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<p>Cell scratch test and Transwell were used to measure the migration abilities of HSVSMCs. NC = Negative control group, only control siRNA transfected; GAS5(-) = lncRNA-GAS5 knockdown group transfected with silence siRNA. <b>A:</b>Cell scratch test was used to measure the migration abilities of HSVSMCs. The results showed that the HSVSMCs have the best migration abilities in the first 24 hours. Values are mean±SE, N = 4. <b>B:</b> The migration abilities of HSVSMCs measured by Transwell. After transfected by lncRNA-GAS5 siRNA for 48 hours, the HSVSMCs were passage into the Transwell Inserts. Then 4 hours, 7 hours, 10 hours later, the migration HSVSMCs were photographed and counted, respectively. Knockdown of lncRNA-GAS5 expression promotes migration of HSVSMCs. Optical microscope images under 200x magnification. <b>C:</b> The migration abilities of HSVSMCs were reflected indirectly by the new migration cells counting with Transwell. Silencing of lncRNA-GAS5 expression increses migration ability of HSVSMCs. Values are mean±SE, N = 10; *, P<0.05.</p

Aberrantly Expressed lncRNAs in Primary Varicose Great Saphenous Veins

<div><p>Long non-coding RNAs (lncRNAs) are key regulatory molecules involved in a variety of biological processes and human diseases. However, the pathological effects of lncRNAs on primary varicose great saphenous veins (GSVs) remain unclear. The purpose of the present study was to identify aberrantly expressed lncRNAs involved in the prevalence of GSV varicosities and predict their potential functions. Using microarray with 33,045 lncRNA and 30,215 mRNA probes, 557 lncRNAs and 980 mRNAs that differed significantly in expression between the varicose great saphenous veins and control veins were identified in six pairs of samples. These lncRNAs were sub-grouped and mRNAs expressed at different levels were clustered into several pathways with six focused on metabolic pathways. Quantitative real-time PCR replication of nine lncRNAs was performed in 32 subjects, validating six lncRNAs (AF119885, AK021444, NR_027830, G36810, NR_027927, uc.345-). A coding-non-coding gene co-expression network revealed that four of these six lncRNAs may be correlated with 11 mRNAs and pathway analysis revealed that they may be correlated with another 8 mRNAs associated with metabolic pathways. In conclusion, aberrantly expressed lncRNAs for GSV varicosities were here systematically screened and validated and their functions were predicted. These findings provide novel insight into the physiology of lncRNAs and the pathogenesis of varicose veins for further investigation. These aberrantly expressed lncRNAs may serve as new therapeutic targets for varicose veins. The Human Ethnics Committee of Shanghai East Hospital, Tongji University School of Medicine approved the study (NO.: 2011-DF-53).</p></div

Validation of microarray data and the Q-RT-PCR data.

<p>Nine lncRNAs were chosen for validation in 32 pairs of VVs samples compared with NVs samples by Q-RT-PCR. seven of the nine lncRNAs showed the same trends with respect to up- or down- regulation as the microarray data and six lncRNAs (AK021444, AF119885, G36810, uc.345, NR_027927 and NR_027830) showed statistically significant differences (<i>P</i><0.05). The heights of the columns in the chart represent the mean expression value of log2 fold changes (VVs/NVs) for each of the nine validated lncRNAs in the microarray and Q-RT-PCR data; The bars represent standard errors. The validation results indicated that the microarray data were closely correlate with the Q-RT-PCR results. *: <i>P</i><0.05, **: <i>P</i><0.01.</p

Top 20 significantly differential expressed mRNAs from the microarray data.

<p>NCBI accession: the reference ID of mRNA in NCBI (National Center for Biotechnology Information).</p

Coding-non-coding gene co-expression network of the four lncRNAs.

<p>The network represents co-expression correlations between the four lncRNAs and significantly differentially expressed mRNAs. Only co-expression gene pairs with Pearson coefficient (|r|)>0.99 are shown. Four separate networks were constructed. They are displayed together in this figure. Gene nodes with a cyan node lines represent lncRNAs and gene nodes without node lines represents a protein-coding RNA (mRNA). Red nodes represent up-regulated RNAs, and blue nodes represent down-regulated RNAs. Solid lines between two nodes indicate positively correlated interactions between RNAs, and dotted lines indicate negatively correlated interactions. Node size represents the node degrees. ☆indicates protein coding RNA transcribed from natural antisense strands of the gene HOXC4.</p

The 11 significantly aberrantly expressed mRNAs correlated with the four validated lncRNAs.

<p>NCBI accession: the standard reference ID of mRNA in NCBI (National Center for Biotechnology Information).</p

Low Expression of lncRNA-GAS5 Is Implicated in Human Primary Varicose Great Saphenous Veins

<div><p>The cellular mechanisms of primary varicose great saphenous veins (GSVs) involve inflammation, apoptosis, and proliferation of local cells and extracellular matrix degradation. Long non-coding RNAs (lncRNAs) play important roles in these cellular processes; however, which and how lncRNAs related to these mechanisms take effect on GSVs remain unclear. By screening lncRNAs that might experience changes in GSV varicosities, we selected the lower expressed lncRNA-GAS5 (growth arrest specific transcript 5) for functional assessments. Silencing of lncRNA-GAS5 promoted cell proliferation and migration, and cell cycle of the human saphenous vein smooth muscle cells (HSVSMCs), whereas overexpressing it inhibited these cellular behaviors and reduced apoptosis of HSVSMCs. RNA pull-down experiment revealed a direct bind of lncRNA-GAS5 to a Ca²⁺-dependent RNA-binding protein, Annexin A2. Further experiments showed that silencing of Annexin A2 reduced the HSVSMCs proliferation and vice versa. In the context of lncRNA-GAS5 knockdown, silencing of Annexin A2 reduced the proliferation of HSVSMCs while overexpression of Annexin A2 increased the proliferation. Thus, the low expression of lncRNA-GAS5 may facilitate HSVSMCs proliferation and migration through Annexin A2 and thereby the pathogenesis of GSV varicosities.</p></div

Top 20 significantly differential expressed LncRNAs from the microarray data.

<p>Sourse: different database each lncRNA was included; LncRNA accession number: the reference ID of lncRNA in each database.</p

Pathways of dysregulated mRNAs with the enrichment scores (−log10 (<i>P</i>-value))>2.

<p>The bar plot shows the enrichment scores (−log10 (<i>P</i>-value)) value of the significant enrichment pathways. The white bar shows the pathway in which the up-regulated mRNAs were found to be involved and the blue bars show the pathways in which the down-regulated mRNAs were found to be involved. Pathway analysis involves mapping genes to KEGG pathways. The <i>P</i>-value denotes the significance of the correlation between the pathway and the conditions. Most of the shown here are related to metabolism, which indicates that the varicose veins may be a metabolic disease.</p