Human polynucleotide phosphorylase selectively and preferentially degrades microRNA-221 in human melanoma cells

Abstract

MicroRNAs (miRNA), small noncoding RNAs, affect a broad range of biological processes, including tumorigenesis, by targeting gene products that directly regulate cell growth. Human polynucleotide phosphorylase (hPNPase(old-35)), a type I IFN-inducible 3'-5' exoribonuclease, degrades specific mRNAs and small noncoding RNAs. The present study examined the effect of this enzyme on miRNA expression in human melanoma cells. miRNA microarray analysis of human melanoma cells infected with empty adenovirus or with an adenovirus expressing hPNPase(old-35) identified miRNAs differentially and specifically regulated by hPNPase(old-35). One of these, miR-221, a regulator of the cyclin-dependent kinase inhibitor p27(kip1), displayed robust down-regulation with ensuing up-regulation of p27(kip1) by expression of hPNPase(old-35), which also occurred in multiple human melanoma cells upon IFN-beta treatment. Using both in vivo immunoprecipitation followed by Northern blotting and RNA degradation assays, we confirm that mature miR-221 is the target of hPNPase(old-35). Inhibition of hPNPase(old-35) by shRNA or stable overexpression of miR-221 protected melanoma cells from IFN-beta-mediated growth inhibition, accentuating the importance of hPNPase(old-35) induction and miR-221 down-regulation in mediating IFN-beta action. Moreover, we now uncover a mechanism of miRNA regulation involving selective enzymatic degradation. Targeted overexpression of hPNPase(old-35) might provide an effective therapeutic strategy for miR-221-overexpressing and IFN-resistant tumors, such as melanoma.

Fig. 1.

hPNPase^old-35 regulates the expression of specific miRNAs. (A) HO-1 cells were either infected with Ad.vec or Ad.hPNPase^old-35 at a multiplicity of infection of 5,000 viral particles per cell for 3 d, and expressions of indicated miRNAs were analyzed by qPCR using primary and mature miRNA-specific Taqman probes. (B) Northern blotting was performed to detect mature miRNAs and its precursor species (pre-) by using specific probes. (C) At 12 h (b) and 24 h (c) after infection with Ad.hPNPase^old-35, cell lysates were prepared as described in Materials and Methods, and hPNPase^old-35 was immunoprecipitated by anti-hPNPase^old-35 antibody. Immunoprecipitation with nonspecific IgG (a) was used as a negative control. Associated miRNAs were extracted, and expression analysis was performed by Northern blotting. (D) HO-1 cells were treated with IFN-β (1,000 U/mL) and subjected to real-time PCR for different miRNAs. Data are presented as mean normalized expression ± SD for triplicate determinations from three independent experiments.

Fig. 2.

hPNPase^old-35 degrades miRNAs in vitro. (A) The authenticity of the in vitro-translated protein, generated as described in Materials and Methods, was analyzed by Western blotting with anti-hPNPase^old-35 antibody. (B) In vitro RNA degradation assays were performed as described in Materials and Methods. At different time points, the levels of indicated miRNAs were determined by qPCR. (C) Comparative degradation of miR-221, miR-222, and miR-106b at a 2-h time point using an in vitro degradation assay. The data represent mean ± SD of two independent experiments, each done in triplicate. (D) A direct interaction between targeted miRNAs with in vitro-translated hPNPase^old-35 was confirmed by immunoprecipitation and Northern blotting.

Fig. 3.

hPNPase^old-35 down-regulates miR-221 expression in multiple melanoma cell lines. At 3 d after infection with either Ad.vec or Ad.hPNPase^old-35 the specified cell type was analyzed for distinct miRNA expression (A) and p27^kip1 and EF1-α proteins (B) as described in Materials and Methods. (C) Cell lines were treated with IFN-β for 24 h, and expression levels of indicated miRNAs were measured by qPCR. (D) Induction of mRNA for hPNPase^old-35 in Ad.hPNPase^old-35-infected or IFN-β-treated cells was determined by qPCR and presented as fold-induction compared with FM-516 cells. The data represent mean ± SD of triplicate determinations for three independent experiments. Data points marked with asterisks indicate significant differences (P < 0.01) from the corresponding control data points.

Fig. 4.

hPNPase^old-35 shRNA confers resistance to IFN-β–mediated miR-221 down-regulation. HO-1 cells infected with a lentivirus expressing the indicated shRNA were treated with IFN-β (500 or 1,000 U/mL) for 24 h, and the expression of hPNPase^old-35 (A) and mda-5 (B) mRNA (Left) and protein (Right) were analyzed by qPCR and Western blotting, respectively. (C) HO-1 cells were treated as in A, and the expression of miR-221 was analyzed by qPCR (Right). HO-1 cells were treated as in A, except that IFN-β treatment was performed for 48 h and the expression of p27^Kip1 analyzed by Western blotting (Right). The data represent mean ± SD of three independent experiments.

Fig. 5.

HO-1 clones expressing miR-221 are resistant to IFN-β–mediated growth inhibition. (A) The stable miR-221–overexpressing HO-1 clones were analyzed for basal miR-221 level and its target p27^kip1 by qPCR and Western blotting, respectively. (B) HO-1 cells either expressing empty vector (HO-1 Con) or miR-221 (Cl.9 and Cl.11) were treated with IFN-β at 500 or 1,000 U/mL. At the indicated times (d3 and d6) cells were trypsinized and counted as described in Materials and Methods. (C) Percentage of apoptotic cells in different clones was determined by Annexin-V binding assays after IFN-β (1,000 U/mL) treatment for the indicated times. (D) Colony formation assays using the indicated clones were performed as described in Materials and Methods. The data represent the mean ± SD of three independent experiments. *Significant difference (P < 0.01) vs. corresponding control points.

Fig. 6.

HO-1 clones expressing miR-221 are resistant to IFN-β–mediated cell cycle arrest. (A) The designated cell types were treated with the indicated doses of IFN-β for different times, and cell cycle was monitored by flow cytometry. Percentages of cell population in the G₁ phase. (B and C) Indicated cells were treated as in A, and p27^kip1 mRNA (B) and protein (C) expression were determined by qPCR and Western blotting, respectively. The data represent mean ± SD of three independent experiments. (D) Schematic model for regulation of miR-221 by IFN-β. See text for details.