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. 2010 Jun;49(6):549-59.
doi: 10.1002/gcc.20765.

Overexpression of dicer as a result of reduced let-7 MicroRNA levels contributes to increased cell proliferation of oral cancer cells

Andrew Jakymiw  1 , Rushi S Patel, Natasha Deming, Indraneel Bhattacharyya, Priya Shah, Richard J Lamont, Carol M Stewart, Donald M Cohen, Edward K L Chan
Affiliations

Overexpression of dicer as a result of reduced let-7 MicroRNA levels contributes to increased cell proliferation of oral cancer cells

Andrew Jakymiw et al. Genes Chromosomes Cancer. 2010 Jun.

Abstract

Recent reports have demonstrated that Dicer, an RNase III endonuclease required for microRNA (miRNA) maturation, is aberrantly expressed in different types of cancer. Furthermore, Dicer has been reported to be regulated by the let-7 family of miRNA genes. We hypothesize that Dicer is aberrantly expressed in oral cancer cells due to altered expressions of let-7 and that Dicer contributes to the development and progression of the disease. Western blot examination of Dicer protein levels in four head and neck squamous cell carcinoma (HNSCC) cell lines, including two oral cancer cell lines, demonstrated that Dicer had between 4- and 24-fold higher expression levels when compared to normal human primary gingival epithelial cells. Furthermore, five of six oral cancer tissues analyzed by indirect immunofluorescence had increased Dicer protein expression, compared to normal gingival epithelial tissue. The Dicer mRNA levels were not found to correlate well with protein expression in the HNSCC cell lines, suggesting that Dicer protein expression was post-transcriptionally regulated. Analysis of let-7a and let-7b levels in HNSCC cell lines by real-time PCR demonstrated that let-7b, but not let-7a, was significantly reduced in the HNSCC cell lines compared to control cells. Lastly, transfection of oral cancer cells with chemically synthesized let-7b and small interfering RNAs targeting Dicer significantly inhibited cell proliferation up to 83% and >100%, respectively, as early as 3 days post-transfection. Together, these data demonstrate that elevated expression levels of Dicer in oral cancer cells correlate with downregulation of let-7b and increased cell proliferation.

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Figures

Figure 1
Figure 1
Overexpression of Dicer in head and neck squamous cell carcinoma cell lines. Western blot analysis of Dicer expression in several head and neck squamous cell carcinoma cell lines (FaDu, SCC-25, CAL 27, and RPMI 2650) compared to normal primary gingival epithelial cells (pGEC). Tubulin antibody was used to check for equal loading of samples. Serial dilutions of RPMI 2650 cell lysates (50%, 25%, and 12.5%) were included to aid in visualizing the degree of Dicer overexpression. The data is representative of two independent experiments. f/d; fold difference, quantitative measurement of the relative Dicer protein fold expression differences using ImageJ software.
Figure 2
Figure 2
Dicer is up-regulated in oral squamous cell carcinomas. Indirect immunofluorescence analysis of Dicer (green) expression in formalin fixed paraffin-embedded normal human gingival epithelial and two oral squamous cell carcinoma (OSCC) tissues. EGFR (red) detection was used to help demarcate the normal basal epithelium from the underlying connective tissue and also help identify epithelial-derived cancerous regions in the OSCC tissues. Inset panels are higher (3×) magnification views of the boxed areas demonstrating the discrete cytoplasmic foci staining of Dicer (arrows). Nuclei (blue) were counterstained with DAPI. Scale bar: 25 μm.
Figure 3
Figure 3
The cellular localization of Dicer in a murine xenograft tumor is consistent with oral squamous cell carcinomas. (a) Hematoxylin and eosin-stained CAL 27 murine xenograft tumor. Arrows demarcate the boundary between the xenograft tumor and the overlying muscle tissue of the mouse tongue. Scale bar: 500 μm. (b) Indirect immunofluorescence analysis of Dicer (green) expression in formalin fixed paraffin-embedded xenograft tumor tissue. The discrete cytoplasmic foci staining of Dicer are indicted by arrows. EGFR (red) detection was used to help demarcate the xenograft tumor. Nuclei (blue) were counterstained with DAPI. Scale bar: 10 μm.
Figure 4
Figure 4
let-7b expression is reduced in head and neck squamous cell carcinoma cell lines. Real-time PCR analysis of (a) Dicer mRNA and (b) let-7 microRNA expression levels in several head and neck squamous cell carcinoma cell lines (FaDu, SCC-25, CAL 27, and RPMI 2650) compared to normal primary gingival epithelial cells (pGEC). Data shown were obtained from three biological replicates. S.E.M.; standard error measurement. *P < 0.05.
Figure 5
Figure 5
Knockdown of Dicer inhibits cell proliferation of oral cancer cells. CAL 27 cells were either mock transfected or transfected with siGFP, siDicer, or let-7b. (a) Western blot analysis of Dicer protein levels 72 hours post-transfection. Serial dilutions of siGFP lysates (50%, 25%, and 12.5%) were used to help comparatively quantitate the degree of protein knockdown. Tubulin was used as a loading control. The experiment was repeated two additional times with similar results. (b) Real-time PCR analysis of Dicer mRNA levels 72 hours post-transfection. The data is representative of three independent experiments. (c) 5,000 cells were seeded on a 96-well plate 24 hours after either mock transfection or transfection with siGFP, siDicer, or let-7b. Cell numbers were assayed 3, 6, and 8 days post-transfection. The results represent the mean ± standard error measurement. Indicated at each time point are also the average percent inhibitory effects on cell proliferation for siDicer and let-7b relative to siGFP transfected cells. The data is representative of three independent experiments performed in triplicate. *P < 0.05 in comparison to control siGFP or mock transfected cells.
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