Integrative Functional Genomics Implicates EPB41 Dysregulation in Hepatocellular Carcinoma Risk  Xinyu Yang, Dianke Yu, Yanli Ren, Jinyu Wei, Wenting Pan, Changchun Zhou, Liqing Zhou, Yu Liu, Ming Yang  The American Journal of Human Genetics  Volume 99, Issue 2, Pages 275-286 (August 2016) DOI: 10.1016/j.ajhg.2016.05.029 Copyright © 2016 American Society of Human Genetics Terms and Conditions

Figure 1 Flowchart of an Integrative Functional Genomics Methodology to Identify Cancer Susceptibility Genetic Variants in c-Myc-Binding Sites across the Whole Genome The American Journal of Human Genetics 2016 99, 275-286DOI: (10.1016/j.ajhg.2016.05.029) Copyright © 2016 American Society of Human Genetics Terms and Conditions

Figure 2 Abolishment of a c-Myc Binding Site in the EPB41 Promoter by the rs157224G>T Genetic Polymorphism Influences Promoter Activities (A) Electrophoretic mobility-shift assay (EMSA) with biotin-labeled c-Myc consensus, rs157224G, or rs157224T probes and SMMC7721 nuclear extract. Left: EMSA with c-Myc consensus or rs157224G probes. Lanes numbered from left to right. Lanes 4 and 8, probe only; lanes 2 and 6, probe and nuclear extracts; lanes 1, 3, 5, and 7, probe and nuclear extracts plus 100× unlabeled rs157224G (lanes 1 and 5) or c-Myc consensus probes (lanes 3 and 7). Right: EMSA with rs157224G or rs157224T probes. Lanes 1 and 6, probe only; lanes 2 and 7, probe and nuclear extracts; lanes 3–5 and 8–10, probe and nuclear extracts plus 100× unlabeled rs157224G (lanes 5 and 8), rs157224T (lanes 3 and 10), or c-Myc consensus probes (lanes 4 and 9). Probes: EPB41-rs157224G, 5′-GAAGCAATTTGACACGTGGTACTGCTCCTAA-3′; EPB41-rs157224T, 5′-GAAGCAATTTGACACTTGGTACTGCTCCTAA-3′; c-Myc-consensus, 5′-CAGGAAGCAGACCACGTGGTCAGGCTATA-3′. (B and C) EMSA with biotin-labeled rs157224G or rs157224T probes and Huh7 (B) or HepG2 (C) nuclear extracts. (D) EMSA competition assay using c-Myc antibody in SMMC7721 and HepG2 cells. (E) ChIP assays using SMMC7721 cells carrying the rs157224GG genotype. The presence of c-Myc-binding EPB41 or P53 promoter was verified by PCR, with GAPDH as the negative control. (F) Transient luciferase reporter gene expression assays with constructs containing different lengths or different rs157224 allele of the region telomeric to EPB41 in SMMC7721 or HepG2 cells. pRL-SV40 were cotransfected with these constructs to standardize transfection efficiency. Fold-changes were detected by defining the luciferase activity of cells co-transfected with pGL3-basic as 1. All experiments were performed in triplicate in three independent transfection experiments and each value represents mean ± SD. Compared with pGL3-Basic transfected cells, ∗p < 0.05; ∗∗p < 0.01. The American Journal of Human Genetics 2016 99, 275-286DOI: (10.1016/j.ajhg.2016.05.029) Copyright © 2016 American Society of Human Genetics Terms and Conditions

Figure 3 Impacts of EPB41 on HCC Cell Proliferation In Vitro and In Vivo (A) Overexpression of EPB41 significantly suppressed cell growth of SMMC7721, HepG2, and Huh7 cells. Cell number was counted at 24 hr, 48 hr, and 72 hr after transfection. (B) Silencing EPB41 with three siRNAs (siEPB41-1, siEPB41-2, and siEPB41-3) accelerates cell proliferation of SMMC7721, HepG2, and Huh7 cells. (C) Enforced EPB41 expression induces HCC cell apoptosis. Apoptosis was determined with FACSCalibur flow cytometer. (D) Silencing EPB41 with siRNAs inhibits HCC cell apoptosis. (E) Colony formation assays. pcDNA3.1-EPB41 or pcDNA3.1 as well as 20 nmol/L NC RNA or EPB41 siRNAs was transfected into HCC cells, respectively. (F and G) EPB41 significantly inhibits growth of HepG2 xenografts compared with control xenografts after 15 days. (H) EPB41 and β-actin protein levels in xenografts. ∗∗p < 0.01. The American Journal of Human Genetics 2016 99, 275-286DOI: (10.1016/j.ajhg.2016.05.029) Copyright © 2016 American Society of Human Genetics Terms and Conditions

Figure 4 EPB41 Reduces Migration and Invasion Ability of HCC Cells (A) Enforced EPB41 expression inhibits wound healing in SMMC7721 and HepG2 cells. Wound fields were observed directly after removal of inserts (0 hr) and cell migration was followed for 24 hr and 48 hr. Wound-healing area in HCC cells was presented by histogram. (B) Silencing EPB41 accelerates wound healing in SMMC7721 and HepG2 cells. Wound fields were observed directly after removal of inserts (0 hr) and cell migration was followed for 18 hr and 36 hr. Wound-healing area in HCC cells was presented by histogram. (C) EPB41 inhibits invasion abilities of SMMC7721 and HepG2 cells. Cells on the lower surface of the chamber were stained by crystal violet at 48 hr after transfection. ∗p < 0.05, ∗∗p < 0.01. The American Journal of Human Genetics 2016 99, 275-286DOI: (10.1016/j.ajhg.2016.05.029) Copyright © 2016 American Society of Human Genetics Terms and Conditions

Figure 5 EPB41 Expression in HCC Tissue Specimens and Public Gene Profiling Databases (A–C) EPB41 mRNA and protein were quantified using qRT-PCR, immunohistochemical analyses, and western blot in 48 tumor-normal pairs. All data of EPB41 expression were normalized to β-actin expression levels. (D) EPB41 mRNA expression in HCC and normal tissues grouped by rs157224G>T genotypes. (E) EPB41 expression in GEO: GSE45267. (F) EPB41 expression in GEO: GSE364, the gene expression profiles of HCC samples with or without metastases. Subjects without metastasis showed much higher EPB41 expression than ones with portal vein metastasis or intrahepatic metastasis. ∗p < 0.05, ∗∗p < 0.01. The American Journal of Human Genetics 2016 99, 275-286DOI: (10.1016/j.ajhg.2016.05.029) Copyright © 2016 American Society of Human Genetics Terms and Conditions