Relationship between TRAF6 and deterioration of HCC: an immunohistochemical and in vitro study

Abstract

ObjectiveTo explore the relationship between tumor necrosis factor receptor-associated factor 6 (TRAF6) and the clinicopathological features in HCC as well as its biological function.MethodsTotally, 412 liver tissues were collected, including 171 hepatocellular carcinoma (HCC) and their corresponding non-tumor tissues, 37 cirrhosis and 33 normal liver tissues. The expression of TRAF6 was assessed by immunohistochemistry. Then, analysis of the correlations between TRAF6 expression and clinicopathological parameters in HCC was conducted. Furtherer, in vitro experiments on HepG2 and Hep3B cells were performed to validate the biological function of TRAF6 on HCC cells. TRAF6 siRNA was transfected into HepG2 and Hep3B cell lines and TRAF6 expression was evaluated with RT-qPCR and western blot. The assays of cell viability, proliferation, apoptosis and caspase-3/7 activity were carried out to investigate the effects of TRAF6 on HCC cells with RNA interference. Cell viability was assessed with Cell Titer-Blue kit. Cell proliferation was tested with MTS kit. Cell apoptosis was checked through morphologic detection with fluorescence microscope, as well as caspase-3/7 activity was measured with fluorogenic substrate detection.ResultsThe positive expression rate of TRAF6 protein was 49.7 % in HCC, significantly higher than that of normal liver (12.1 %), cirrhosis (21.6 %) and adjacent non-cancerous tissues (36.3 %, all P < 0.05). Upregulated TRAF6 was detected in groups with metastasis (Z = −2.058, P = 0.04) and with low micro-vessel density (MVD) expression (Z = −2.813, P = 0.005). Spearman correlation analysis further showed that the expression of TRAF6 was positively correlated with distant metastasis (r = 0.158, P = 0.039) and negatively associated with MVD (r = −0.249, P = 0.004). Besides, knock-down of TRAF6 mRNA in HCC cell lines HepG2 and Hep3B both resulted in cell viability and proliferation inhibition, also cell apoptosis induction and caspase-3/7 activity activation.ConclusionsTRAF6 may contribute to metastasis and deterioration of the HCC via influencing cell growth and apoptosis. Thus, TRAF6 might become a predictive and therapeutic biomarker for HCC.

Genetic Alterations of TRAF Proteins in Human Cancers

“…Corroborating initial evidence, intestinal epithelial cell-specific TRAF6 −/− mice exhibit exacerbated DSS-induced colitis ( 87 ). In line with the in vivo data, knockdown of TRAF6 or inhibition of TRAF6 E3 ligase activity suppresses the survival, proliferation, migration, and metastasis of many human epithelial cancers, including breast, lung, liver, and colon cancers as well as HNSCC ( 230 – 232 , 236 – 240 ). In the majority of these cases, the TRAF6-NF-κB axis is identified as the main oncogenic pathway, which is constitutively activated by TRAF6 overexpression or hyperactivated by upstream receptors such as TNFα, RANK, and TLR4/3 ( 236 – 240 ).…”

Tumor Necrosis Factor Receptor–Associated Factor 6 Promotes Hepatocarcinogenesis by Interacting With Histone Deacetylase 3 to Enhance c‐Myc Gene Expression and Protein Stability

“…TRAF6 is well known as an E3 ubiquitin ligase important for inflammatory signaling . Recent studies have indicated that TRAF6 is involved in HCC development . However, the regulatory mechanisms of TRAF6 in HCC remain unknown.…”

“…Recent studies have indicated that TRAF6 plays critical roles in cancer . Elevated TRAF6 expression is observed in HCC . However, the mechanisms of the oncogenic function of TRAF6 in HCC remain uncharacterized.…”

Diagnostic and prognostic roles of IRAK1 in hepatocellular carcinoma tissues: an analysis of immunohistochemistry and RNA-sequencing data from the cancer genome atlas

“…The IRAK1 antibody (F-4, sc-5228, 1:50 dilution) provided by Santa Cruz Biotechnology (Heidelberg, Germany) was used for IHC detection. The detailed protocol of IHC was shown in our previous studies 20,21. The IRAK1 staining in HCC cells was analyzed through blind evaluation, which was performed by two pathologists who independently identified the intensity of staining and the positive staining ratio of IRAK1.…”