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. 2016 May 31;7(22):32774-84.
doi: 10.18632/oncotarget.9047.

COMMD7 is correlated with a novel NF-κB positive feedback loop in hepatocellular carcinoma

Lu Zheng  1 , Chang-Lin Deng  1 , Liang Wang  1 , Xiao-Bing Huang  1 , Nan You  1 , Yi-Chen Tang  1 , Ke Wu  1 , Ping Liang  1 , Na Mi  1 , Jing Li  1
Affiliations

COMMD7 is correlated with a novel NF-κB positive feedback loop in hepatocellular carcinoma

Lu Zheng et al. Oncotarget. .

Abstract

The correlation between nuclear factor-kappa B (NF-κB) and COMMD7 in hepatocellular carcinoma (HCC) development remained unclear. Here, our clinicopathological data showed that COMMD7 is overexpressed in HCC with a correlation to NF-κB. Using HepG2 and SMMC-7721 cells that aberrantly overexpressed COMMD7, we found that NF-κB directly binds with COMMD7 promoter and serves as an activator for COMMD7 transcription by luciferase reporter assay, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA). In both HepG2 cells and SMMC-7721 cells, the silencing of COMMD7 significantly inhibited the cell proliferation, whereas NF-κB silencing inhibited the expression of COMMD7 and further inhibited cell proliferation. In addition, cell apoptosis was promoted by COMMD7 silencing, and further promoted by NF-κB silencing. Cell migration and invasion were also inhibited by COMMD7 silencing, and further inhibited by NF-κB silencing. Thus, COMMD7 is correlated with a novel NF-κB positive feedback loop in hepatocellular carcinoma. Developing strategies for the treatment of HCC should consider the correlation between NF-κB and COMMD7, so as to improve the specificity and sensitivity of therapy and to reduce toxicity.

Keywords: COMMD7; apoptosis; hepatocellular carcinoma; nuclear factor-kappa B; proliferation.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. Correction between COMMD7 and NF-κB in hepatocellular carcinoma
(A) COMMD7 and (B) p65 expression in hepatocellular carcinoma and para-carcinoma tissues using immunohistochemistry (IHC). Representative images at different magnifications from independent experiments are shown. Tumor: hepatocellular carcinoma; Normal: para-carcinoma tissue. (C) qRT-PCR and (D) Western Blot assays revealed the up-regulation of COMMD7 in hepatocellular carcinoma (tumor, T), compared with para-carcinoma tissues (normal, N). (E) qRT-PCR also revealed the up-regulation of NF-κB in hepatocellular carcinoma. (F) Correlation analysis revealed a significant correlation between COMMD7 and the expression of p65 subunit of NF-κB. **p < 0.01 vs. normal.
Figure 2
Figure 2. Expression of COMMD7 is also up-regulated in hepatocellular carcinoma cells
(A) Immunofluorescence of COMMD7 and (B) expression of COMMD7 mRNA levels in HL7702 human liver cells, and HepG2, SMMC-7721, Huh7 and Hep3B human hepatocellular carcinoma cells were detected by qRT-PCR, and (C) protein level was detected by Western Blot, respectively. *p < 0.05; **p < 0.01 vs. HL7702.
Figure 3
Figure 3. NF-κB binds to the endogenous COMMD7 promoters
(A) Oligonucleotide sequences corresponding to the potential COMMD7 promoters are shown, with distance from the transcription start site (TSS) indicated. Arrows denote site orientation. (B) 293T cells were transfected with the indicated reporter plasmids and co-transfected with NF-κB-expressing or empty pcDNA3 plasmids. Mean values (n = 3) of luciferase reporter activity ± standard deviation are shown. *p < 0.05; **p < 0.01 mutation (MUT) vs. wide-type (WT). (C) Binding of NF-κB to the endogenous COMMD7 promoters. Chromatin-immunoprecipitated NF-κB-binding non-enriched (rabbit IgG) DNA, enriched (NF-κB) DNA, and 0.01% whole cell extract DNA in HepG2 or SMMC-7721 were amplified by PCR. (D) Electrophoretic mobility shift assay (EMSA). 32P-labeled oligonucleotide probes corresponding to the COMMD7 promoter 1, 4, 5 and 6, complexed with NF-κB in the presence or absence of anti-NF-κB antibody or specific/mutant competitors were detected by EMSA.
Figure 4
Figure 4. Stable transfection of NF-κB shRNA or COMMD7 shRNA inhibited the proliferation of hepatocellular carcinoma cells
(A–F): HepG2; (G–L): SMMC-7721 cells. The expression of p65 subunit of NF-κB and COMMD7 were detected by confocal microscopy and western blot. Expression of p65 subunit (A, C and G, I) of NF-κB and COMMD7 (B, C and H, I) after NF-κB shRNA transfection. Expression of COMMD7 (D, E and J, K) after COMMD7 shRNA transfection. (F and L) Cell proliferation was detected by MTT assay. *p < 0.05, vs. NC-shRNA; #p < 0.05, vs. COMMD7-shRNA.
Figure 5
Figure 5. Stable transfection of NF-κB shRNA or COMMD7 shRNA enhanced apoptosis of hepatocellular carcinoma cells
(A–C): HepG2; (D–F): SMMC-7721 cells. (A and D) Cell apoptosis in each group was determined using the Annexin V-FITC/PI flow cytometry, and (B and E) proportion of apoptosis cells was measured. *p < 0.05, vs. NC-shRNA; #p < 0.05, vs. COMMD7-shRNA. (C and F) The roles of COMMD7 and NF-κB in cell apoptosis were further confirmed by Hoechest staining.
Figure 6
Figure 6. Stable transfection of NF-κB shRNA or COMMD7 shRNA suppressed the invasion of hepatocellular carcinoma cells
(A and B): HepG2; (C and D): SMMC-7721 cells. (A and C) Invasion assay was assessed by transwell chamber and migrated cells were counted (B and D). *p < 0.05, vs. NC-shRNA; #p < 0.05, vs. COMMD7-shRNA.
Figure 7
Figure 7. Stable transfection of NF-κB shRNA or COMMD7 shRNA suppressed tumorigenicity in vivo
Tumor volume (V) was measured by caliper daily and calculated using the formula V = (L × W2)/2, where L was the length and W was the width of the tumor. Growth curves were plotted using average tumor volume within each experimental group every week after respective inoculation of HepG2 cells (A–C) or SMMC-7721 cells (D–F) that were stable transfected with COMMD7 shRNA or NF-κB shRNA, and control cells. Five weeks later, the mice were euthanized, and the dissected tumors were collected (A and D), the tumor growth curves (B and E), and the expression of p65 or COMMD7 in tumors (C and F) were performed. *p < 0.05, vs. NC-shRNA; #p < 0.05, vs. COMMD7-shRNA.
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