Dear Reader

<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

PRISMA-P (Preferred Reporting Items for Systematic review and Meta-Analysis Protocols) 2015 checklist: Recommended items to address in a systematic review protocol.

(DOCX)</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

Knockdown and over-expression of lncRNA-GAS5 in HSVSMCs.

<p><b>A:</b> The lncRNA-GAS5 expression level was knockdowned by siRNA. Three siRNAs knockdowned the lncRNA-GAS5 expression level in HSVSMCs effectively. mean±SE, N = 3; P<0.05. <b>B:</b> The lncRNA-GAS5 expression level were over-expressed by constructing and transfecting expression plasmid. The lncRNA-GAS5 expression plasmid (pCMV-GAS5-OVER) increased the lncRNA-GAS5 expression level in HSVSMCs effectively. mean±SE, N = 4; P<0.05.</p

The expression of lncRNAs-GAS5 by Q-RT-PCR with the increase of tested sample pairs.

<p>lncRNA-GAS5 show significantly different expressions consistently with the increasing of tested samples. ΔΔCT show the actual relative expression fold change as 2^ΔΔCT. Values are mean±SE. *: P<0.05; **: P<0.01.</p

The aberrant expression of the 15 lncRNAs by Q-RT-PCR with the increase of tested sample pairs.

<p>After exclusion of 17 lncRNAs for invalid primers and 7 lncRNAs for very low relative lncRNAs expressions, the expression differences between the varicose GSVs and control veins of 15 lncRNAs were measured by Q-RT-PCR. ΔΔCT show the actual relative expression fold change as 2^-ΔΔCT. The positive value means down-regulated expression of the lncRNA between the varicose GSVs and control veins, conversely, the negative value means up-regulated expression of the lncRNA between the varicose GSVs and control veins. *: P<0.05.</p

The effects of lncRNA-GAS5 mediated by Annexin A2.

<p>A2 = Annexin A2; Mock = control group, only empty plasmid transfected; NC = Negative control group, only control siRNA transfected; A2 siRNA = Annexin A2 knockdown group transfected with silence siRNA; A2 over = Annexin A2 overexpression group transfected with Annexin A2 expression plasmid; GAS5 down = lncRNA-GAS5 knockdown group transfected with silence siRNA. <b>A:</b>Silver stains for protein gels obtained by lncRNA-GAS5 RNA Pulldown. Bio-GAS5 (sense): treatment group; Bio-GAS5 (antisense): control groups; GAS5 (sense): negative control groups. The GAS5- specific-binding protein gels (in Red box) was identified by MALDI-TOF-MS. Results show that Annexin A2 isoform 2 is a direct binding protein to lncRNA-GAS5. N = 2. <b>B:</b> The Annexin A2 expression level was knockdowned in HSVSMCs effectively by siRNA. Values are mean±SE, N = 3; *, P<0.05. <b>C:</b> The Annexin A2 expression level was over-expressed by constructing and transfecting expression plasmid. Values are mean±SE, N = 3; *, P<0.05. <b>D:</b> The proliferation abilities of HSVSMCs were reflected indirectly by the OD450 values measured using CCK-8 kit when Annexin A2 expression level were knockdowned or over-expressed in HSVSMCs. Values are mean±SE, N = 3; *, P<0.05. <b>E:</b> The proliferation abilities of HSVSMCs were reflected indirectly by the OD450 values measured using CCK-8 kit when Annexin A2 and lncRNA-GAS5 expression levels were knockdowned or over-expressed simultaneously in HSVSMCs. Firstly Annexin A2 siRNA or expression plasmid were transfected into the HSVSMCs, six hours later, the cell culture medium were changed and then lncRNA-GAS5 siRNA were transfected into the HSVSMCs. Another 48 hours later, the OD450 values were measured by CCK-8 kit. Values are mean±SE, N = 3; *, P<0.05.</p