Unknown,Transcriptomics,Genomics,Proteomics

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SiRNA Administration Against AFP Inhibits Hepatocellular Carcinoma Proliferation in Vitro and Progression in Mice

ABSTRACT: Increased ?-fetoprotein (AFP) levels have been reported to predict a poor prognosis in hepatocellular carcinoma (HCC). We assessed the mechanism of AFP involvement in the progression of HCC and determined whether AFP could be a molecular target. We used human HCC cell lines to assess proliferation and apoptosis response to exogenous AFP. We introduced AFP small interfering RNA (siRNA) into HCC cell lines to examine whether it could inhibit cell proliferation and anti-apoptotic properties. The effects of systemically introduced AFP siRNA were assessed using a tumor xenotransplantation model. The effects of AFP on gene expression in HCC cell lines and human HCC specimens were examined. Exogenous AFP induced cell proliferation dose-dependently and inhibited apoptosis induced by 5-fluorouracil (5-FU) in all cell lines examined. AFP siRNA inhibited the proliferation of AFP-producing HCC cell lines and induced apoptosis in co-cultures with 5-FU. Tumor sizes in mice treated with AFP siRNA were significantly smaller than those in controls. AFP siRNA administration in mice induced a low proliferation index and a high apoptosis index in tumors. cDNA microarray analysis, reverse transcription-polymerase chain reaction (RT-PCR), and Western blot using HepG2 and HLE cells with AFP showed that AFP reduced expression of genes related to apoptosis (DFFB) and tumor suppression (NDRG2). Expression of these molecules was also suppressed in human HCC tissues that overexpress AFP. AFP is not only a passive tumor marker, but also an active tumor stimulator through several mechanisms. AFP siRNA introduction may be of possible therapeutic use for HCC. Keywords: Genetic modification Two-condition experiment, Control vs. AFP-stimulated cells.

ORGANISM(S): Homo sapiens

SUBMITTER: Noboru Mitsuhashi

PROVIDER: E-GEOD-12307 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Tumor necrosis factor α-induced protein 1 (TNFAIP1) is frequently downregulated in cancer cell lines and promotes cancer cell apoptosis. However, its role, clinical significance and molecular mechanisms in hepatocellular carcinoma (HCC) are unknown. Methods: The expression of TNFAIP1 in HCC tumor tissues and cell lines was measured by Western blot and immunohistochemistry. The effects of TNFAIP1 on HCC proliferation, apoptosis, metastasis, angiogenesis and tumor formation were evaluated by Cell Counting Kit-8 (CCK8), Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL), transwell, tube formation assay in vitro and nude mice experiments in vivo. The interaction between TNFAIP1 and CSNK2B was validated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), Co-immunoprecipitation and Western blot. The mechanism of how TNFAIP1 regulated nuclear factor-kappaB (NF-κB) pathway was analyzed by dual-luciferase reporter, immunofluorescence, quantitative Real-time polymerase chain reaction (RT-qPCR) and Western blot. Findings: The TNFAIP1 expression is significantly decreased in HCC tissues and cell lines, and negatively correlated with the increased HCC histological grade. Overexpression of TNFAIP1 inhibits HCC cell proliferation, metastasis, angiogenesis and promotes cancer cell apoptosis both in vitro and in vivo, whereas the knockdown of TNFAIP1 in HCC cell displays opposite effects. Mechanistically, TNFAIP1 interacts with CSNK2B and promotes its ubiquitin-mediated degradation with Cul3, causing attenuation of CSNK2B-dependent NF-κB trans-activation in HCC cell. Moreover, the enforced expression of CSNK2B counteracts the inhibitory effects of TNFAIP1 on HCC cell proliferation, migration, and angiogenesis in vitro and in vivo. Interpretation: Our results support that TNFAIP1 can act as a tumor suppressor of HCC by modulating TNFAIP1/CSNK2B/NF-κB pathway, implying that TNFAIP1 may represent a potential marker and a promising therapeutic target for HCC. Fund: This work was supported in part by the financial support from the China Natural Science Foundation (No. 81972642, No. 81601122 and No. 81770389), Hunan Natural Science Foundation (No. 2017JJ3205), Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (No. 20134486), and the Scientific Research Fund of Hunan Provincial Education Department (17B162).

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SiRNA Administration Against AFP Inhibits Hepatocellular Carcinoma Proliferation in Vitro and Progression in Mice

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