Effects of RNF2 and SIK1 depletion on HCC Cell viability determined by MTT assays on HCC cells transfected with indicated plasmids or control(left panel). and increases tumor growth and metastasis in the mouse xenograft model [10]. To further investigate the mechanism by which SIK1 was downregulated in HCC, we sought to identify previously unknown cellular interaction partners of SIK1 by affinity purification and mass-spectrometry (MS) analysis of Flag-tagged-SIK1. The MS Rabbit Polyclonal to Cytochrome P450 39A1 analysis indicated that RNF2 was specifically present in complex with Flag-SIK1. Since very little was known about the effect of RNF2 on HCC, we firstly investigated the clinical role of RNF2 in HCC using clinical data from TCGA Data Portal. It was found that the percentages of HCC samples with RNF2 mRNA amplification were 19.7%, implying that this amplification of RNF2 mRNA is associated with poor prognosis in HCC. Kaplan-Meier analysis further confirmed that RNF2 amplification is usually closely correlated with higher risk in HCC patients (Physique ?(Figure1A1A). Open in a separate windows Physique 1 Expression patterns of RNF2 and SIK1 in human HCCA. Relationship between RNF2 amplification and HCC prognosis. Kaplan-Meier curves based on data from TCGA show that RNF2 amplification in HCC was associated with shorter overall survival ( 0.05). B. 3-deazaneplanocin A HCl (DZNep HCl) RNF2 and SIK1 expression in HCC tissues and paired normal liver tissues. Western blots of RNF2 and SIK1 in six representative paired samples of non-tumor tissue (N) and HCC tissue (T) are shown. -Actin was used as a control for protein load. C. Expression patterns of RNF2 and SIK1 immunoreactivity in HCC samples and in non-tumor tissues from a representative case. D. RNF2 expression levels in noncancerous liver tissues were lower than those in HCC tissues (left) ( 0.05). Conversely, SIK1 expression levels in noncancerous liver tissues were higher than those in HCC tissues (right) ( 0.05). E. Low SIK1 levels were correlated with high RNF2 expression in HCC ( 0.05). The median expression levels of SIK1 and RNF2 were used as the cutoff values. F. Kaplan-Meier curves show that HCC patients with high RNF2 and low SIK1 immunoreactivity had a poorer prognosis than patients with low RNF2 and high SIK1 immunoreactivity did. The data are expressed as the mean SD. The results are representative of three impartial experiments. * 0.05, ** 0.01. To further investigate the expression patterns of RNF2 and SIK1 in HCC, we randomly examined six pairs of human HCC samples and matched non-tumor tissues. It was observed 3-deazaneplanocin A HCl (DZNep HCl) that RNF2 levels in all tested HCC samples, were consistently higher compared with adjacent normal liver tissues. Concurrently, SIK1 levels in tumors were significantly lower compared with levels in adjacent normal liver tissues (Physique ?(Figure1B).1B). These data indicated that RNF2 overexpression correlates with SIK1 3-deazaneplanocin A HCl (DZNep HCl) down-regulation in HCC. To further quantitate the expression of RNF2 and SIK1, we used Immunohistochemical staining to analyze another cohort of 67 HCC patients with clinical records by correlating expression levels with overall survival (Supplementary Table 1). Our data further confirmed the inverse correlations between RNF2 and SIK1 in HCC tissues (Physique ?(Physique1C).1C). The staining score of RNF2 was higher in HCC tissues (4.46 0.13) than in non-tumor tissues (3.02 0.23) (Physique ?(Physique1D,1D, P 0.009). Conversely, SIK1 levels were higher in non-tumor tissues (5.15 0.41) than in HCC tissues (3.12 0.29, 0.006) (Figure ?(Figure1D).1D). Obviously, RNF2 expression in HCC tissues was inversely correlated with SIK1 levels (Physique ?(Physique1E;1E; = ? 0.304, = 0.013). We further investigated the effect of RNF2 and SIK1 expression pattern on the outcome of HCC patients. The Kaplan-Meier Survival analysis of the 67 patients suggests that expression of high RNF2 level with concurrent low SIK1 tended to correlate with a poor prognosis (Physique ?(Figure1F1F). Effect of RNF2 on HCC cell proliferation The enhancement.