14 research outputs found
Changes in the expression of proteins associated with aerobic glycolysis and cell migration are involved in tumorigenic ability of two glioma cell lines
<p>Abstract</p> <p>Background</p> <p>The most frequent and malignant brain cancer is glioblastoma multiforme (GBM). In gliomas, tumor progression and poor prognosis are associated with the tumorigenic ability of the cells. U87MG cells (wild-type p53) are known to be tumorigenic in nude mice, but T98G cells (mutant p53) are not tumorigenic. We investigated the proteomic profiling of these two cell lines in order to gain new insights into the mechanisms that may be involved in tumorigenesis.</p> <p>Results</p> <p>We found 24 differentially expressed proteins between T98G and U87MG cells. Gene Ontology supports the notion that over-representation of differentially expressed proteins is involved in glycolysis, cell migration and stress oxidative response. Among those associated with the glycolysis pathway, TPIS and LDHB are up-regulated in U87MG cells. Measurement of glucose consumption and lactate production suggests that glycolysis is more effective in U87MG cells. On the other hand, G6PD expression was 3-fold higher in T98G cells and this may indicate a shift to the pentose-phosphate pathway. Moreover, GRP78 expression was also three-fold higher in T98G than in U87MG cells. Under thapsigargin treatment both cell lines showed increased GRP78 expression and the effect of this agent was inversely correlated to cell migration. Quantitative RT-PCR and immunohistochemistry of GRP78 in patient samples indicated a higher level of expression of GRP78 in grade IV tumors compared to grade I and non-neoplastic tissues, respectively.</p> <p>Conclusions</p> <p>Taken together, these results suggest an important role of proteins involved in key functions such as glycolysis and cell migration that may explain the difference in tumorigenic ability between these two glioma cell lines and that may be extrapolated to the differential aggressiveness of glioma tumors.</p
Placenta-Enriched LincRNAs <i>MIR503HG</i> and <i>LINC00629</i> Decrease Migration and Invasion Potential of JEG-3 Cell Line
<div><p><i>LINC00629</i> and <i>MIR503HG</i> are long intergenic non-coding RNAs (lincRNAs) mapped on chromosome X (Xq26), a region enriched for genes associated with human reproduction. Genes highly expressed in normal reproductive tissues and cancers (CT genes) are well known as potential tumor biomarkers. This study aimed to characterize the structure, expression, function and regulation mechanism of <i>MIR503HG</i> and <i>LINC00629</i> lincRNAs. According to our data, <i>MIR503HG</i> expression was almost exclusive to placenta and <i>LINC00629</i> was highly expressed in placenta and other reproductive tissues. Further analysis, using a cancer cell lines panel, showed that <i>MIR503HG and LINC00629</i> were expressed in 50% and 100% of the cancer cell lines, respectively. <i>MIR503HG</i> was expressed predominantly in the nucleus of JEG-3 choriocarcinoma cells. We observed a positively correlated expression between <i>MIR503HG</i> and <i>LINC00629</i>, and between the lincRNAs and neighboring miRNAs. Also, both <i>LINC00629</i> and <i>MIR503GH</i> could be negatively regulated by DNA methylation in an indirect way. Additionally, we identified new transcripts for <i>MIR503HG</i> and <i>LINC00629</i> that are relatively conserved when compared to other primates. Furthermore, we found that overexpression of <i>MIR503HG2</i> and the three-exon <i>LINC00629</i> new isoforms decreased invasion and migration potential of JEG-3 tumor cell line. In conclusion, our results suggest that lincRNAs <i>MIR503HG</i> and <i>LINC00629</i> impaired migration and invasion capacities in a choriocarcinoma <i>in vitro</i> model, indicating a potential role in human reproduction and tumorigenesis. Moreover, the <i>MIR503HG</i> expression pattern found here could indicate a putative new tumor biomarker.</p></div
5-Aza-2-deoxycytidine (5-Aza-dC) treatment does not affect methylation status of the CpG islands near to putative promoter region of both genes.
<p>Representative normalized melt curves from samples treated with demethylating agent 5-Aza-dC. A-B. DNA analyzed from the CpG island at the promoter region of <i>MIR503HG</i> gene in DLD1 (a) and HCC1954 (b) cell lines. C. DNA analyzed from the CpG island at the promoter region of <i>LINC00629</i> gene in DLD1 cell line. Arrows indicate curves that correspond the percentage of methylation from reference, treatment, and control DNA samples. Images were obtained from High-ReSolution Melt Software v2.</p
<i>MIR503HG</i> and <i>LINC00629</i> lncRNAs expression are related to each other and to their neighboring microRNAs, in normal tissue panel.
<p>A-J. Pearson Correlation (r) test between one of the lncRNAs and a neighbor microRNA.K. Pearson Correlation (r) test between <i>MIR503HG</i> and <i>LINC00629</i> genes.</p
<i>MIR503HG2</i> or <i>LINC00269</i> were overexpressed in JEG-3 cell line.
<p>Plasmidial expression vectors containing <i>MIR503HG2</i> or <i>LINC00629</i> full-length sequences were transfected into JEG-3 cell line. Relative expression was analyzed by RT-qPCR normalized by the geometric mean from <i>GAPDH</i> and <i>HPRT</i> endogenous genes. The empty plasmid was used as a control. *p <0.05 and **p <0.01 (<i>t</i>-test).</p
Evolutionary conservation of the new isoforms.
<p>A. Sequence obtained from <i>MIR503HG2</i> isoform (<i>H</i>. <i>sapiens</i>) aligned to human genome using BLAT tool (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu</a>. <i>version</i>: <i>Feb</i>. <i>2009</i>, <i>CRCh37/hg19)</i> with similar sequences in <i>Saimiri boliviensis</i> (S. <i>boliviensis</i>), <i>Callithrix jacchus</i> (<i>C</i>. <i>jacchus</i>), <i>Nomascus leucogenys</i> (<i>N</i>. <i>leucogenys</i>) and <i>Papio anubis</i> (<i>P</i>. <i>anubis</i>) found in NCBI-BLAST. B. Sequences obtained from <i>LINC0026</i> isoforms comprising two exons (<i>H</i>. <i>sapiens</i>. 2) and three exons (<i>H</i>. <i>sapiens</i>. 3) aligned to human genome using BLAT tool (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu</a>. <i>version</i>: <i>Feb</i>. <i>2009</i>, <i>CRCh37/hg19)</i> with similar sequences in <i>Gorilla gorilla</i> (<i>G</i>. <i>gorilla</i>), <i>Pan troglodytes</i> (<i>P</i>. <i>troglodytes</i>) and <i>Pan paniscus</i> (<i>P</i>. <i>paniscus</i>) found in NCBI-BLAST.</p
Cellular location of <i>MIR503HG</i> and <i>LINC00269</i> RNAs.
<p>RNA samples were extracted from nuclear and cytoplasmic fractions of JEG3 cells. RT-qPCR obtained relative expression from three experiments normalized to 18S ribosomal RNA. GAPDH fits as a control of predominantly cytoplasmic genes. **p <0.01 and ***p <0.001 (<i>t</i>-test).</p
Myosin-Va Contributes to Manifestation of Malignant-Related Properties in Melanoma Cells
<i>MIR503HG</i> and <i>LINC00269</i> expression can be regulated by methylation.
<p>Expression levels of <i>MIR503HG</i>, <i>LINC00269</i>,and their neighbouring miRNAs—miR-424 and miR-503, were determined in RNA samples from breast and colon adenocarcinoma cell lines treated with 5-aza-2-deoxycytidine (5-Aza-dC) by RT-qPCR normalized to the geometric mean from <i>GAPDH</i> and <i>HPRT</i> genes (for <i>MIR503HG</i> and <i>LINC0026</i> genes) or snoRNAs <i>RNU24</i> and <i>RNU48</i> (for miRNAs). Relative expression was obtained from three independent experiments using samples without treatment as reference samples (2<sup>-ΔΔCT</sup>). *p <0.05 (<i>t</i>-test).</p