Briefly, lentivirus-infected HCC cells were seeded in 24-well plates at a density of 2000 cells/well

Briefly, lentivirus-infected HCC cells were seeded in 24-well plates at a density of 2000 cells/well. FOXM1 and KIF4A are shown as box plots. The number of samples for each grade is usually shown below the group. Data were analyzed with the KruskalCWallis H test. h, i Overall survival rate associated with FOXM1 (h) and KIF4A (i) based on records in TCGA. Data in Kaplan-Meier curves were analyzed with the log-rank test. (TIF 5320 kb) 13046_2019_1202_MOESM2_ESM.tif (5.1M) GUID:?AF5204D7-E5CE-4D5D-B86A-0019F88070E0 Additional file 3: Figure S2. Effect confirmation of the lentivirus infected HCC cell lines. a HepG2 cells infected with lentivirus of FOXM1 or KIF4A overexpression. b Huh7 cells infected with FOXM1 or KIF4A knockdown lentivirus and Hep3B cells infected with ADU-S100 ammonium salt FOXM1 knockdown lentivirus. (TIF 802 kb) 13046_2019_1202_MOESM3_ESM.tif (803K) GUID:?AB6D0747-23F1-4DBD-8E50-9AAF9AF20474 Data Availability StatementThe datasets analyzed during the current study are available from the corresponding author on reasonable request. Abstract Background Forkhead box M1 (FOXM1) is usually a proliferation-associated transcription factor of the forkhead box proteins superfamily, which includes four isoforms FOXM1a, b, c, and d. FOXM1 has been implicated in hepatocellular carcinoma (HCC) progression, but the underlying molecular mechanism remains elusive. In this study, we aim to clarify the molecular basis for FOXM1-mediated HCC progression. Methods Bioinformatic analysis was used to explore the differentially expressed genes predicting HCC proliferation. The expression of FOXM1 and kinesin family member (KIF)4A was confirmed by western blotting and immunohistochemistry in HCC tissues. Kaplan-Meier survival analysis was conducted to analyze the clinical impact of FOXM1 and KIF4A on HCC. The effect of FOXM1 around the regulation of KIF4A expression was studied in cell biology experiments. The conversation between KIF4A and FOXM1 was analyzed by chromatin immunoprecipitation and luciferase experiments. A series of experiments was performed to explore the functions of FOXM1/KIF4A in HCC progression, such as cell proliferation, cell growth, cell viability, and cell cycle. A xenograft mouse model was used to explore the regulatory effect of FOXM1-KIF4A axis on HCC tumor growth. Results FOXM1 and KIF4A were overexpressed in human primary HCC tissues compared to that in matched adjacent normal liver tissue and ADU-S100 ammonium salt are significant risk factors for HCC recurrence and shorter survival. We found that KIF4A was dominantly regulated by FOXM1c among the Rabbit polyclonal to GAPDH.Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) is well known as one of the key enzymes involved in glycolysis. GAPDH is constitutively abundant expressed in almost cell types at high levels, therefore antibodies against GAPDH are useful as loading controls for Western Blotting. Some pathology factors, such as hypoxia and diabetes, increased or decreased GAPDH expression in certain cell types four isoforms, and further identified KIF4A as a direct downstream target of FOXM1c. Inhibiting FOXM1 decreased KIF4A expression in HCC cells, whereas its overexpression had the opposite effect. FOXM1-induced HCC cell proliferation was dependent on elevated KIF4A expression as KIF4A knockdown abolished FOXM1-induced proliferation of HCC cells both in vitro and in vivo. Conclusion The FOXM1CKIF4A axis mediates human HCC progression and is a potential therapeutic target for HCC treatment. Electronic supplementary material The online version of this article (10.1186/s13046-019-1202-3) contains supplementary material, which is available to authorized users. for 10?min, and western blotting was performed. Total RNA was extracted using TRIzol reagent, and 1?g was used to prepare cDNA by reverse transcription using PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio; RR047A). Quantitative real-time PCR was carried out on an ABI StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq Tli RNaseH Plus (Takara Bio; RR820A) and the primers were?shown in Additional file 1: Table S4. Data are presented as mean??SD of at least three independent experiments. ADU-S100 ammonium salt ChIP and luciferase assays HepG2 cells produced to 90% confluence were cross-linked with 1% (v/v) formaldehyde. Chromatin was sonicated into fragments of 100 to 400?bp over six cycles of 10?s on /10?s off using a Bioruptor Sonicator (Diagenode, Denville, NJ, USA). The lysates were pre-cleared in bovine serum albumin-blocked protein A/G beads and incubated overnight with specific anti-FOXM1 antibody or control IgG. After washing, the DNA was eluted, and reverse cross-linked overnight at 65?C. Eluted DNA was used as a template for semi-quantitative PCR. The input control was the supernatant before precipitation. The predicted binding sequences and primers used to amplify KIF4A promoter sequences are listed in Additional file 1: Table S5. For the luciferase reporter assay, pGL4.2-basic-Luc reporter plasmids and the internal control plasmid pRL-TK were transfected into HepG2 cells grown to 70% confluence in 24-well plates. The FOXM1 expression plasmid or vacant vector were co-transfected for 48?h, and reporter gene activity was assayed using the Dual Luciferase Assay System (Promega; E1910) according to the manufacturers instructions. The activity of the pGL4.2-basic-KIF4A promoter-luciferase reporter normalized to that.