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• W3005471014 abstract "Inflammation and proliferation of vascular smooth muscle cells (VSMCs) are the key events in intimal hyperplasia. This study aimed to explore the mechanism by which long non-coding RNA (lncRNA) KCNQ1OT1 affects VSMC inflammation and proliferation in this context. A vein graft (VG) model was established in mice to introduce intimal hyperplasia. Isolated normal VSMCs were induced with platelet-derived growth factor type BB (PDGF-BB), and the cell proliferation, migration, and secretion of inflammatory factors were determined. The results showed that KCNQ1OT1 was downregulated in the VSMCs from mice with intimal hyperplasia and in the PDGF-BB-treated VSMCs, and such downregulation of KCNQ1OT1 resulted from the increased methylation level in the KCNQ1OT1 promoter. Overexpressing KCNQ1OT1 suppressed PDFG-BB-induced VSMC proliferation, migration, and secretion of inflammatory factors. In VSMCs, KCNQ1OT1 bound to the nuclear transcription factor kappa Ba (IκBa) protein and increased the cellular IκBa level by reducing phosphorylation and promoting ubiquitination of the IκBa protein. Meanwhile, KCNQ1OT1 promoted the expression of IκBa by sponging miR-221. The effects of KCNQ1OT1 knockdown on promoting VSMC proliferation, migration, and secretion of inflammatory factors were abolished by IκBa overexpression. The roles of KCNQ1OT1 in reducing the intimal area and inhibiting IκBa expression were proved in the VG mouse model after KCNQ1OT1 overexpression. In conclusion, KCNQ1OT1 attenuated intimal hyperplasia by suppressing the inflammation and proliferation of VSMCs, in which the mechanism upregulated IκBa expression by binding to the IκBa protein and sponging miR-221. Inflammation and proliferation of vascular smooth muscle cells (VSMCs) are the key events in intimal hyperplasia. This study aimed to explore the mechanism by which long non-coding RNA (lncRNA) KCNQ1OT1 affects VSMC inflammation and proliferation in this context. A vein graft (VG) model was established in mice to introduce intimal hyperplasia. Isolated normal VSMCs were induced with platelet-derived growth factor type BB (PDGF-BB), and the cell proliferation, migration, and secretion of inflammatory factors were determined. The results showed that KCNQ1OT1 was downregulated in the VSMCs from mice with intimal hyperplasia and in the PDGF-BB-treated VSMCs, and such downregulation of KCNQ1OT1 resulted from the increased methylation level in the KCNQ1OT1 promoter. Overexpressing KCNQ1OT1 suppressed PDFG-BB-induced VSMC proliferation, migration, and secretion of inflammatory factors. In VSMCs, KCNQ1OT1 bound to the nuclear transcription factor kappa Ba (IκBa) protein and increased the cellular IκBa level by reducing phosphorylation and promoting ubiquitination of the IκBa protein. Meanwhile, KCNQ1OT1 promoted the expression of IκBa by sponging miR-221. The effects of KCNQ1OT1 knockdown on promoting VSMC proliferation, migration, and secretion of inflammatory factors were abolished by IκBa overexpression. The roles of KCNQ1OT1 in reducing the intimal area and inhibiting IκBa expression were proved in the VG mouse model after KCNQ1OT1 overexpression. In conclusion, KCNQ1OT1 attenuated intimal hyperplasia by suppressing the inflammation and proliferation of VSMCs, in which the mechanism upregulated IκBa expression by binding to the IκBa protein and sponging miR-221. Intimal hyperplasia is a common phenomenon that occurs in the process of artery remodeling after vascular injury, and it is often observed in the treatment of various vascular diseases, such as atherosclerosis, angioplasty, stent implantation, and bypass operation.1He M. Wang C. Sun J.H. Liu Y. Wang H. Zhao J.S. Li Y.F. Chang H. Hou J.M. Song J.N. et al.Roscovitine attenuates intimal hyperplasia via inhibiting NF-κB and STAT3 activation induced by TNF-α in vascular smooth muscle cells.Biochem. Pharmacol. 2017; 137: 51-60Crossref PubMed Scopus (12) Google Scholar,2Subbotin V.M. Excessive intimal hyperplasia in human coronary arteries before intimal lipid depositions is the initiation of coronary atherosclerosis and constitutes a therapeutic target.Drug Discov. Today. 2016; 21: 1578-1595Crossref PubMed Scopus (24) Google Scholar In response to vascular injury, vascular smooth muscle cells (VSMCs) can undergo increased inflammation, proliferation, and migration, as well as decreased expression of smooth muscle markers, developing into intimal hyperplasia.3Wang X.W. He X.J. Lee K.C. Huang C. Hu J.B. Zhou R. Xiang X.Y. Feng B. Lu Z.Q. MicroRNA-221 sponge therapy attenuates neointimal hyperplasia and improves blood flows in vein grafts.Int. J. Cardiol. 2016; 208: 79-86Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Therefore, inhibiting the proliferation and inflammation of VSMCs is a crucial step in delaying vein graft (VG) intimal hyperplasia, and research on the regulatory mechanism of the function and phenotype of VSMCs is desperately needed. Platelet-derived growth factor type BB (PDGF-BB) is a strong stimulant for VSMC proliferation and migration,4Lu Q.B. Wan M.Y. Wang P.Y. Zhang C.X. Xu D.Y. Liao X. Sun H.J. Chicoric acid prevents PDGF-BB-induced VSMC dedifferentiation, proliferation and migration by suppressing ROS/NFκB/mTOR/P70S6K signaling cascade.Redox Biol. 2018; 14: 656-668Crossref PubMed Scopus (112) Google Scholar and it is generally used in intimal hyperplasia-related study. Nuclear transcription factor κB (NF-κB) is a key transcription factor implicated in the control of inflammation, cell migration, proliferation, and apoptosis, and it is reported to be greatly involved in intimal hyperplasia.1He M. Wang C. Sun J.H. Liu Y. Wang H. Zhao J.S. Li Y.F. Chang H. Hou J.M. Song J.N. et al.Roscovitine attenuates intimal hyperplasia via inhibiting NF-κB and STAT3 activation induced by TNF-α in vascular smooth muscle cells.Biochem. Pharmacol. 2017; 137: 51-60Crossref PubMed Scopus (12) Google Scholar The inhibitors of NF-κB (IκB), including IκBa, IκBb, IκBg, and IκBe, comprise a protein family that binds with NF-κB to prevent its nuclear translocation.5Napetschnig J. Wu H. Molecular basis of NF-κB signaling.Annu. Rev. Biophys. 2013; 42: 443-468Crossref PubMed Scopus (646) Google Scholar Once the NF-κB-bound IκB is degraded, which may result from phosphorylation and the subsequent ubiquitination-dependent degradation, NF-κB is released and able to translocate to the nucleus; at this point, the NF-κB pathway is activated.6Mitchell S. Vargas J. Hoffmann A. Signaling via the NFκB system.Wiley Interdiscip. Rev. Syst. Biol. Med. 2016; 8: 227-241Crossref PubMed Scopus (542) Google Scholar NF-κB is a critical regulator of VSMC proliferation under inflammation.7Yang D. Sun C. Zhang J. Lin S. Zhao L. Wang L. Lin R. Lv J. Xin S. Proliferation of vascular smooth muscle cells under inflammation is regulated by NF-κB p65/microRNA-17/RB pathway activation.Int. J. Mol. Med. 2018; 41: 43-50PubMed Google Scholar It has been reported that the phosphorylation of IκB plays a key role in human VSMC proliferation,8Sasu S. Beasley D. Essential roles of IkappaB kinases alpha and beta in serum- and IL-1-induced human VSMC proliferation.Am. J. Physiol. Heart Circ. Physiol. 2000; 278: H1823-H1831Crossref PubMed Google Scholar and the overexpression of IκBa inhibits VSMC proliferation and intimal hyperplasia formation.9Zuckerbraun B.S. McCloskey C.A. Mahidhara R.S. Kim P.K. Taylor B.S. Tzeng E. Overexpression of mutated IkappaBalpha inhibits vascular smooth muscle cell proliferation and intimal hyperplasia formation.J. Vasc. Surg. 2003; 38: 812-819Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar However, the regulation of PDGF-induced downregulation of IκB is not fully elucidated. Interestingly, microRNA-221 (miR-221) has been identified to be transcriptionally induced upon PDGF treatment in primary VSMCs, and it is critical for cell proliferation,10Davis B.N. Hilyard A.C. Nguyen P.H. Lagna G. Hata A. Induction of microRNA-221 by platelet-derived growth factor signaling is critical for modulation of vascular smooth muscle phenotype.J. Biol. Chem. 2009; 284: 3728-3738Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar while the decrease in the expression and activity of miR-221 represses neointimal hyperplasia in VGs.3Wang X.W. He X.J. Lee K.C. Huang C. Hu J.B. Zhou R. Xiang X.Y. Feng B. Lu Z.Q. MicroRNA-221 sponge therapy attenuates neointimal hyperplasia and improves blood flows in vein grafts.Int. J. Cardiol. 2016; 208: 79-86Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Importantly, we predicted complementary bases between miR-221 and IκBa (encoded by the NFKBIA gene) using the bioinformatics method (TargetScan), implying potential binding between them. Therefore, we speculated that miR-221 may affect VSMC proliferation and intimal hyperplasia development by targeting IκBa. Long non-coding RNAs (lncRNAs), serving as the sponge of the miRNAs, have garnered extensive attention.11Liu Y. Wu B. Knockdown of Long Non-coding RNA TUG1 Suppresses Osteoblast Apoptosis in Particle-induced Osteolysis by Up-regulating BMP-7.Clinical Surgery Research Communications. 2018; 2: 19-25Google Scholar Increasingly, lncRNAs like RNCR312Shan K. Jiang Q. Wang X.Q. Wang Y.N. Yang H. Yao M.D. Liu C. Li X.M. Yao J. Liu B. et al.Role of long non-coding RNA-RNCR3 in atherosclerosis-related vascular dysfunction.Cell Death Dis. 2016; 7: e2248Crossref PubMed Scopus (166) Google Scholar and ANRIL13Congrains A. Kamide K. Oguro R. Yasuda O. Miyata K. Yamamoto E. Kawai T. Kusunoki H. Yamamoto H. Takeya Y. et al.Genetic variants at the 9p21 locus contribute to atherosclerosis through modulation of ANRIL and CDKN2A/B.Atherosclerosis. 2012; 220: 449-455Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar have been noted to play a role in regulating the VSMCs’ proliferation or growth. We used an online database (DIANA tools) to search for the candidate lncRNAs and found that lncRNA KCNQ1OT1 was predicted to have binding sites with miR-221. Meanwhile, by using RNA pull-down assay and mass spectrometry, we found that KCNQ1OT1 could bind with IκBa protein in VSMCs. Notably, KCNQ1OT1 is involved in cardiac development, and KCNQ1OT1 gene variants could be associated with the risk of developing long QT syndrome or a prolonged QT interval,14Coto E. Calvo D. Reguero J.R. Morís C. Rubín J.M. Díaz-Corte C. Gil-Peña H. Alosno B. Iglesias S. Gómez J. Differential methylation of lncRNA KCNQ1OT1 promoter polymorphism was associated with symptomatic cardiac long QT.Epigenomics. 2017; 9: 1049-1057Crossref PubMed Scopus (23) Google Scholar,15Jiang Y. Du W. Chu Q. et al.Downregulation of Long Non-Coding RNA Kcnq1ot1: An Important Mechanism of Arsenic Trioxide-Induced Long QT Syndrome.Cell Physiol Biochem. 2018; 45: 192-202Crossref PubMed Scopus (37) Google Scholar suggesting that KCNQ1OT1 may play a role in cardiovascular diseases. Taken together, we inferred that KCNQ1OT1 may regulate the expression of IκBa by binding the protein and targeting miR-221, resulting in the inflammation and proliferation of VSMCs and intimal hyperplasia pathogenesis. This study aimed to clarify this hypothesis and explore the impact of KCNQ1OT1 on intimal hyperplasia progression. First, the VG model was constructed in mice (VG, n = 25) to introduce the intimal hyperplasia. At 0, 1, 2, 3, and 4 weeks (n = 5 at each time point), detection on the intimal area indicated that the surface area was increased in a time-dependent manner (Figure 1A). At the same time, the VSMCs were isolated from the model mice at 0, 1, 2, 3, and 4 weeks, and it was interesting to find that the expression of KCNQ1OT1 in VSMCs declined in a time-dependent way (Figure 1B). We assumed that KCNQ1OT1 could be implicated in the pathogenesis of intimal hyperplasia. For investigating the expression level of KCNQ1OT1 during the proliferation of VSMCs, we used PDGF-BB to stimulate the VSMCs isolated from the normal mice. With the concentration of PDGF-BB increased in a gradient (0, 5, 10, and 20 ng/mL), the expression of KCNQ1OT1 at 48 h in VSMCs was reduced in a dose-dependent way (Figure 1C). In addition, when treated with PDGF-BB (10 ng/mL) for different durations (24, 48, and 72 h), the expression of KCNQ1OT1 in VSMCs was decreased in a time-dependent manner (Figure 1D). These data implied some relationship between KCNQ1OT1 expression and VSMC proliferation induced by PDGF-BB. To clarify the potential role of KCNQ1OT1 in affecting VSMC proliferation, we overexpressed KCNQ1OT1 in VSMCs treated with PDGF-BB (10 ng/mL) by transfecting the Ad-KCNQ1OT1 vector, with the Ad-GFP acting as the negative control. As shown in Figure 2A, the proliferation of VSMCs was promoted by PDGF-BB, but it was attenuated by KCNQ1OT1 overexpression. The cell migration enhanced by PDGF-BB was also almost abolished by Ad-KCNQ1OT1 transfection (Figure 2B). In VSMCs, the secretion of inflammatory factors, such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α), was markedly increased with PDGF-BB treatment, while overexpression of KCNQ1OT1 repressed the secretion of these inflammatory factors (Figure 2C). Collectively, we demonstrated that overexpression of KCNQ1OT1 abolished the effects of PDGF-BB on stimulating VSMCs proliferation, migration, and secretion of inflammatory factors. To explain the downregulation of KCNQ1OT1 in the VSMCs of mice with intimal hyperplasia and the PDGF-BB-treated VSMCs from normal mice, we determined the methylation level of its promoter. Compared with the VSMCs from the mice in the sham group (n = 7), the methylation level of KCNQ1OT1 promoter in the VSMCs of mice that underwent VG (n = 7) was notably elevated (Figure 3A). In the VSMCs isolated from normal mice, PDGF-BB treatment clearly augmented the methylation level in the promoter of KCNQ1OT1 (Figure 3B). However, the high level of methylation in the promoter of KCNQ1OT1 induced by PDGF-BB was diminished after DNA methyltransferase 1 (DNMT1) knockdown, while silencing of DNMT3a or DNMT3b had no influence on the methylation level (Figure 3C). Herein, we revealed that the downregulation of KCNQ1OT1 in the VSMCs of mice with intimal hyperplasia and the PDGF-BB-treated normal VSMCs could be attributed to the increased methylation level in its promoter. Next, we used RNA pull-down and mass spectrometry to screen for proteins that may bind to KCNQ1OT1 in VSMCs, and we found that the IκBa protein was expressed in the pull-down compounds (data not shown). Then, RNA pull-down and RNA immunoprecipitation (RIP) were employed to confirm the binding and the interaction between KCNQ1OT1 and IκBa protein. VSMCs were transfected with biotinylated KCNQ1OT1 or the antisense RNA probe, and RNA pull-down was carried out, followed by western blot with the IκBa antibody. Figure 3D validates the binding between biotinylated KCNQ1OT1 and IκBa rather than between the antisense RNA probe and IκBa; however, the binding between biotinylated KCNQ1OT1 and IκBa was reduced after PDGF-BB treatment. In the RIP assay, abundant KCNQ1OT1 was detected in the complex precipitated by the antibody against IκBa, suggesting the binding between KCNQ1OT1 and IκBa, which was diminished in cells treated with PDGF-BB (Figure 3E). Then, we illustrated that the level of IκBa protein was reduced in VSMCs treated with PDGF-BB; however, the overexpression of KCNQ1OT1 abolished this inhibitory effect on IκBa protein level (Figure 3F). These findings proved that KCNQ1OT1 upregulated the expression of IκBa protein by binding with it, while PDGF-BB lowered the IκBa level through downregulating KCNQ1OT1. Except for binding with IκBa protein, the other pathways by which KCNQ1OT1 regulated the IκBa level were also explored. Via transfecting si-KCNQ1OT1 in VSMCs, KCNQ1OT1 expression was inhibited and p-IκBa was upregulated, whereas IκBa was significantly downregulated (Figure 3G), indicating the increased phosphorylation level of IκBa. In contrast, overexpression of KCNQ1OT1 boosted the p-IκBa expression but decreased the expression of IκBa (Figure 3H), suggesting that the phosphorylation level of IκBa was lowered. It could be deduced that the expression of KCNQ1OT1 inhibited the phosphorylation of IκBa. Afterward, we illustrated that knockdown of KCNQ1OT1 promoted the ubiquitination of IκBa protein (Figure 3I), and the CHX assay further verified that KCNQ1OT1 downregulation facilitated the degeneration of IκBa protein (Figure 3J). Taken together, it could be elucidated that KCNQ1OT1 restrained the ubiquitination-dependent degradation of IκBa protein. Via bioinformatics software, the binding sites of miR-221 (miR-221-3p) on the IκBa 3′ untranslated region (3′ UTR) were predicted (TargetScan), as well as the complementary bases between KCNQ1OT1 and miR-221 (DIANA tools; Figure 4A). There was a total of 25 binding sites between KCNQ1OT1 and miR-221, and Figure 4A showed the site at 32403-32427. By co-transfecting pGL3-KCNQ1OT1-WT (wild-type) or pGL3-KCNQ1OT1-mut (mutant) and the miR-221 mimic into 293 cells, we found that overexpression of miR-221 inhibited the luciferase activity in KCNQ1OT1-WT-transfected cells, while it had no influence on that in KCNQ1OT1-mut-transfected cells (Figure 4B). The RNA-binding protein AGO2 participates in the maturation of miRNAs and mediates the binding between miRNAs and their target RNAs. The result of the RIP assay using the anti-AGO2 showed that AGO2 could bind with KCNQ1OT1 and miR-221 in VSMCs (Figure 4C). Moreover, overexpression of KCNQ1OT1 in VSMCs downregulated miR-221 (Figure 4D) but increased the expression of IκBa protein (Figure 4E). Knockdown of KCNQ1OT1 in VSMCs suppressed the IκBa protein level, which was reversed by miR-221 inhibition (Figure 4F); in contrast, the upregulation of IκBa protein caused by KCNQ1OT1 overexpression was reversed by miR-221 expression (Figure 4G). These data proved that KCNQ1OT1 sponged miR-221 to elevate the expression of its target, IκBa. For exploring the mechanism by which KCNQ1OT1 affected VSMC proliferation, migration, and inflammation, knockdown of KCNQ1OT1 and simultaneous upregulation of IκBa were performed in VSMCs. VSMCs were allocated into several groups, which were as follows: si-control, si-KCNQ1OT1-1, si-KCNQ1OT1-1+pcDNA-IκBa, si-KCNQ1OT1-2, and si-KCNQ1OT1-2+pcDNA-IκBa. We illustrated that silencing of KCNQ1OT1 in VSMCs promoted cell proliferation (Figure 5A) and migration (Figure 5B), which were largely abolished with IκBa overexpression. In addition, interference of KCNQ1OT1 enhanced the secretion of inflammatory factors in VSMCs, including IL-1β, IL-6, and TNF-α, but the secretion of inflammatory factors was inhibited by overexpression of IκBa (Figure 5C). The results revealed that KCNQ1OT1 suppressed VSMC proliferation, migration, and secretion of inflammatory factors via upregulating IκBa. Finally, we validated the function of KCNQ1OT1 in affecting intimal hyperplasia in mice undergoing VG. Figure 6A showed that the thickness and surface area of the intima was reduced in mice injected with lentivirus vector expressing KCNQ1OT1 (Lv-KCNQ1OT1) (n = 7) compared with those injected with empty vector (n = 7). The upregulation of KCNQ1OT1 in VSMCs isolated from the Lv-KCNQ1OT1-injected mice was verified (Figure 6B). Furthermore, the expression of miR-221 was reduced (Figure 6C), while p-IκBa protein was reduced and IκBa protein was increased after overexpressing KCNQ1OT1 (Figure 6D). The overexpression of KCNQ1OT1 also reduced the mRNA levels of IL-1β, IL-6, and TNF-α (Figure 6E), suggesting that KCNQ1OT1 overexpression could attenuate the inflammatory response. These findings demonstrated that overexpression of KCNQ1OT1 attenuated intimal hyperplasia in mice undergoing VG. In this study, we considered the regulatory mechanism of lncRNA KCNQ1OT1 on the inflammation and proliferation of VSMCs and investigated its influence on the development and progression of intimal hyperplasia. The results revealed that KCNQ1OT1 upregulates IκBa, suppressing the inflammation and proliferation of VSMCs; this results in a reduced intimal area (Figure 6F). Our study provides a promising therapeutic target for the treatment of VG intimal hyperplasia. VSMCs, which have a major presence in the media of vessels, are the dominant cellular constituents of arteries and the crucial determinants in vascular disorders.16Sun H.J. Zhao M.X. Ren X.S. Liu T.Y. Chen Q. Li Y.H. Kang Y.M. Wang J.J. Zhu G.Q. Salusin-β Promotes Vascular Smooth Muscle Cell Migration and Intimal Hyperplasia After Vascular Injury via ROS/NFκB/MMP-9 Pathway.Antioxid. Redox Signal. 2016; 24: 1045-1057Crossref PubMed Scopus (80) Google Scholar In response to vascular injury, VSMCs will engage in phenotypic regulation featuring augmented inflammation, proliferation, and migration; thus, they form a neointima.1He M. Wang C. Sun J.H. Liu Y. Wang H. Zhao J.S. Li Y.F. Chang H. Hou J.M. Song J.N. et al.Roscovitine attenuates intimal hyperplasia via inhibiting NF-κB and STAT3 activation induced by TNF-α in vascular smooth muscle cells.Biochem. Pharmacol. 2017; 137: 51-60Crossref PubMed Scopus (12) Google Scholar Proinflammatory cytokines are pivotal regulators of arterial inflammation and intimal hyperplasia, and they participate in pathological vascular remodeling.17Li Y.Q. Wang J.Y. Qian Z.Q. Li Y.L. Li W.N. Gao Y. Yang D.L. Osthole inhibits intimal hyperplasia by regulating the NF-κB and TGF-β1/Smad2 signalling pathways in the rat carotid artery after balloon injury.Eur. J. Pharmacol. 2017; 811: 232-239Crossref PubMed Scopus (16) Google Scholar The present study demonstrated that PDGF-BB induced VSMC proliferation and migration, accompanied by increased secretion of inflammatory factors, including IL-1β, IL-6, and TNF-α, which was consistent with the previous study by Pi et al.18Pi Y. Zhang L.L. Li B.H. Guo L. Cao X.J. Gao C.Y. Li J.C. Inhibition of reactive oxygen species generation attenuates TLR4-mediated proinflammatory and proliferative phenotype of vascular smooth muscle cells.Lab. Invest. 2013; 93: 880-887Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar However, these noticeable facilitating effects on the VSMC proliferative phenotype and inflammation were largely abolished by KCNQ1OT1 overexpression, implying an enormous potential of KCNQ1OT1 as an effective target in the treatment of intimal hyperplasia. Abnormal expression of imprinted genes is often associated with various human disorders with complex mutations and phenotypic defects. As an imprinted gene, KCNQ1OT1 is generally methylated in its promoter, which has been proven to be associated with embryonic developmental failure19Khoueiry R. Ibala-Romdhane S. Al-Khtib M. Blachère T. Lornage J. Guérin J.F. Lefèvre A. Abnormal methylation of KCNQ1OT1 and differential methylation of H19 imprinting control regions in human ICSI embryos.Zygote. 2013; 21: 129-138Crossref PubMed Scopus (16) Google Scholar and the risk of symptomatic cardiac long QT.14Coto E. Calvo D. Reguero J.R. Morís C. Rubín J.M. Díaz-Corte C. Gil-Peña H. Alosno B. Iglesias S. Gómez J. Differential methylation of lncRNA KCNQ1OT1 promoter polymorphism was associated with symptomatic cardiac long QT.Epigenomics. 2017; 9: 1049-1057Crossref PubMed Scopus (23) Google Scholar In the current study, the downregulation of KCNQ1OT1 in the VSMCs from mice with intimal hyperplasia and in the PDGF-BB-induced VSMCs was attributed to the increase of methylation level at its promoter, indicating some inner link between KCNQ1OT1 expression and intimal hyperplasia pathogenesis. Except in the abnormal expression change of KCNQ1OT1 itself, it is devoted to modulating the expression of ubiquitous genes to participate in diverse biological processes. For example, Mohammad et al.20Mohammad F. Mondal T. Guseva N. Pandey G.K. Kanduri C. Kcnq1ot1 noncoding RNA mediates transcriptional gene silencing by interacting with Dnmt1.Development. 2010; 137: 2493-2499Crossref PubMed Scopus (225) Google Scholar found that KCNQ1OT1 mediates transcriptional gene silencing by interacting with the DNMT1, and they confirmed the direct interaction between KCNQ1OT1 and DNMT1 via RIP assay. Interestingly, our study showed that the knockdown of DNMT1 reduced the methylation level of the KCNQ1OT1 promoter, affecting the KCNQ1OT1 expression. These data indicated that there may be feedback regulation between KCNQ1OT1 and DNMT1, although more evidence on this is still needed. Another study conducted by Chen et al.21Chen B. Ma J. Li C. Wang Y. Long noncoding RNA KCNQ1OT1 promotes proliferation and epithelial-mesenchymal transition by regulation of SMAD4 expression in lens epithelial cells.Mol. Med. Rep. 2018; 18: 16-24PubMed Google Scholar indicated that KCNQ1OT1 promotes lens epithelial cell proliferation and the epithelial-mesenchymal transition via upregulating SMAD4. Although they did not elucidate the specific interactions between KCNQ1OT1 and SMAD4, they determined that KCNQ1OT1 positively regulated SMAD4 at both the mRNA and protein levels. Consistent with their study, our research explains the regulation of KCNQ1OT1 on IκBa expression at both the mRNA and protein levels. On the one hand, KCNQ1OT1 directly binds to IκBa protein, which is similar to its effect on β-catenin protein,22Gao X. Ge J. Li W. Zhou W. Xu L. LncRNA KCNQ1OT1 promotes osteogenic differentiation to relieve osteolysis via Wnt/β-catenin activation.Cell Biosci. 2018; 8: 19Crossref PubMed Scopus (55) Google Scholar to restrain the phosphorylation and ubiquitination-dependent degradation of IκBa protein. On the other, KCNQ1OT1 functions as a competing endogenous RNA (ceRNA) of miR-221, leading to the upregulation of its target gene, IκBa. The influence of miR-221 on VSMC proliferation and intimal hyperplasia was previously reported.3Wang X.W. He X.J. Lee K.C. Huang C. Hu J.B. Zhou R. Xiang X.Y. Feng B. Lu Z.Q. MicroRNA-221 sponge therapy attenuates neointimal hyperplasia and improves blood flows in vein grafts.Int. J. Cardiol. 2016; 208: 79-86Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar,10Davis B.N. Hilyard A.C. Nguyen P.H. Lagna G. Hata A. Induction of microRNA-221 by platelet-derived growth factor signaling is critical for modulation of vascular smooth muscle phenotype.J. Biol. Chem. 2009; 284: 3728-3738Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar However, it is surprising that we identified IκBa as a novel target of miR-221 in this study, which further elucidates the action mechanism of miR-221 contributing to VSMC proliferation and intimal hyperplasia occurrence. NF-κB is a key transcription factor that is implicated in the control of inflammation, cell migration, proliferation, and apoptosis; hence, its inhibitory subunit IκBa is also widely focused on. Recently, it has been claimed that phosphorylation and degradation of IκBa abolishes PDGF-BB-evoked NF-κB nuclear translocation and influences VSMC phenotypic switching, proliferation, migration, and neointima formation.4Lu Q.B. Wan M.Y. Wang P.Y. Zhang C.X. Xu D.Y. Liao X. Sun H.J. Chicoric acid prevents PDGF-BB-induced VSMC dedifferentiation, proliferation and migration by suppressing ROS/NFκB/mTOR/P70S6K signaling cascade.Redox Biol. 2018; 14: 656-668Crossref PubMed Scopus (112) Google Scholar VSMC migration and vascular injury-induced intimal hyperplasia are also accompanied by IκBa phosphorylation and degradation.16Sun H.J. Zhao M.X. Ren X.S. Liu T.Y. Chen Q. Li Y.H. Kang Y.M. Wang J.J. Zhu G.Q. Salusin-β Promotes Vascular Smooth Muscle Cell Migration and Intimal Hyperplasia After Vascular Injury via ROS/NFκB/MMP-9 Pathway.Antioxid. Redox Signal. 2016; 24: 1045-1057Crossref PubMed Scopus (80) Google Scholar Our study demonstrated that the increase of IκBa induced by KCNQ1OT1 suppresses VSMC proliferation, migration, and secretion of inflammatory factors, further confirming the intrinsic function of IκBa in controlling VSMC proliferation, migration, and inflammation via the inhibition of NF-κB activation. Taken together, the study results highlight the significance and mechanism of KCNQ1OT1 involved in the VSMC proliferative phenotype and inflammation, and the subsequent intimal hyperplasia, which provides a novel insight into the prevention and treatment of vascular inflammatory disorders following VG. Animal housing and procedures were approved by the local Animal Care and Use Committee at the First Affiliated Hospital of Wenzhou Medical University and complied with humane animal care standards. VG surgeries were performed with a VG introduced via end-to-end anastomoses.23Cooley B.C. Nevado J. Mellad J. Yang D. St Hilaire C. Negro A. Fang F. Chen G. San H. Walts A.D. et al.TGF-β signaling mediates endothelial-to-mesenchymal transition (EndMT) during vein graft remodeling.Sci. Transl. Med. 2014; 6: 227ra34Crossref PubMed Scopus (269) Google Scholar Prior to surgery, all the mice were anesthetized with intraperitoneal pentobarbital (50 mg/kg). After an aseptic incision made in the ventral neck, the posterior facial branch of the jugular vein was exposed and veins were harvested from donor mice and placed in isotonic saline. For recipient mice, the femoral arteries were aseptically exposed through an inguinal incision; they were then dissected and occluded temporarily using nontraumatic clamps. After resection of a small portion of the artery, a VG was introduced via end-to-end anastomoses using 8–10 interrupted stitches of 11-0 nylon suture for each anastomosis. After blood flow was restored, the clamps were removed and skin wounds were closed using 5-0 nylon suture. Heparin (100 U/mL) in 0.2 mL of saline was used locally during the grafting procedure. Patency was verified 3 days following grafting. Five mice each were sacrificed at the time points of 0, 1, 2, 3, and 4 weeks after VG. The intimal surface area was calculated by subtracting t" @default.
• W3005471014 created "2020-02-14" @default.
• W3005471014 creator A5000419989 @default.
• W3005471014 creator A5007883693 @default.
• W3005471014 creator A5014743742 @default.
• W3005471014 creator A5029579190 @default.
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• W3005471014 date "2020-06-01" @default.
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• W3005471014 title "lncRNA KCNQ1OT1 Suppresses the Inflammation and Proliferation of Vascular Smooth Muscle Cells through IκBa in Intimal Hyperplasia" @default.
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