PARP6 suppresses the proliferation and metastasis of hepatocellular carcinoma by degrading XRCC6 to regulate the Wnt/β-catenin pathway

Abstract

PARP6 belongs to the mono-ADP-ribosyltransferase family and has been shown to be involved in the genesis and development of some tumours. However, the role of PARP6 in hepatocellular carcinoma (HCC) development remains to be fully elucidated. In the current study, we demonstrated that PARP6 was expressed at a low level in HCC cells and was negatively related to the degree of tumour differentiation. Additionally, silencing PARP6 led to an increase in the proliferation, invasion and migration ability of HCC cells in both in vitro and in vivo assays. Conversely, an elevation in the PARP6 expression level had the opposite effect. Through gene chip analysis combined with experimental verification, we confirmed that PARP6 can inhibit the expression of XRCC6 by inducing degradation and thus affect the Wnt/β-Catenin signalling pathway, which contributes to the suppression of HCC. Further mechanistic investigation demonstrated that the ubiquitin ligase HDM2 can interact with PARP6 and XRCC6, and mediated the regulatory effect of PARP6 on XRCC6 degradation. Taking together, PARP6 appears to inhibit HCC progression through the XRCC6/Wnt/β-catenin signal axis and could be used as a biomarker for the clinical monitoring of HCC development.

Keywords: HCC; PARP6; XRCC6; cell proliferation; metastasis.

Figure 1

Expression of PARP6 was downregulated in HCC tissues. A. The expression of PARP6 in HCC and adjacent tissues of tumours was analysed by IHC (×400). B. The relative positive cells of PARP6 in 50 pairs of HCC and adjacent tissues of tumours. C. The protein expression of PARP6 was analysed by Western blot analysis in 9 pairs of HCC (T) and adjacent tissues (N). D. The mRNA expression of PARP6 was analysed by qRT-PCR in 98 pairs of HCC (T) and adjacent tissues (N). E. PARP6 expression was assessed using data from the TCGA databases. (**, P<0.01).

Figure 2

Detection of PARP6 expression levels in HCC cells with PARP6 overexpression or depletion. A, B. The protein and mRNA expression of PARP6 were analysed by Western blot and qRT-PCR in different HCC and normal cell lines. C. Efficiency of four PARP6 siRNAs was detected by western blotting in HCC cells. D. The expression of PARP6 was detected by western blot after PARP6 plasmid transfection.

Figure 3

PARP6 inhibits the proliferation of HCC in vivo and in vitro. A, B. CCK-8 kit detection of cell proliferation after depletion or overexpression of PARP6. C. Plate cloning experiments were used to detect colony formation after depletion or overexpression of PARP6. D. Xenograft nude mouse model of tumorigenesis was used to confirm cell growth after depletion or overexpression of PARP6. E. The volume and weight of tumours were assessed in nude mice injected with PARP6-overexpressing LM3 and vector-transfected LM3. F. The volume and weight of tumours were assessed in nude mice injected with PARP6-silenced Sk-Hep-1 and control Sk-Hep-1. The data are expressed as the mean ± SD (*, P<0.05; **, P<0.01).

Figure 4

PARP6 inhibits the metastasis of HCC cells in vitro and in vivo. A, B. Migration and invasion were assessed by Transwell in LM3 cells overexpressing PARP6. C, D. Migration and invasion were assessed by Transwell in Sk-Hep-1 cells with PARP6 knockdown. E. Western blotting was used to analyse the expression of EMT markers in HCC cells with PARP6 overexpression or knockdown. F. Live imaging to observe lung metastasis. G, H. Representative images of the histological assessment of the lungs via H&E staining (×200). The metastatic lesion is indicated by an arrow. The number of metastatic lesions in the lung was significantly decreased in the mice injected with PARP6-overexpressing LM3 cells (*, P<0.05; **, P<0.01).

Figure 5

PARP6 regulates the XRCC6/Wnt/β-Catenin signalling axis. A. Gene chip analysis were applied to examine the differential expression genes between PARP6-overexpressing LM-3 cells and vector-transfected LM-3 cells (FC>3). B. Gene microarray analysis revealed that these genes were involved in a variety of signalling pathways. C, D. Western blotting was used to analyse the protein levels of XRCC6, β-catenin, c-Myc, MMP-7 and cyclin D1 in PARP6-overexpressing LM-3 cells and PARP6-silenced SK-Hep-1 cells. E. Western blot was used to analyse the protein levels of XRCC6, β-Catenin, c-Myc, MMP-7 and Cyclin D1 in LM-3 cells transfected with PARP6 and XRCC6 plasmids (*, P<0.05; **, P<0.01).

Figure 6

PARP6 regulates HCC progression through XRCC6. A, B. The viability of PARP6-overexpressing LM3 cells was examined by CCK8 and colony formation assay after treatment with XRCC6 overexpression plasmid or XAV-939 with or without XRCC6 overexpression. C, D. The invasion and migration abilities were measured by Transwell assay in PARP6-overexpressing LM3 cells after treatment with XRCC6 overexpression plasmid or XAV-939 (*, P<0.05; **, P<0.01).

Figure 7

PARP6 interacts with HDM2 to degrade XRCC6. A. LM3 cells were co-transfected with PARP6 and XRCC6 overexpression plasmids and treated with 50 lg/ml CHX before collection at designated time points. Relative quantification of XRCC6 protein levels was assessed at different time points. B. Reciprocal Co-immunoprecipitation of PARP6 and XRCC7 in LM3 cells. C. Before collection, the protein level was examined in PARP6-overexpressing LM3 cells with or without treatment with 10 μΜ MG132 for 4 h. D. Whole lysates were immunoprecipitates with anti-HDM2 beads, then products were blotted with anti-PARP6, and anti-XRCC6 antibodies. E. Western blotting was used to analyse the protein levels of XRCC6, PARP6 and HDM2 in LM3 cells treated with or without PARP6 overexpression plasmid, and in PARP6-overexpressed LM3 cells transfected with HDM2 siRNA, or Nutlin-3 (*, P<0.05; **, P<0.01).