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. 2019 Apr 29;70(1):259–275. doi: 10.1002/hep.30613

Figure 7.

Figure 7

Lnc‐UCID promotes CDK6 expression and cell proliferation by sequestering DHX9 from the CDK6‐3′UTR. (A) DHX9 knockdown abrogated the siUCID‐induced down‐regulation of CDK6. HepG2 or SF cells were co‐transfected with the indicated siRNAs for 48 hours. (B) DHX9 knockdown abrogated the siUCID‐induced G1 population accumulation. The siRNA‐transfected cells were synchronized with nocodazole before FACS analysis. (C) The lnc‐UCID‐induced up‐regulation of CDK6 was attenuated when its 850‐1030‐nt sequence was deleted. HepG2 or QGY‐7703 cells were transfected with the indicated plasmid for 24 hours, cultured with puromycin for 48 hours, and then subjected to western blotting. (D) The lnc‐UCID‐induced increases in DNA replication and cell growth were abrogated when the 850‐1030‐nt sequence was deleted. QGY‐7703 cells were transfected twice with the indicated plasmids at 24‐hour intervals. Twenty‐four hours after the last transfection, the cells were subjected to BrdU incorporation or cell counting assays. (E) Lnc‐UCID overexpression diminished the association of DHX9 with CDK6‐DBE1. HepG2 cells were transfected twice at 24‐hour intervals with the indicated plasmids and then subjected to RIP assays 48 hours after the last transfection. RIP assays were conducted with a DHX9 antibody or isotype‐matched control IgG, and the precipitated RNAs were detected by quantitative real‐time PCR. RNA enrichment indicates the levels of CDK6‐DBE1, CDK6‐non‐DBE, or lnc‐UCID in the anti‐DHX9 precipitates relative to those in the IgG precipitates. (F) Expression of DHX9 abolished the lnc‐UCID‐increased luciferase activity of the CDK6‐DBE1‐containing reporter, and silencing DHX9 rescued the siUCID‐reduced luciferase activity of this reporter. HepG2 cells were co‐transfected with psi‐CDK6‐DBE1 and the indicated plasmids or siRNA. For (C)‐(F), Ctrl, pc3‐puro was used as a NC. *P < 0.05; ***P < 0.01; ***P < 0.001. Abbreviations: LE, long exposure; SE, short exposure; UCID, pc3‐puro‐lnc‐UCID containing the full‐length lnc‐UCID; and UCID‐Δcore, pc3‐puro‐lnc‐UCID‐Δcore containing mutant lnc‐UCID with the 850‐1030‐nt deletion.