<i>PVT1</i> and local miRNA expression in cervical cancer tissue.

<p>(A) <i>PVT1</i> expression was significantly higher in primary cervical tumors (n = 127) compared to adjacent normal cervical tissue (n = 30; p<0.001). Expression of miRNAs 1204 (B; p<0.05) and 1206 (C; p<0.001), but not other local miRNAs, was also significantly higher in cervical tumor tissue (n = 38) compared to adjacent normal tissue (n = 18). (D) Kaplan-Meier plots revealed an association of higher tumor <i>PVT1</i> levels with significantly poorer survival times compared to lower <i>PVT1</i> levels (p = 0.03).</p

Disruption of Axonal Transport Perturbs Bone Morphogenetic Protein (BMP) - Signaling and Contributes to Synaptic Abnormalities in Two Neurodegenerative Diseases

<div><p>Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases.</p></div

The lncRNA <i>PVT1</i> Contributes to the Cervical Cancer Phenotype and Associates with Poor Patient Prognosis

<div><p>The plasmacytoma variant translocation 1 gene (<i>PVT1</i>) is an lncRNA that has been designated as an oncogene due to its contribution to the phenotype of multiple cancers. Although the mechanism by which <i>PVT1</i> influences disease processes has been studied in multiple cancer types, its role in cervical tumorigenesis remains unknown. Thus, the present study was designed to investigate the role of <i>PVT1</i> in cervical cancer <i>in vitro</i> and <i>in vivo</i>. <i>PVT1</i> expression was measured by quantitative PCR (qPCR) in 121 invasive cervical carcinoma (ICC) samples, 30 normal cervix samples, and cervical cell lines. Functional assays were carried out using both siRNA and LNA-mediated knockdown to examine <i>PVT1’</i>s effects on cervical cancer cell proliferation, migration and invasion, apoptosis, and cisplatin resistance. Our results demonstrate that <i>PVT1</i> expression is significantly increased in ICC tissue versus normal cervix and that higher expression of <i>PVT1</i> correlates with poorer overall survival. In cervical cancer cell lines, <i>PVT1</i> knockdown resulted in significantly decreased cell proliferation, migration and invasion, while apoptosis and cisplatin cytotoxicity were significantly increased in these cells. Finally, we show that <i>PVT1</i> expression is augmented in response to hypoxia and immune response stimulation and that this lncRNA associates with the multifunctional and stress-responsive protein, Nucleolin. Collectively, our results provide strong evidence for an oncogenic role of <i>PVT1</i> in cervical cancer and lend insight into potential mechanisms by which <i>PVT1</i> overexpression helps drive cervical carcinogenesis.</p></div

<i>PVT1</i> expression in cervical cancer cells.

<p>(A) <i>PVT1</i> expression in commercially available cervical cell lines. Lowest <i>PVT1</i> expression was observed in HPV 16 E6/E7-transformed cells derived from normal ectocervix (E6/E7-Ecto), while SiHa cervical cancer cells displayed the highest <i>PVT1</i> expression compared to 2 other cervical cancer-derived lines (HeLa and DoTc2). (B) Expression of <i>PVT1</i> in SiHa cells was further increased upon 48h treatment with INF-α (10 μM) or the hypoxia mimetic cobalt chloride (CoCl₂; 150 μM). Representative images of RNA FISH experiments in SiHa cells transfected with either control (C) or <i>PVT1</i> (D) LNAs. Control LNA-transfected cells exhibited punctate signals for <i>PVT1</i> (red) in both the nucleus (stained with DAPI in blue) and the cytoplasm. Both nuclear and cytoplasmic <i>PVT1</i> staining was absent in <i>PVT1</i> LNA-transfected cells. Phase image (bottom left panel) depicts cell morphology. Scale bar (white) = 10 μm. *p<0.05, **p<0.01.</p

<i>PVT1</i> interacts with nucleolin in SiHa cells.

<p>(A) SiHa cells stained for <i>PVT1</i> (green) and nucleolin (red), and DAPI. Boxed cells (top panel) are at higher magnification (bottom panel). White arrows show <i>PVT1</i>-nucleolin colocalization. Scale bars = 20μM. (B) Western blot for Nucleolin of SiHa protein captured by <i>PVT1</i> RNA affinity chromatography. (C) <i>PVT1</i> RNA was immunoprecipitated with anti-Nucleolin, but not the negative control rabbit IgG (Cont). **p<0.01</p

<i>PVT1</i> promotes cervical cancer cell proliferation, migration and invasion.

<p>(A) Transfection of SiHa cervical cancer cells with siRNAs targeting <i>PVT1</i> (siPVT1) resulted in an approximate 70% knockdown in <i>PVT1</i> lncRNA expression as compared to cells transfected with a scrambled control siRNA (siCONT). (B) SiHa cells transfected with siPVT1 exhibited a significant decrease in proliferation compared to siCONT cells. Transfected SiHa cells were also assessed for changes in (C) migration and (D) invasion 6 h or 48 h following introduction of chemoattractant (FBS), respectively. siPVT1 cells showed a significant decrease in both cell migration and invasion compared to siCONT cells. Quantitative results are graphed on the left, while representative images are on the right. ***p<0.001, ****p<0.0001</p

<i>PVT1</i> inhibits cervical cancer cell death and cisplatin sensitivity.

<p>siPVT1 cells exhibited a significant increase in cell death (A) and apoptosis (B) compared to siCONT cells. (C) Proliferation of siPVT1 SiHa cells was significantly decreased in response to multiple doses of cisplatin. (D) Dose-response curve for control and <i>PVT1</i> LNA-transfected SiHa cells following 4 h treatment with cisplatin. The IC₅₀ value of the <i>PVT1</i> knockdown cells was significantly decreased compared to control knockdown cells (p<0.0001), suggesting a role for this lncRNA in cisplatin resistance. **p<0.01, ***p<0.001, ****p<0.0001</p

Components of the BMP signaling pathway genetically interact with motor proteins.

<p><b>A:</b> Larval segmental nerves from heteroallelic combinations of kinesin or dynein mutations with mutations in the BMP signaling pathway were examined using CSP. Khc−/+, klc−/+, dhc−/+, roblk−/+, mad−/+, med−/+, sax−/+, thv−/+ and wit−/+ were comparable to wild type and their larval nerves showed smooth staining. In contrast axonal blockages are observed in khc−/+;mad−/+, klc−/+;mad−/+, dhc−/+;mad−/+ and roblk−/+;mad−/+ (arrows). No blockages are seen in mad−/+, khc−/+, klc−/+, dhc−/+ and roblk−/+. Axonal blockages (arrows) are seen in khc−/+;med−/+, klc−/+;med−/+, dhc−/+;med−/+ and roblk−/+;med−/+. No blockages are seen in med−/+. Axonal blockages are seen in khc−/+;sax−/+ and klc−/+;sax−/+ (arrows) but not in dhc−/+;sax−/+ and roblk−/+;sax−/+. Axonal blockages are seen in dhc−/+;thv−/+ (arrows), but not in khc−/+;thv−/+, klc−/+;thv−/+ and roblk−/+;thv−/+. Axonal blockages are also seen in khc−/+;wit−/+, dhc−/+;wit−/+ and roblk−/+;wit−/+ (arrows) but not klc−/+;wit−/+. Bar = 10 µm. <b>B:</b> Quantification analysis of the % of the average number of axonal blockages in 50 µm nerve length show significant amounts of axonal blockages in khc−/+;mad−/+,khc−/+;med−/+, khc−/+;sax−/+, khc−/+;wit−/+, klc−/+;mad−/+, klc−/+;med−/+, klc−/+;sax−/+, dhc−/+;mad−/+, dhc−/+;med−/+, dhc−/+;thv−/+, dhc−/+;wit−/+, roblk−/+;mad−/+, roblk−/+;med−/+, and roblk−/+;wit−/+, compared to wild type, khc−/+, klc−/+, dhc−/+, roblk−/+, mad−/+, med−/+, sax−/+, thv−/+ or wit−/+. N = 10 larvae. For statistical analysis ANOVA was used followed by post-hoc analysis using the Bonferroni's test.</p

Summary of <i>in vivo</i> measurements from the customized single particle tracking software program obtained from genotypes TKV-GFP, TKV-GFP;klc−/+ and TKV-GFP;roblk−/+.

<p>Arrows indicate direction of change with 50% reduction of motors.</p><p>*Significance <0.05,</p><p>**significance <0.01,</p><p>***significance <0.001 as determined by nonparametric Wilcoxon-Mann-Whitney test. Distributions were determined to be non-normal by Lilliefors test.</p