Fig 1: Triple upregulation of TCF7/c-Myc/FOSL1 was a sensitive marker predicting unfavorable prognosis. (A–C) In cohort 1, the overall survival curves were stratified according to c-Myc (A) and FOSL1 (B) expression or triple overexpression of TCF7/c-Myc/FOSL1 (C). (D–F) In cohort 2, the overall survival curves were stratified according to c-Myc (D) and FOSL1 (E) expression or triple overexpression of TCF7/c-Myc/FOSL1 (F). Patients with high expression of TCF7 had poor prognoses, while patients with triple upregulation of TCF7/c-Myc/FOSL1 had much worse prognoses than other patients in both cohort 1 and cohort 2 (P < 0.001 and P = 0.001).
Fig 2: FOSL1 was required for TCF7-induced proliferation of PHCC. (A) Effects of Wnt agonist 1 on the expression of TCF7 and FOSL1 in QBC939 and FRH0201 cells. (B) FOSL1 knockdown in QBC939/FRH0201 cells or stable QBC939/FRH0201 cell overexpressing TCF7. (C) CCK8 assays showing the effects of FOSL1, Wnt agonist 1, and TCF7 on the proliferation of QBC939 and FRH0201 cells. (D) Effects of FOSL1 knockdown on the proliferation of QBC939 and FRH0201 cells in soft agar colony assays. (E) FOSL1 was overexpressed by lentivirus infection of FOSL1 plasmid after silencing TCF7 in QBC939 and FRH0201 cells. (F and G) CCK8 (F) and soft agar (G) assays revealed that FOLS1 overexpression rescued the reduced proliferation induced by TCF7 knockdown. (H) Establishment of a stable QBC939 cell line with FOSL1 knockdown using a lentivirus carrying FOSL1 shRNA. (I) Xenografts from nude mice were generated with or without FOSL1 knockdown. Mice were intravenously injected in presence or absence of Wnt agonist 1 at 3.5 mg/kg. (J and K) FOSL1 knockdown decreased the tumor volume and weight of xenografts.
Fig 3: FOSL 1 transcription was promoted by TCF7 in PHCC. (A) FOSL1 mRNA was decreased in TCF7-silenced QBC939 cells among the 31 candidate genes screened with qRT-PCR. (B) Effects of TCF7 knockdown or overexpression on FOSL1 expression. (C) Representative FOSL1 IHC staining of xenograft tumors derived from cells transfected with empty vector or shTCF7. Scale bar: 50 μm. (D) FOSL1 mRNA in xenografts transfected with shTCF7 or empty vector. (E) Association of IHC scores for FOSL1 and TCF7 in human PHCC tissues. (F) Correlations of mRNA levels of TCF7 and FOSL1 in fresh PHCC tissues. (G) Luciferase assays for analysis of the effects of TCF7 on the transcription of c-Myc and FOSL1. *, **, and *** represent P < 0.05, P < 0.01, and P < 0.001, respectively, between the indicated subgroups. Statistical significance was analyzed using Student's t-tests.
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