Abstract
BackgroundMisregulation of the p53-mdm2 loop function is a major mechanism to promote hepatocellular carcinoma (HCC). RBM38, a member of the RNA recognition motif (RRM) family of RNA binding proteins (RBPs), plays a fundamental role in the posttranscriptional control of gene expression and regulatory functions in human tumors. A novel RBM38-p53-mdm2 autoregulatory feedback loop has been demonstrated. However, its mechanistic role in HCC remains unclear.MethodsIn the present study, we investigated the role and molecular mechanism of misregulation in the p53-mdm2 loop function by RBM38 in HCC. First we investigated the correlation of RBM38 activity and p53-mdm2 loop function in liver cancer cells and HCC tissues by western blot and quantitative RT-PCR. We then conducted functional assays to investigate the molecular roles of RBM38 in inhibiting liver cancer cells aggressiveness in vitro and suppressing tumorigenicity in vivo.ResultsWe observed RBM38 protein expression was commonly silenced coupled with increased mdm2 and decreased wild type (wt) p53 in liver cancer cells and HCC tissues compared to the corresponding normal liver cells and adjacent liver tissues. RBM38 mRNA level was significantly lower in HCC than adjacent liver tissues, whereas mdm2 and wtp53 mRNA levels were similar between HCC and adjacent liver tissues. This implied that deactivation of RBM38 could disrupt the p53-mdm2 loop and promote HCC, even though p53 and mdm2 transcript amounts were stable. Then, we generated stable liver cancer cell lines with overexpressed RBM38 (RBM38-OE) and found that up-regulation of RBM38 could inhibit mdm2 and restore wtp53 expression. Luciferase assay shown that RBM38 destabilized the mdm2 transcript through binding to multiple AU-/U-rich elements in mdm2 3’-UTR. Furthermore, functional assays showed that ectopic expression of RBM38 could induce liver cancer cell apoptosis and senescence, inhibit proliferation and colony growth, and suppress migration and invasion in vitro. Lastly, RBM38 could suppress HCC tumorigenicity in vivo.ConclusionOur findings suggested that RBM38 may be a core contributor in stabilizing the p53-mdm2 loop function to prevent HCC, and a potential novel target to provide a therapeutic strategy for HCC by inhibiting mdm2 and rescuing p53 from inactivation.
Highlights
Misregulation of the p53-mdm2 loop function is a major mechanism to promote hepatocellular carcinoma (HCC)
Our results strongly suggested that RNA binding motif protein 38 (RBM38) is a potential novel target to provide a therapeutic strategy for HCC by inhibiting mdm2 and rescuing p53 from inactivation
RBM38 protein was commonly expressed lower in human liver cancer cells and human HCC tissue, coupled with wtp53 inhibition and mdm2 amplification First, RBM38, wtp53, and mdm2 protein expression in eight liver cancer cell lines and one normal liver cell line were quantified by western blot (Fig. 1a)
Summary
Misregulation of the p53-mdm loop function is a major mechanism to promote hepatocellular carcinoma (HCC). P53 and mdm interact with each other to form an autoregulatory feedback loop [7, 8], in which the balance between p53 and mdm is critical for regulating cell growth and death in normal cells, and preventing tumors under non-stress conditions or various stimuli [9, 10]. It has been well accepted that misregulation of the p53-mdm loop usually leads to mdm stabilization and p53 degradation, which plays important and unique roles in tumorigenesis and progression of cancers. Dharel [16] demonstrated that the 309 T > G polymorphism (SNP309, rs2279744), which is located in the intronic p53-responsive promoter of the mdm gene, may increase mdm stabilization and accelerate p53 degradation in the early onset of HCC in patients with chronic HCV infection. Disruption of the mdm2-p53 interaction by introducing molecules that inhibit mdm, restore wtp and stabilize the active conformation of the p53 protein [14, 18] may offer an effective therapeutic approach, attracting more attention for HCC over recent years [19,20,21]
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