Dysregulation of MicroRNA-34a Expression in Head and Neck Squamous Cell Carcinoma Promotes Tumor Growth and Tumor Angiogenesis

MicroRNAs (miRs) are small non-coding RNAs that play an important role in cancer development where they can act as oncogenes or as tumor-suppressors. miR-34a is a tumor-suppressor that is frequently downregulated in a number of tumor types. However, little is known about the role of miR-34a in head and neck squamous cell carcinoma (HNSCC).miR-34a expression in tumor samples, HNSCC cell lines and endothelial cells was examined by real time PCR. Lipofectamine-2000 was used to transfect miR-34a in HNSCC cell lines and human endothelial cells. Cell-proliferation, migration and clonogenic survival was examined by MTT, Xcelligence system, scratch assay and colony formation assay. miR-34a effect on tumor growth and tumor angiogenesis was examined by in vivo SCID mouse xenograft model. Our results demonstrate that miR-34a is significantly downregulated in HNSCC tumors and cell lines. Ectopic expression of miR-34a in HNSCC cell lines significantly inhibited tumor cell proliferation, colony formation and migration. miR-34a overexpression also markedly downregulated E2F3 and survivin levels. Rescue experiments using microRNA resistant E2F3 isoforms suggest that miR-34a-mediated inhibition of cell proliferation and colony formation is predominantly mediated by E2F3a isoform. In addition, tumor samples from HNSCC patients showed an inverse relationship between miR-34a and survivin as well as miR-34a and E2F3 levels. Overexpression of E2F3a completely rescued survivin expression in miR-34a expressing cells, thereby suggesting that miR-34a may be regulating survivin expression via E2F3a. Ectopic expression of miR-34a also significantly inhibited tumor growth and tumor angiogenesis in a SCID mouse xenograft model. Interestingly, miR-34a inhibited tumor angiogenesis by blocking VEGF production by tumor cells as well as directly inhibiting endothelial cell functions.Taken together, these findings suggest that dysregulation of miR-34a expression is common in HNSCC and modulation of miR34a activity might represent a novel therapeutic strategy for the treatment of HNSCC

MiR-103 Controls Milk Fat Accumulation in Goat (<i>Capra hircus</i>) Mammary Gland during Lactation

<div><p>Milk is the primary source of nutrition for young mammals including humans. The nutritional value of milk is mainly attributable to fats and proteins fractions. In comparison to cow milk, goat milk contains greater amounts of total fat, including much higher levels of the beneficial unsaturated fatty acids. MicroRNAs (miRNAs), a well-defined group of small RNAs containing about 22 nucleotides (nt), participate in various metabolic processes across species. However, little is known regarding the role of miRNAs in regulating goat milk composition. In the present study, we performed high-throughput sequencing to identify mammary gland-enriched miRNAs in lactating goats. We identified 30 highly expressed miRNAs in the mammary gland, including miR-103. Further studies revealed that miR-103 expression correlates with the lactation. Further functional analysis showed that over-expression of miR-103 in mammary gland epithelial cells increases transcription of genes associated with milk fat synthesis, resulting in an up-regulation of fat droplet formation, triglyceride accumulation, and the proportion of unsaturated fatty acids. This study provides new insight into the functions of miR-103, as well as the molecular mechanisms that regulate milk fat synthesis.</p></div

Elevated miR-103 expression decreases gene expression associated with lipolysis and β-oxidation.

<p>Over-expression of miR-103 down-regulates the mRNA levels of <i>HSL</i> (A), <i>ATGL</i> (B), <i>ACSL1</i>(C), <i>CPT1</i> (D), <i>PPARα</i> (E) and <i>ACOX1</i> (F). <i>HSL</i> (A) and <i>ATGL</i> (B) are key regulators of lipolysis. <i>ACSL1</i>(C), <i>CPT1</i> (D), <i>PPARα</i> (E) and <i>ACOX1</i> (F) are key regulators of β-oxidation. Gene expression in Ad(control)-infected, Ad-miR-103-infected, and uninfected cells was assessed at 0, 24, 48, and 72 h. qRT-PCR measurement of gene expression expressed as fold change compared to their respective level at 0 h, normalized to 1. Columns, average of 12 experiments; bars, SEM. *, <i>p</i><0.05, **, <i>p</i><0.01.</p

MiR-103 promotes milk fat accumulation in GMEC.

<p>A: Over-expression of miR-103 promotes fat accumulation. Cells in the left column were stained by Oil Red. The fluorescence of the cells was in the right column. Uninfected (first row) and Ad-infected cells (second row) were both used as controls. Arrows indicate Ad- and Ad-miR-103-infected cells; B: Over-expression of miR-103 increases fat droplet content. Fat droplets were extracted using isopropanol in un-, Ad-control without any miRNA sequences and Ad-miR-103-infected cells at 72 h post infection. The data (absorbance, 510 nm) were expressed as fold change as compared to uninfected cells, normalized to 1. Columns, average of 3 experiments; bars, SEM. *, <i>p</i><0.05; C: Elevated miR-103 expression up-regulates triglyceride content. Triglyceride content was determined by using a Serum Triglyceride Determination Kit in un-, Ad-(control) and Ad-miR-103-infected cells. Columns, average of 12 experiments; bars, SEM. *, <i>p</i><0.05; D: Over-expression of miR-103 increases total fatty acids content in GMEC. Fatty acid content expressed as fold change compared to internal control C19:0. Columns, average of 9 experiments; bars, SEM. *, <i>p</i><0.05; E: Over-expression of miR-103 reduces total saturated fatty acids and promotes total unsaturated fatty acids. Columns, average of 9 experiments; bars, SEM. *, <i>p</i><0.05; F: Over-expression of miR-103 alters the composition of major types of fatty acids in GMEC. Columns, average of 9 experiments; bars, SEM. *, <i>p</i><0.05, **, <i>p</i><0.01.</p

Relative mRNA expression in miR-103 over-expression or suppression background.

<p>Genes were clustered based on main functions relative to milk fat synthesis. Ad(control)-infected cells were used as control for Ad-miR-103-infected cells. Inhibitor-control was used as control for miR-103-inhibitor-control. MRNA expression levels were determined at 72 h post infection or at 48 h post transfection. MRNA levels were measured by qRT-PCR, Data expressed as fold change compared to controls, normalized to 1. Columns, average of 12 experiments; *, <i>P</i><0.05; **, <i>P</i><0.01.</p

MiR-103-1 transcriptionally regulates its co-regulated host gene <i>PANK3</i>.

<p>A: A positive correlation between miR-103-1 and <i>PANK3</i> expression in mammary gland of 30 lactating goats. This correlation suggests that miR-103-1 and its host gene <i>PANK3</i> are co-regulated in goat. Intensity scatter plot shows comparison of expression of miR-103 and <i>PANK3</i>; B: The optimal MOI (200) of Ad-miR-103 for infection and dose of miR-103-inhibitor for transfection (60 nM). Ad-control without any miRNAs sequences or inhibitor-control was used as control. GMEC was used as a model. The data (miR-103 levels) were expressed as fold change as compared to controls, normalized to 1. Columns, average of 12 experiments; bars, SEM. *, <i>p</i><0.05; C: MiR-103-1 transcriptionally regulates its host gene <i>PANK3</i>. Over-expression of miR-103 down-regulates the expression of PANK3 in GMEC, whereas suppression of miR-103 leads to up-regulation of <i>PANK3</i>. Over-expression or suppression of miR-103 was conducted by using Ad-miR-103 or miR-103 inhibitor as controls, respectively. MOI of Ad-miR-103 and concentration of inhibitor is shown under X coordinate axis. The data (<i>PANK3</i> levels) were expressed as fold change as compared to controls, normalized to 1. Columns, average of 9 experiments; bars, SEM. *, <i>p</i><0.05.</p

The top 30 miRNAs with maximum number of reads count in goat mammary gland during mid-lactation.

<p>RNA samples were pooled from 10 individuals during mid-lactation (120 days after parturition). The top 30 miRNAs in mammary gland were identified by Solexa sequencing and clustered based on their reported function (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079258#pone.0079258.s005" target="_blank">Table S2</a>). Values on X-axis indicate the number of reads count of miRNAs. All goat miRNAs are named after bovine (<i>Bos taurus</i>). “R+1” and“L-1”: compared to bovine's sequence, goat miRNA sequence has addition or reduction of one base on the 3′ or 5′end.</p

MiR-103 expression correlates with lactation stages.

<p>MiR-103 is differentially regulated at mid-lactation and dry period. qRT-PCR measurement of miR-103 level at mid-lactation expressed as fold change compared to that at dry period, normalized to 1. Columns, average of 12 experiments; bars, SEM. *, <i>p</i><0.05.</p