Silencing of C9orf86 induces G1 arrest and apoptosis in breast cancer cells.

<p>(A) Cell cycle distribution was analyzed by flow cytometry 72 h after transfection. Bars are shown as the mean ± SD of cells in G1 phase of the cell cycle. (B) Apoptosis was determined by flow cytometric detection of Annexin-V-FITC-positive/PI-negative cells 72 h after infection. Bars are shown as the mean ± SD of cells with Annexin-V-FITC-positive and PI-negative. All data are shown as mean ± SD of two or three independent experiments, *P<0.05. NC, negative control.</p

C9orf86 expression in breast cancer cells and tissues.

<p>Expression of C9orf86 was quantified in human breast cancer (lanes 2–6), and normal (lane 1) breast epithelial cells by Western blot (A) and qRT-PCR (B). (C) QRT-PCR shows that expression of C9orf86 is increased in invasive BC tissues compared with NATs (P<0.05). Western blotting and RT-PCR were performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control.</p

Silencing of C9orf86 expression inhibits invasion ability of MCF-7 cells and SK-BR-3 cells.

<p>Cell invasion was assayed in a transwell coated with Matrigel. Cells that crossed the Matrigel-coated filter were fixed, stained, and counted. Six random microscopic fields were counted for each group. The results presented are an average of six random microscopic fields from three independent experiments. Significant reduction of invasion was observed after silencing C9orf86 expression in MCF-7 cells and SK-BR-3 cells. *P<0.05. NC, negative control.</p

C9orf86 expression and survival in breast cancer patients.

<p>(A) and (B) Representative staining of C9orf86 in the cytoplasm of breast cancer cells, by IHC staining at 40× and 200× magnifications. (C) Kaplan-Meier estimates of overall survival curves for different C9orf86 expression levels in breast cancer patients, stratified by clinical stage.</p

Effect of C9orf86 knockdown on MCF-7 and SK-BR-3 cell growth in nude mice.

<p>(A) Photographs of nude mice and tumors extracted from C9orf86 knockdown and NC groups (MCF-7 and SK-BR-3). (B) Tumors were weighed after animals were killed 7 weeks post-tumor cell injection. There was a decreasing trend in both the number of cells and size of tumors in the C9orf86 knockdown and NC group of mice for MCF-7 (P<0.01) and SK-BR-3 (P = 0.261) cells. (C) Growth curves for tumors in MCF-7-C9orf86-siRNA-treated group (n = 6) <i>versus</i> the MCF-7-NC-treated group (n = 8) (all P values <0.01) and growth curves for tumors in SK-BR-3-C9orf86-siRNA-treated group (n = 2) <i>versus</i> SK-BR-3-NC-treated group (n = 7) (all P values >0.05). (D) The level of C9orf86 mRNA from the tumor 7 weeks after the injection in NC-group was higher than C9orf86-siRNA group (MCF-7 and SK-BR-3, all P values <0.05). Data are shown as the mean ± SD. *P<0.05. NC, negative control.</p

Relationship between C9orf86 expression level and clinicopathological variables of BC patients.

<p>Abbreviations: BC, breast cancer; AJCC, American Joint Committee on Cancer.</p

Silencing of miR-214 induces apoptosis in NPC cells.

<p>(A) Forty-eight hours post-transfection, miR-214 expression levels (normalized to <i>U6 RNA</i>) were significantly decreased by 82% (<i>P</i><0.01) in CNE2/LNA-antimiR-214 and 87.7% (<i>P</i><0.01) in SUNE1/LNA-antimiR-214 cells, relative to the LNA-control. (B) Forty-eight hours post-transfection, Caspase 3 activity in the cells were measured by spectrophotometry. The relative activity of Caspase 3 was significantly increased in CNE2/LNA-antimiR-214 cells and SUNE1/LNA-antimiR-214 cells. (C) Forty-eight hours post-transfection, apoptosis was determined by flow cytometric detection of Annexin-V-FITC-positive/PI-negative cells. Bars are shown as the mean ± SD of cells with Annexin-V-FITC-positive and PI-negative. All data are shown as mean ± SD of triplicate experiments. ** <i>P</i><0.01.</p

Knockdown of miR-214 inhibits tumor growth in nude mice: <i>in vivo</i> functional studies.

<p>(A) Photographs of tumors extracted from CNE2/LNA-antimiR-214 (n = 9) and CNE2/LNA-control groups. (B) Growth curves for CNE2/LNA-antimiR-214 (n = 9) vs. CNE2/LNA-control (n = 10) cells in an <i>in vivo</i> proliferation assay. (C) Tumors were weighed after animals were killed at 14 days post-tumor-cell injection. The weight of tumors was significantly decreased in CNE2/LNA-antimiR-214 group compared to CNE2/LNA-control group, (<i>P</i> = 0.006, Mann-Whitney test). (D) Representative photomicrographs of FISH for miR-214 on xenograft tumor sections obtained from mice bearing CNE2/LNA-antimiR-214 (n = 9) and CNE2/LNA-control groups (×400). (E) qRT-PCR analysis of miR-214 expression in CNE2 cells 48 h post post-transfection and in xenograft tumors after mice sacrifice. Data in (B), (C) and (E) indicate the mean ± SD. * <i>P</i><0.05, ** <i>P</i><0.01.</p

Clinicopathological characteristics and follow-up data of 210 NPC patients.

<p>Abbreviations: NPC, nasopharyngeal carcinoma; WHO, World Health Organization; NKUC, non-keratinizing undifferentiated carcinoma; NKDC, non-keratinizing differentiated carcinoma; KSCC, keratinizing squamous cell carcinoma; OS, overall survival.</p

Expression of miR-214 in NPC tissues and NPC cell lines.

<p>(A) Representative picture of fluorescence density for miR-214 expression in 5 NPC patients compared to adjacent normal tissues (400× magnification). MiR-214 positive signals detected by FISH are in green, which is upregulated in NPC tumor cells compared to the adjacent epithelial cells. (B) Relative quantitative analysis by qRT-PCR showed that expression of miR-214 is significantly higher in NPC tissues than healthy control (<i>P</i> = 0.001). (C) FISH detection of miR-214 in three NPC cell lines (CNE2, SUNE1, and HONE1) and one non-tumorigenic epithelial cell line (NPEC2 Bmi-1). MiR-214 positive signals are visualized in green (D). Relative qRT-PCR analysis shows that CNE2, SUNE1, and HONE1 cells express higher levels of miR-214 compared with NPEC2 Bmi-1 cells. All data are shown as mean ± SD of triplicate experiments (** <i>P</i><0.01). Abbreviations: NPC, Nasopharyngeal carcinoma; NATs, normal adjacent tissues, qRT-PCR, quantitative RT-PCR, SD, standard deviation.</p