouse liver as determined by analysis of the ChIP-seq (mTOR dataset: GSM1067407; H3K4me3 dataset: GSM1970920). Boxed region shows Neat1 locus. (E) mTOR binds to the NEAT1 promoter in a rapamycin-sensitive manner in HCC cells. SNU423 cells were treated without or with 100 nM rapamycin for 2 h. Anti-mTOR ChIP was performed and the results were analyzed by qRT-PCR. Blue boxes indicate NEAT1 coding, promoter and upstream region used for ChIP analysis. GAPDH promoter was used as a negative control. % Input = 100*2^(Adjusted input - Ct (IP). Mean ± SEM (n = 3), unpaired two-tail t test. (F) mTOR binding to NEAT1 promoter is blocked by mTOR knockdown in HCC cells. SNU423 cells were transfected with mTOR siRNA (simTOR) or control siRNA (siNC). mTOR binding to NEAT1 promoter was assayed by anti-mTOR ChIP. (G) mTORC1 binds to NEAT1 promoter in a rapamycin-sensitive manner. HA-mTOR or HA-Raptor was transiently expressed in SNU423 cells and then treated without or with rapamycin. HA-mTOR and HA-Raptor binding to NEAT1 promoter was assayed by anti-HA ChIP. (H) mTORC2 does not bind to NEAT1 promoter in HCC cells. Myc-Rictor was transiently expressed in SNU423 cells and then treated without or with rapamycin. Myc-Rictor binding to NEAT1 promoter was assayed by anti-Myc ChIP. Mean ± SEM (n = 3), unpaired two-tail t test. *** p < 0.001. ns, not significant.