Fig 1.
Schematic representation of the members of the LUBAC complex, RBCK1, RNF31 and SHARPIN, including domain structures and active sites.
RNF31, the catalytic subunit of the LUBAC complex, interacts with SHARPIN and RBCK1. SHARPIN and RBCK1 bind to the NZF2 and UBA domains of RNF31 via their UBL domains. The RBR domain of RNF31, but not that of RBCK1, plays a key role for the linear ubiquitin chain generating property of LUBAC. Arrows show known interactions between the proteins. Abbreviations: ZnF, zinc finger; NZF, Npl4 zinc finger; UBL, ubiquitin-like domain; IBR, in-between RING domain; RBR, RING-IBR-RING domain.
Fig 2.
Model showing some pathways that have been suggested to be related to breast cancer.
Pathway 1; The LUBAC complex, consisting of RBCK1, RNF31 and SHARPIN, has been shown to activate NFkB signaling. Pathway 2; RBCK1 has been shown to regulate ERalpha transcription. Pathway 3; RNF31 has been shown to regulate ERalpha stability presumably via monoubiquitination.
Table 1.
Clinicopathological data of patients.
Fig 3.
Expression of genes constituting the linear ubiquitin assembly complex comparing tumor and adjacent tissues.
The expression levels of RBCK1 (A) and SHARPIN (B) were significantly higher in tumors compared with adjacent tissues (p < 0.001). A non-paired model was applied. Gene expression (y-axis) was quantified by real-time PCR and normalized to UBC.
Fig 4.
ROC Curve analyses comparing tumor and adjacent tissues.
Up-regulated mRNA expression of RNF31, RBCK1 and SHARPIN exhibited high predictive distinction abilities to distinguish tumor tissues from adjacent non-tumor tissues with Area Under the Curve (AUC) equal to 0.95, 0.94 and 0.91, respectively.
Fig 5.
Genes involved in LUBAC complex show strong correlations to each other in adjacent non-tumor tissues.
Correlations among the expression of genes involved in the LUBAC complex revealed good pair wise correlations among non-tumor tissues (r > 0.9) (A, C and E), while the correlation diminished or disappeared among tumors (B, D and F). Gene expression was quantified by real-time PCR and normalized to UBC.
Table 2.
Correlation of ERalpha, RBCK1, RNF31 and SHARPIN mRNA expression levels in breast tumors.
Pearson correlation was performed pairwise for all assayed mRNAs in breast tumors and the subgroup of ERalpha-positive tumors.
Table 3.
ORs and 95% CIs for the mRNA expression of investigated genes in tumors with clinicopathological parameters.
ORs and CIs for RBCK1, RNF31 and SHARPIN from the logistic regression model, stratified on some parameters including ER, PR and HER2 statuses.
Table 4.
ORs and 95% CIs for the mRNA expression of investigated genes in tumors with clinicopathological parameters.
ORs and CIs for RBCK1, RNF31and SHARPIN from the logistic regression model, stratified on some parameters including menopausal and lymph node statuses, stage and grade.
Fig 6.
Western blot analysis of RNF31, RBCK1 and SHARPIN for breast tumors.
A) Equal amounts of protein (40 μg) from 17 tumor tissues were loaded. Beta-actin was used as loading control. RNF31, RBCK1 and SHARPIN displayed bands with apparent molecular weights of approximately 120, 55 and 40 kDa, respectively. B) ImageJ software was used to quantify the Western blot analysis results of protein expression of interest. The protein expression levels were normalized to beta-actin as a loading control. Curve estimation regression was fitted with the protein expression levels of interest and their corresponding mRNA expression levels. Among three members of LUBAC complex only SHARPIN protein expression levels were correlated moderately with its corresponding mRNA levels (r = 0.59).
Table 5.
The correlation of RBCK1, RNF31 and SHARPIN protein expression levels with clinicopathological parameters.
ImageJ software was used to quantify the western blot analysis results of protein expression of interest. The protein expression levels were normalized to beta-actin as a loading control. Median was used to classify the protein expression levels into two subgroups, as low and high. Chi-square test used to show significant differences between protein expression levels and some clinicopathological parameters including ER, PR, lymph node, stage and grade statuses.