The online version of this article (https://doi.org/10.1186/s13046-018-0890-4) contains supplementary material, which is available to authorized users.
Wuhua Zhou, Li Gong and Qinchuan Wu contributed equally to this work.
A correction to this article is available online at https://doi.org/10.1186/s13046-018-0944-7.
Plant homeodomain finger protein 8 (PHF8) serves an activator of epithelial-mesenchymal transition (EMT) and is implicated in various tumors. However, little is known about PHF8 roles in hepatocellular carcinoma (HCC) and regulating E-cadherin expression.
PHF8 expression pattern was investigated by informatic analysis and verified by RT-qPCR and immunochemistry in HCC tissues and cell lines. CCK8, xenograft tumor model, transwell assay, and tandem mCherry-GFP-LC3 fusion protein assay were utilized to assess the effects of PHF8 on proliferation, metastasis and autophagy of HCC cells in vitro and in vivo. ChIP, immunoblot analysis, rescue experiments and inhibitor treatment were used to clarify the mechanism by which PHF8 facilitated EMT, metastasis and autophagy.
PHF8 upregulation was quite prevalent in HCC tissues and closely correlated with worse overall survival and disease-relapse free survival. Furthermore, PHF8-knockdown dramatically suppressed cell growth, migration, invasion and autophagy, and the expression of SNAI1, VIM, N-cadherin and FIP200, and increased E-cadherin level, while PHF8-overexpression led to the opposite results. Additionally, FIP200 augmentation reversed the inhibited effects of PHF8-siliencing on tumor migration, invasion and autophagy. Mechanistically, PHF8 was involved in transcriptionally regulating the expression of SNAI1, VIM and FIP200, rather than N-cadherin and E-cadherin. Noticeably, E-cadherin degradation could be accelerated by PHF8-mediated FIP200-dependent autophagy, a crucial pathway complementary to transcriptional repression of E-cadherin by SNAI1 activation.
These findings suggested that PHF8 played an oncogenic role in facilitating FIP200-dependent autophagic degradation of E-cadherin, EMT and metastasis in HCC. PHF8 might be a promising target for prevention, treatment and prognostic prediction of HCC.
Additional file 1: Table S1. Oligonucleotide sequences of primers for quantitative real time PCR. (DOCX 13 kb)13046_2018_890_MOESM1_ESM.docx
Additional file 2: Table S2. Details of primary antibodies. (DOCX 15 kb)13046_2018_890_MOESM2_ESM.docx
Additional file 3: Figure S1. Representative PHF8 IHC images with different stainingintensity. Magnification, × 40, × 200. (TIF 3414 kb)13046_2018_890_MOESM3_ESM.tif
Additional file 4: Table S3. Oligonucleotide sequences of primers for ChIP. (DOCX 16 kb)13046_2018_890_MOESM4_ESM.docx
Additional file 5: Table S4. Association of PHF8 expression with clinicopathologic features. (DOCX 17 kb)13046_2018_890_MOESM5_ESM.docx
Additional file 6: Table S5. Univariate- and Multivariate- analysis of risk factors for relapse-free survival (RFS) and overall survival (OS). (DOCX 18 kb)13046_2018_890_MOESM6_ESM.docx
Additional file 7: Figure S2. Exogenous overexpression of PHF8 enhances proliferation, migration, invasion and autophagy of HepG2 and SK-Hep-1 cells in vitro. a qRT-PCR and western-blot analysis of transfection efficiency of Flag-PHF8 plasmid in HepG2 and SK-Hep-1 cells. Empty plasmid (Vector) was used for negative control. b Enhanced proliferation of HepG2 and SK-Hep-1 cells in PHF8 overexpression group by CCK8 assasy (n = 6). c, d Representative images and quantification of migrated and invasive cells by transwell assay in HepG2 and SK-Hep-1 cells (n = 3, magnification, × 100). e Representative immunoblot results of LC3B and p62 in HepG2 and SK-Hep-1 cells transfected with indicated plasmids, and then cultured in complete medium with 10% FBS or EBSS starvation condition with or without CQ (100 μmol) for 8-h (n = 3). f Representative fluorescence images of autophagosomes and autolysosomes in HepG2 and SK-Hep-1 cells with PHF8 overexpression by tandem mCherry-GFP-LC3 fusion protein assay (magnification, × 400). g Quantification of autophagosomes and autolysosomes from random 5 high-power fields of the merged images of each group. * p < 0.05, ** P < 0.01, *** P < 0.001. Data were presented by mean ± SD. (TIF 6912 kb)13046_2018_890_MOESM7_ESM.tif
Additional file 8: Figure S3. The blockage of PHF8 inhibits tumorigenesis and metastasis in vivo. a – d Appearance of primary tumor, tumor growth curves and tumor weight in two groups (n = 6). d Overview of lung metastatic lesions (upper panel, white arrow indicated the metastatic colonization) and HE images (lower panel, magnification, × 100). e The number of lung metastatic nets of each group was counted in a low power field (n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001. Data were presented by mean ± SD. (TIF 5523 kb)13046_2018_890_MOESM8_ESM.tif
Additional file 9: Table S6. Correlation of PHF8 expression with expression of ATG17/ FIP200 and E-cadherin based on immunohistochemistry analysis. (DOCX 14 kb)13046_2018_890_MOESM9_ESM.docx
Additional file 10: Figure S4. Representative images of HE and IHC staining of HCC tissues with or without vascular invasion. IHC staining for PHF8, FIP200 and E-cadherin. Magnification, × 40 and × 200. (TIF 6378 kb)13046_2018_890_MOESM10_ESM.tif
Additional file 11: Figure S5. CQ blocks the migration and invasion of HCC cells. a, b SMMC-7721 and Huh7 cells pre-treated by CQ (100 μmol) for 12-h were subjected to transwell migration or invasion assay. Magnification, × 100. *** P < 0.001. Data were presented by mean ± SD. (TIF 2688 kb)13046_2018_890_MOESM11_ESM.tif
Additional file 12: Figure S6. Relationship between PHF8 expression and overall survival of human cancers from Protein Atlas Database (https://www.proteinatlas.org/). Patients were divided into two groups by the line of best separation of mRNA expression. (TIF 3076 kb)
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