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Fig. 5 | Molecular Cancer

Fig. 5

From: ALKBH5 suppresses malignancy of hepatocellular carcinoma via m6A-guided epigenetic inhibition of LYPD1

Fig. 5

ALKBH5 impairs the stability of LYPD1 mRNA via an IGF2BP1-m6A-dependent pattern. a m6A abundance on LYPD1 mRNA in negative control or ALKBH5-overexpressing HCCLM3 cells was plotted by the IGV. Green and pink colors show the m6A signals of input samples, while red and blue stand for signals of IP samples. The range of signals in all groups was normalized to a 0–560 scale. At the same position, m6A peaks of IP group over input group were recognized as the genuine m6A level. Black blocks below figure indicated the sites where the m6A level differed between two groups, and the most remarkable location was highlighted with a gray pane. b Relative enrichment of LYPD1 mRNA associated with ALKBH5 protein was identified by RIP assays using anti-IgG and anti-ALKBH5 antibodies. The IgG group was a negative control to preclude nonspecific binding. The Y axis represented the percent of input for each IP sample according to the formula: %Input =1/10*2Ct [IP] – Ct [input]. c m6A modification of LYPD1 was detected by MeRIP-qPCR analysis using anti-IgG and anti-m6A antibodies. Relative m6A enrichment of LYPD1 mRNA for each IP group was normalized to input. Silencing of ALKBH5 induced an increase m6A abundance on LYPD1 compared with control group, while ALKBH5 overexpression led to the opposite result; d Graphical explanation for construction of luciferase reporters. The wild-type (full-length) or mutant (m6A motif mutated) sequence of LYPD1–3’UTR was inserted into a pcDNA3.1 vector between Firefly and Renilla elements. Relative luciferase activity was computed by the ratio of Firefly and Renilla luciferase values. e Relative luciferase activity of Huh7, MHCC97H and HCCLM3 cells transfected with the LYPD1-wild type or -mutated construct was measured, with normal or altered expression of ALKBH5; f ALKBH5-silenced or -overexpressed cells were treated with actinomycin D and harvested at 0, 3 and 6 h. RNA decay rate was determined to estimate the stability of LYPD1 (normalized to the expression at 0 h); g IGF2BP1 was knockdown in two HCC cells followed by the measurement of LYPD1 expression via qPCR; h RIP-qPCR validated that IGF2BP1 could bind to LYPD1 mRNA. Relative enrichment of LYPD1 mRNA in each group was showed with the normalization to input; i Rescue assays were employed to verify the impact of IGF2BP1 on ALKBH5-mediated modulation of LYPD1

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