LIN28 Expression in malignant germ cell tumors downregulates let-7 and increases oncogene levels - PubMed (original) (raw)

. 2013 Aug 1;73(15):4872-84.

doi: 10.1158/0008-5472.CAN-12-2085. Epub 2013 Jun 17.

Harpreet K Saini, Charlotte A Siegler, Jennifer E Hanning, Emily M Barker, Stijn van Dongen, Dawn M Ward, Katie L Raby, Ian J Groves, Cinzia G Scarpini, Mark R Pett, Claire M Thornton, Anton J Enright, James C Nicholson, Nicholas Coleman; CCLG

Affiliations

LIN28 Expression in malignant germ cell tumors downregulates let-7 and increases oncogene levels

Matthew J Murray et al. Cancer Res. 2013.

Abstract

Despite their clinicopathologic heterogeneity, malignant germ cell tumors (GCT) share molecular abnormalities that are likely to be functionally important. In this study, we investigated the potential significance of downregulation of the let-7 family of tumor suppressor microRNAs in malignant GCTs. Microarray results from pediatric and adult samples (n = 45) showed that LIN28, the negative regulator of let-7 biogenesis, was abundant in malignant GCTs, regardless of patient age, tumor site, or histologic subtype. Indeed, a strong negative correlation existed between LIN28 and let-7 levels in specimens with matched datasets. Low let-7 levels were biologically significant, as the sequence complementary to the 2 to 7 nt common let-7 seed "GAGGUA" was enriched in the 3' untranslated regions of mRNAs upregulated in pediatric and adult malignant GCTs, compared with normal gonads (a mixture of germ cells and somatic cells). We identified 27 mRNA targets of let-7 that were upregulated in malignant GCT cells, confirming significant negative correlations with let-7 levels. Among 16 mRNAs examined in a largely independent set of specimens by quantitative reverse transcription PCR, we defined negative-associations with let-7e levels for six oncogenes, including MYCN, AURKB, CCNF, RRM2, MKI67, and C12orf5 (when including normal control tissues). Importantly, LIN28 depletion in malignant GCT cells restored let-7 levels and repressed all of these oncogenic let-7 mRNA targets, with LIN28 levels correlating with cell proliferation and MYCN levels. Conversely, ectopic expression of let-7e was sufficient to reduce proliferation and downregulate MYCN, AURKB, and LIN28, the latter via a double-negative feedback loop. We conclude that the LIN28/let-7 pathway has a critical pathobiologic role in malignant GCTs and therefore offers a promising target for therapeutic intervention.

©2013 AACR.

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Figures

Figure-1

Figure-1. Let-7 and LIN28 expression in malignant-GCTs

A) The left and middle panels respectively show median levels of all nine let-7 family members and levels of LIN28 in the 20 samples from set-1 with matching microRNA and mRNA data. The right panel shows levels of LIN28 in sample set-2. B) Linear-regression analysis of median let-7 family levels versus LIN28 levels in the set-1 samples with matching data. In A) and B), all values are referenced to the mean of the normal gonadal samples. C) qRT-PCR validation in sample set-3, referenced to the pooled normal gonadal control sample. Error-bars= standard error of the mean (SEM). For one sample (asterisked) there was insufficient RNA for let-7e quantification. The color-code for all panels is shown in the key. For details of the normal gonadal controls, see Materials and Methods.

Figure-2

Figure-2. Significance of let-7 down-regulation in malignant-GCTs

A) Sylamer landscape plots for SCR words corresponding to the common seed of the nine let-7 microRNA family members in the combined analysis of pediatric (set-1) and adult (set-2) malignant-GCTs. Log10-transformed _p_-values for each SCR word are plotted on the _y_-axis, against the ranked gene list on the _x_-axis. A negative _y_-axis deflection on the right-hand side of the plot signifies SCR enrichment in up-regulated genes. The left-hand plot shows data for the hexamer complementary to the core 2-7nt component of the common seed-region, the central plot the two heptamers (1-7nt; 2-8nt), and the right-hand plot the octamer (1-8nt). The single summed significance score and _p_-value for all four SCR words is shown. B) Levels of the six selected let-7 mRNA targets in sample set-3, determined by qRT-PCR. Error-bars=SEM. C) Correlations between each mRNA and let-7e in set-3. _P_-values were determined by linear-regression.

Figure-3

Figure-3. LIN28 depletion in 2102Ep malignant-GCT cells

A) Depletion of LIN28, measured by qRT-PCR over d1-d7 (left) and by Western blot over d3-d5 (right). NTC=non-targeting-control siRNA, kd=knockdown. B) Cell numbers following LIN28 depletion. C) Levels of let-7e over d1-d7 following LIN28 depletion. In A) to C), statistical comparisons are versus NTC-treated cells. d=day. D) Levels of let-7 family members (let-7b, let-7d and let-7e) and the six selected let-7 mRNA targets at d2, d4 and d7 following LIN28 depletion. Expression values are referenced to NTC-treated cells. Statistical comparisons are for _LIN28_kd cells at d4 and d7, versus _LIN28_kd cells at d2. Error-bars=SEM. In panels B, C and D, *=p<0.05;**=p<0.005;***=_p_≤0.0001.

Figure-4

Figure-4. Correlations between LIN28, MYCN and cell numbers following LIN28 depletion

The graphs show data for all four malignant-GCT cell-lines at d4 following LIN28 depletion, compared with NTC-treated cells. Panel A shows cell numbers versus the levels of LIN28 (red) and MYCN (blue), while panel B shows levels of MYCN versus LIN28. NTC=non-targeting-control siRNA, kd=knockdown. Correlation _p_-values were determined by linear-regression. Error-bars=SEM.

Figure-5

Figure-5. LIN28B depletion in malignant-GCT cells

A) Cell numbers and qRT-PCR expression levels of LIN28B, LIN28 and MYCN on d4 following LIN28B depletion in 2102Ep (left) and TCam2 (right). B) Western blots showing expression of LIN28B on d4 following LIN28B depletion in 2102Ep (left) and TCam2 (right), compared with NTC-treated cells. C) Levels of representative let-7 family members (let-7b, let-7d and let-7e) on d4 following LIN28B depletion in 2102Ep (left) and TCam2 (right). All values are referenced to NTC-treated cells. NTC=non-targeting-control siRNA, kd=knockdown, error-bars=SEM. *=p<0.05;**=p<0.005;***=_p_≤0.0001.

Figure-6

Figure-6. Effects of let-7e mimic in malignant-GCT cells

A) Cell numbers and expression levels of MYCN and LIN28 in 2102Ep (left) and 1411H (right), at d2 post-transfection of let-7e mimic, relative to cells treated with mimic-negative-control (MNC) RNA. B) Western blots showing expression of LIN28, AURKB, MYCN and LIN28B proteins at d2 and d3 following let-7e mimic transfection of 2102Ep cells, corresponding to Supplementary Figure-S9A. The lower row shows the beta-tubulin loading-control. C) The graph shows protein levels at d3, as determined by densitometry of the western blots shown in B), normalized to beta-tubulin and referenced to MNC-treated cells. D) Cell numbers versus let-7e levels at d2 in four different malignant-GCT cell-lines, compared to MNC-treated cells. The _p_-value was determined by linear-regression. E) Levels of other representative let-7 family members (let-7b and let-7d) and control miR-492, at d2 following let-7e transfection in 2102Ep (left) and 1411H (right), relative to MNC-treated cells.

Figure-7

Figure-7. Luciferase assay confirmation of let-7 targets in malignant-GCT cells

Luciferase assay data at d2 for 2102Ep cells transfected with a reporter containing the full-length 3′UTR for LIN28 (red), MYCN (blue) or AURKB (orange). Cells were also transfected with either let-7e or let-7e-mutant (let-7e-mut). Luminescence values were normalized to cells treated with non-targeting oligonucleotides (NT2), then referenced to cells containing a no 3′UTR control reporter and treated with let-7e/let-7e-mutant, as appropriate. Error-bars=SEM. All correlation _p_-values were determined by linear-regression. *=p<0.05;**=p<0.005;***=_p_≤0.0001.

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