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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

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

miR-10a reduces proliferation of hCMPCs.

<p>A. miR-10a mimics decrease the BrdU incorporation rate of hCMPCs, whereas a miR-10a inhibitor does not significantly affect the process. *P<0.05, n = 6. B. miR-10a inhibits EdU incorporation of hCMPCs. Representative image of hCMPCs transfected with mock, miR-10a mimics or inhibitor stained with DAPI (blue) and EdU (red) (×200). *P<0.05, n = 5. C. Representative flow cytometry results of hCMPC manipulated with mock, miR-10a mimics or miR-10a inhibitor. hCMPCs overexpressing miR-10a show G1/S blocking, and the inhibition of miR-10a promotes G1/S transition compare with mock. D. Data collected from C. *P<0.05, n = 3. E. Relative expression of cell cycle regulatory genes in hCMPCs transfected with miR-10a mimics. F. The same set of genes was measured in hCMPCs with inhibited miR-10a. The expression level of GAPDH was used as the control. *P<0.05, n = 5.</p

miR-10a does not influence hCMPC differentiation toward cardiomyocytes.

<p>A. Representative image of differentiating hCMPCs with manipulated expression of miR-10a or mock. Cells were stained with DAPI, α-actinin and Trop I (×200). B. Data collected from A. *P<0.05, n = 6. C. Relative expression of cardiomyocyte markers in differentiating hCMPCs transfected with miR-10a mimics. D. The same markers were tested in hCMPCs with inhibited miR-10a. The expression level of GAPDH was used as the control. *P<0.05, n = 6.</p

miR-10a binds to GATA6 and suppresses GATA6 expression.

<p>A. Pmir- glo dual luciferase plasmid containing a wild type or mutant GATA6-3′UTR was cotransfected with miR-10a mimics or mock into 293 cells. The relative luciferase activity was shown. *P<0.05, n = 5. B. Western blot shows the expression of GATA6 in hCMPCs transfected with mock and miR-10a mimics. C. The same experiment with mock and inhibitor. Quantification of protein expression was normalized to GAPDH. *P<0.05, n = 3. D. Overexpression of GATA6 rescues the miR-10a inhibitory effect on hCMPC proliferation. Protein expression of GATA6 with different treatment was showed. BrdU absorbance of hCMPCs transfected with mock, miR-10a mimics and siRNA that downregulated GATA6 are shown. Similar to miR-10a, the downregulation GATA6 in hCMPCs reduces proliferation. miR-10a mimics and a plasmid that overexpresses GATA6 were cotransfected into hCMPCs, and a reduced BrdU absorbance was not observed. *P<0.05, n = 6.</p