Additional file 3 of EYA2 suppresses the progression of hepatocellular carcinoma via SOCS3-mediated blockade of JAK/STAT signaling
Creators: name: Liu, Ze-Kun
name: Li, Can
name: Zhang, Ren-Yu
name: Wei, Ding
name: Shang, Yu-Kui
name: Yong, Yu-Le
name: Kong, Ling-Min
name: Zheng, Nai-Shan
name: Liu, Ke
name: Lu, Meng
name: Liu, Man
name: Hu, Cai-Xia
name: Yang, Xiao-Zhen
name: Chen, Zhi-Nan
name: Bian, Huijie
Publisher: name: figshare
Year: 2021
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 3: Fig. S1. The classification and number of somatic mutations identified by whole exome sequencing. Fig. S2. Somatic mutations identified in candidate gene GPR98, EYA2 and UBE2S, which were confirmed by IGV visualization from HCC and case-matched adjacent tissues. The bold numbers in the figures represent allelic fraction of the altered base. Fig. S3. The mRNA expression and function of EYA2 in pan-cancer. (A) mRNA expression of EYA2 in pan-cancer and their respective adjacent based on TCGA database. (B) mRNA expression of EYA2 in pan-cancer cells based on CCLE database. (C) Western blot analysis of the expression of EYA2 in four cancer cells transiently transfected with overexpression vectors. (D) The effect of pcDNA3.1-mediated overexpression of constructs encoding EYA2 wild-type on cell proliferation of cancer cells in vitro. (E) The effect of EYA2 wild-type on cell invasion of cancer cells in vitro. ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma. *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001. (A, D, E) mean ± SEM, Student’s t-tests. Fig. S4. EYA2 inhibits the malignant phenotypes of HCC cells in vitro. (A) Western blot analysis of the expression of EYA2 in five liver cancer cells. (B) Expression of EYA2 in MHCC-97H cells or Hep3B transfected or infected with si-EYA2 or sh-EYA2 detected by qRT-PCR analysis. (C) Western blot analysis of the expression of EYA2 in MHCC-97H or Hep3B cells stably transfected with shRNA or EYA2 lentivirus and EYA2(A510E) mutant lentivirus. The effect of shRNA-mediated knockdown of EYA2 and lentivirus-mediated EYA2 and EYA2(A510E) overexpression on cell proliferation (D) and cell invasion (E) HCC cells in vitro. The flow cytometry analysis of the effect of EYA2 on cell cycle progression (F) and apoptosis (G) of HCC cells. (H) Western blot analysis of P27, BAX, cyclin D1 and CDK2 expression in HCC cells stably transfected with sh-EYA2 or the overexpression vectors. *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001. (B, D, E–G) mean ± SEM, Student’s t-tests. Fig. S5. Expression of EYA2 in tumor tissues detected by western blot analysis. *P &lt; 0.05, mean ± SEM, Student’s t-tests. Fig. S6. Association of the mRNA expression of EYA2 with its methylation level and copy number analyses in different types of tumors. (A) Potential CpG islands of EYA2 were predicted from 5000 bp upstream to 1000 bp downstream of the transcription start site. (B) The methylation level of EYA2 in pan-cancer and their respective adjacent tissues based on TCGA database. (C) Correlation between EYA2 methylation and the mRNA expression in pan-cancer. (D) Copy number distribution of the known genes and EYAs family gene based on TCGA_LIHC (The blue line in the inner circle represented the deletion and the black line in the inner circle represented the gain). (E) Correlation between EYA2 copy number and the mRNA expression. BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; COAD, colon adenocarcinoma; ESCA, esophageal carcinoma; HNSC, head and neck squamous cell carcinoma; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; PAAD, pancreatic adenocarcinoma; PRAD, prostate adenocarcinoma; THCA, thyroid carcinoma; UCEC, uterine corpus endometrial carcinoma. *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001. (B) mean ± SEM, Student’s t-tests; (C) Pearson’s correlation test; (E) Kruskal-Wallis test. Fig. S7. EYA2(A510E) mutation reduces EYA2 protein level. qRT-PCR (A) and western blot (B) analysis of EYA2 expression in HEK293T and Huh-7 transiently transfected with overexpression and mutant vectors. qRT-PCR (C) and western blot (D) analysis of EYA2 expression in HEK293T and two HCC cell lines MHCC-97H, Huh-7 stably infected with lentivirus-EYA2 and lentivirus-EYA2(A510E). *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001. (A, C) mean ± SEM, Student’s t-tests. Fig. S8. Western blot analysis of unfold protein response-related protein expression in MHCC-97 cells treated with MG132. Fig. S9. Association of EYA2 with DACH1, SOCS1, SOCS2 and SOCS3 expression in human HCC tissues. (A) qRT-PCR verification of eight representative genes in Huh-7 cells with EYA2 overexpression. Expression of SOCS1, SOCS2, SOCS3 and DACH1 in HCC and adjacent tissues (B) and correlation of EYA2 with SOCS2, SOCS3 and DACH1 in human HCC tissues (C) based on GSE22058 and GSE14520 database. (D) The network interaction diagram of crucial genes EYA2, DACH1 and SOCS3. Fig. S10. Expression and prognostic significance of SOCS3 in HCC tissues. (A) A representative immunohistochemical staining of SOCS3 in HCC and case-matched adjacent tissues. (B) Immunohistochemistry scores associated to the expression of SOCS3 in HCC and paired adjacent tissues (n = 60 pairs). (C) Correlation analysis between EYA2 and SOCS3 protein expression (n = 60 cases). (D) Kaplan-Meier analysis of overall survival of HCC patients with high or low SOCS3 expression (n = 60 cases). ***P &lt; 0.001. (B) mean ± SEM, Student’s t-tests; (C) Spearman’s correlation test. (D) Kaplan-Meier analysis. Fig. S11. The combination of EYA2 and DACH1 regulates the SOCS3-mediated blockade of JAK/STAT signaling. (A) SOCS3 promoter constructs (− 2000/− 1) co-transfected with pcDNA3.1-EYA2 or/and GV141-DACH1 and the relative luciferase activity measured in MHCC-97H cell line. (B) Western blot analysis of expression of SOCS3, p-STAT3, p-JAK2, STAT3 and JAK2 in MHCC-97H cells co-transfected with si-DACH1/pcDNA3.1-EYA2. (C) Western blot analysis of expression of SOCS3 in MHCC-97H and Huh-7 cells infected with lentivirus-EYA2 and lentivirus-EYA2(A510E) mutant. *P &lt; 0.05. (A, B) mean ± SEM, Student’s t-tests. Fig. S12. Detection of Eya2−/− and Eya2+/+ mice of non-induced group at 11 months. (A) DNA electrophoretic map of genotype identification of Eya2−/− mice. Electrophoresis of the PCR products of the Alb-Cre gene (left) and EYA2 (right). Eya2+/+ represented wild-type mouse with a band size of 278 bp and Eya2−/− represented hepatocyte-specific EYA2 knockout mouse with a band size of 358 bp. (B) Images of the liver from non-induced Eya2−/− and Eya2+/+ mice. (C) H&amp;E staining and immunohistochemistry analysis of the expression of EYA2, SOCS3 and PCNA in liver tissues from the non-induced mice. Fig. S13. The level of STAT3 phosphorylation in response to IL-6 in Eya2−/− hepatocytes. STAT3 phosphorylation induced by IL-6 was significantly higher in EYA2-deficient hepatocytes than control littermates at each point of time.