Tumor suppressor miR-375 regulates MYC expression via repression of CIP2A coding sequence through multiple miRNA-mRNA interactions

Abstract

MicroRNAs (miRNAs) are small, noncoding RNAs involved in posttranscriptional regulation of protein-coding genes in various biological processes. In our preliminary miRNA microarray analysis, miR-375 was identified as the most underexpressed in human oral tumor versus controls. The purpose of the present study is to examine the function of miR-375 as a candidate tumor suppressor miRNA in oral cancer. Cancerous inhibitor of PP2A (CIP2A), a guardian of oncoprotein MYC, is identified as a candidate miR-375 target based on bioinformatics. Luciferase assay accompanied by target sequence mutagenesis elucidates five functional miR-375-binding sites clustered in the CIP2A coding sequence close to the C-terminal domain. Overexpression of CIP2A is clearly demonstrated in oral cancers, and inverse correlation between miR-375 and CIP2A is observed in the tumors, as well as in NCI-60 cell lines, indicating the potential generalized involvement of the miR-375-CIP2A relationship in many other cancers. Transient transfection of miR-375 in oral cancer cells reduces the expression of CIP2A, resulting in decrease of MYC protein levels and leading to reduced proliferation, colony formation, migration, and invasion. Therefore this study shows that underexpression of tumor suppressor miR-375 could lead to uncontrolled CIP2A expression and extended stability of MYC, which contributes to promoting cancerous phenotypes.

FIGURE 1:

miR-375 is underexpressed in oral cancer. (A) Expression levels of miR-375 in human oral tissues (five normal controls and 17 tumors) and in seven head and neck cancer cell lines measured by quantitative real-time PCR (qRT-PCR). Tumors were subgrouped by early-stage (n = 6) and advanced-stage (n = 11) tumors. (B) The overall level of miR-375 was further analyzed to determine statistical differences. All qRT-PCR results are expressed as mean ± SEM from at least three independent experiments. *p < 0.05; **p < 0.01.

FIGURE 2:

miR-375 suppresses oral cancer cell proliferation and colony formation. (A) The cells were counted 72 h posttransfection by trypan blue exclusion staining assay. Representative images are CAL 27 cells transfected with miR-375-mimic or miR-375 inhibitor. Arrowheads indicate sparse areas only observed in the plate with miR-375-mimic–transfected cells. (B) Cell proliferation rate was measured 72 h posttransfection of miR-375-mimic in different doses (25, 50, 100 nM) using MTS assay. The proliferation rates of miR-375-mimic–transfected cells were compared with that of NS control–transfected cells. (C) Transfected cells were seeded on 1:1 diluted Matrigel, and images of live-cell colonies were taken after growth in Matrigel for 4 d. Diameters of 150 colonies from three independent experiments were measured using AxioVision, release 4.7, software. (D) Soft agar colony formation assay–determined anchorage-independent tumor growth was reduced by miR-375-mimic transfection. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3:

miR-375 inhibits oral cancer cell migration and invasion. (A) Representative images of CAL 27 cells were taken at 0, 24, 36, 40, and 48 h during scratch wound-healing assay. Dash lines indicate the initial boundaries of the scratches (0 h) and cell movement leading edges at subsequent time points. (B) Quantitative analysis of the relative wounded area at different time points. The average of the respective wound area was calculated from at least five different random images. (C) Transwell migration assay was performed to examine the effect of miR-375-mimic or inhibitor on CAL 27 cells. After 24 h, the cells on the upper surface were removed and the cells on the lower surface were fixed and stained. Five fields were counted per each filter. (D) Invasion assay was performed using Matrigel-coated Transwell filter. Transfected cells were incubated for 48 h in the filter, and the cells on the upper surface were removed and the cells on the lower surface were fixed and stained the cells on the lower surface were fixed and stained. Five fields were counted per each filter. All results are expressed as mean ± SD from at least three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 4:

CIP2A is directly regulated by miR-375 through multiple binding sites. (A) Five individual miR-375–binding sites were predicted on the CDS of CIP2A. A 1.3-kb CIP2A cDNA containing all putative binding sites was subcloned downstream of FL reporter construct (pMiR-Target). Four nucleotide sequences per seed-binding region for individual sites on CIP2A-CDS were mutated to disrupt the putative miRNA–mRNA interactions. Lines between CIP2A and miR-375 complementary nucleotides are the typical Watson–Crick interactions (A-U and G-C, respectively), and colons are the weak, nontypical base-pair interactions. (B) Luciferase reporter analyses of CIP2A in the presence of miR-375 showing percentages of fold change and the p values for each comparison. Dual luciferase assay was used to determine the direct regulation of CIP2A by miR-375. FL constructs were cotransfected with miR-375-mimic or inhibitor or NS control. Renilla luciferase was used as an internal control to normalize the expression of FL activity. The mutations are designated as A–E, as indicated. Results were compared with either FL-CIP2A alone or cotransfected with miR-375-mimic for the statistical analyses. All results are expressed as mean ± SD from at least three independent experiments. N/A, not available.

FIGURE 5:

miR-375 regulates CIP2A translation by binding to the CDS. (A) Generation of FL-CIP2A in-frame fusion constructs. GATTC sequences inserted upstream of the stop codon of FL shift the frame and generate the fusion construct, creating a new BamHI restriction enzyme site. (B) The BamHI site on the vector backbone and the de novo BamHI site yielded two restricted DNA fragments of 5.0 and 4.2 kb. Electrophoresis was performed on 1% agarose gel. “FL-CIP2A fusion WT” represents the construct with intact miR-375–binding sites, and “FL-CIP2A fusion MUT” represents the construct with mutation of all five miR-375–binding sites. (C) Luciferase reporter analyses of FL-CIP2A in the presence of miR-375-mimic. The WT (white bars) or MUT (black bars) version of FL-CIP2A fusion constructs was cotransfected with miR-375-mimic or NS control. Three different concentrations of miR-375-mimic were used (12.5, 25, 100 nM). Renilla luciferase was cotransfected and used as an internal control to normalize the expression of FL activity. Results were compared with either FL-CIP2A transfection alone (mock) or NS control for the statistical analyses. All results are expressed as mean ± SD from at least three independent experiments. ****p < 0.0001.

FIGURE 6:

miR-375 regulates CIP2A and MYC expression. (A) Oral tumors had significantly higher CIP2A mRNA level than normal tissues. (B) Advanced-stage tumors had significantly higher CIP2A level compared with normal. (C) Significant inverse correlation was observed in linear regression analyses between miR-375 and CIP2A in normal controls and advanced-stage tumors (R = 0.53, p = 0.03). Closed and open circles indicate 11 advanced tumors and five normal controls, respectively. (D) Significant inverse correlation was observed between CIP2A and miR-375 expression in NCI-60 cell lines (R = 0.35, p = 0.01). A similar comparison in seven head and neck cancer cell lines did not reveal a significant p value (ns), despite a similar inverse correlation pattern (inset). (E) Reduced cytoplasmic expression of CIP2A in HeLa cells transfected with 25 nM miR-375-mimic compared with cells transfected with 25 nM miR-375 inhibitor for 48 h. CIP2A staining was detected by mouse monoclonal anti-CIP2A, followed by Alexa 488 goat anti-mouse immunoglobulin (green). Representative images for 40× magnification. Nuclei were counterstained with DAPI (blue). (F) The mRNA level of CIP2A in CAL 27 cells transfected with miR-375-mimic for 72 h were significantly reduced compared with other control groups. All results are expressed as mean ± SD from at least three independent experiments. (G) Significant repression of protein levels of CIP2A were observed, 27 and 31%, respectively, in the miR-375-mimic–transfected cells compared with the untreated cells. To calibrate the semiquantitative measurement, 25, 50, 75, and 100% of untreated cell lysates were included in the Western blot. (H) Western blot analysis of CIP2A and MYC protein levels 24 and 72 h after si-CIP2A transfection in CAL 27 cells. si-CIP2A was transfected at 5 and 80 nM, respectively. si-GFP was used as a control, and 25, 50, and 100% of si-GFP transfected cell lysate were loaded for semiquantitative analysis. (I) Live CAL 27 cells 72 h posttransfection with si-GFP or si-CIP2A counted by trypan blue exclusion staining. CAL 27 cells transfected with si-GFP or si-CIP2A for 24 h were trypsinized and analyzed in a Transwell migration assay after 24 h (J) and Matrigel-coated Transwell invasion assay after 48 h (K). All results are expressed as mean ± SD from three independent experiments. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 7:

Overall schematic model of miR-375 as a tumor suppressor by regulating CIP2A and MYC. In normal physiological condition in which miR-375 levels are high, CIP2A protein translation is negatively regulated by miR-375–mediated posttranscriptional regulation on CIP2A transcripts. As a result, unprotected MYC proteins are subjected to proteolytic degradation and cell proliferation is controlled. In contrast, cells under carcinogenic conditions express low levels of or no miR-375 and fail to efficiently regulate CIP2A expression. MYC proteins are secured by CIP2A proteins from degradation. As a consequence, an increase of MYC stability promotes uncontrolled cell growth and proliferation, resulting into accelerating cancer development.