Abstract
BackgroundPoly C Binding Protein 1 (PCBP1) is an RNA-binding protein that binds and regulates translational activity of subsets of cellular mRNAs. Depletion of PCBP1 is implicated in various carcinomas, but the underlying mechanism in tumorigenesis remains elusive.MethodsWe performed a transcriptome-wide screen to identify novel bounding mRNA of PCBP1. The bind regions between PCBP1 with target mRNA were investigated by using point mutation and luciferase assay. Cell proliferation, cell cycle, tumorigenesis and cell apoptosis were also evaluated in ovary and colon cancer cell lines. The mechanism that PCBP1 affects p27 was analyzed by mRNA stability and ribosome profiling assays. We analyzed PCBP1 and p27 expression in ovary, colon and renal tumor samples and adjacent non-tumor tissues using RT-PCR, Western Blotting and immunohistochemistry. The prognostic significance of PCBP1 and p27 also analyzed using online databases.ResultsWe identified cell cycle inhibitor p27Kip1 (p27) as a novel PCBP1-bound transcript. We then demonstrated that binding of PCBP1 to p27 3’UTR via its KH1 domain mainly stabilizes p27 mRNA, while enhances its translation to fuel p27 expression, prior to p27 protein degradation. The upregulated p27 consequently inhibits cell proliferation, cell cycle progression and tumorigenesis, whereas promotes cell apoptosis under paclitaxel treatment. Conversely, knockdown of PCBP1 in turn compromises p27 mRNA stability, leading to lower p27 level and tumorigenesis in vivo. Moreover, forced depletion of p27 counteracts the tumor suppressive ability of PCBP1 in the same PCBP1 over-expressing cells. Physiologically, we showed that decreases of both p27 mRNA and its protein expressions are well correlated to PCBP1 depletion in ovary, colon and renal tumor samples, independent of the p27 ubiquitin ligase Skp2 level. Correlation of PCBP1 with p27 is also found in the tamoxifen, doxorubincin and lapatinib resistant breast cancer cells of GEO database.ConclusionOur results thereby indicate that loss of PCBP1 expression firstly attenuates p27 expression at post-transcriptional level, and subsequently promotes carcinogenesis. PCBP1 could be used as a diagnostic marker to cancer patients.
Highlights
C Binding Protein 1 (PCBP1) is an RNA-binding protein that binds and regulates translational activity of subsets of cellular mRNAs
We focused on whether Poly C Binding Protein 1 (PCBP1) modulates p27 expression with three reasons: first, cell cycle plays a crucial role in tumorigenesis and cancer progression; second, p27 is one of the most important guardians in cell cycle; third, PCBP1 has been confirmed to interact with p21 mRNA, it is reasonable to inquire the existence of an coordinated interaction between PCBP1 and p27 mRNA to modulate cell cycle
To further confirm whether endogenous PCBP1 binds to p27 mRNA, we carried out the RNA immunoprecipitation (RIP) experiment again and showed that p27 mRNA was really precipitated by PCBP1-specific antibody with a well-known c-myc mRNA bound to PCBP1, but not by normal IgG control (Fig. 1c)
Summary
C Binding Protein 1 (PCBP1) is an RNA-binding protein that binds and regulates translational activity of subsets of cellular mRNAs. Depletion of PCBP1 is implicated in various carcinomas, but the underlying mechanism in tumorigenesis remains elusive. PCBP1 is ubiquitously expressed and functionally plays multiple roles in transcriptional and posttranscriptional regulations, including mRNA stabilization [5, 6]. PCBP1 regulates gene expression via binding to specific elements of target mRNAs with AU-rich elements (AREs) or U-rich elements located in 3’-untranslated regions (3’-UTR) (e.g. AR, p21, p63, eNOS) [7,8,9,10] or in 5’-UTRs (e.g. c-myc, PRL-3, EV71) [6, 11, 12]. PCBP1 can bind with RACK1 to regulate MOR expression [24]
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