| BACKGROUND AND OBJECTIVEBased on the results of genomic sequencing, there are only 2% genes could translate to proteins, and the rest 98% genes transcribed to non-coding RNAs. Among them, long non-coding RNAs (lncRNAs) attracted continuous attentions. LncRNAs are classified as RNA transcripts greater than 200 nucleotides in length that do not code for a functional protein. The proportion of lncRNAs in mammal transcripts is 4%-9%. According to the recent researches, lncRNAs play important roles in regulating gene transcription, epigenetics, stem cells differentiation and other biological processes.In our previous studies, by using a new chemical small molecule 3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one (3BDO), we identified a new lncRNA, TGFB2-OT1 derived from 3’UTR of transforming growth factor beta 2 (TGFB2). Because of the specific location, we cannot specifically knockdown TGFB2-OT1 expression by tranditional genetics methods. Our previous results showed that 3BDO could inhibit TGFB2-OT1 production by promoting T-cell restricted intracellular antigen-1 (TIAl) phosphorylation, thus suggesting TIA1 acted as the key modulator of TGFB2-OT1 expression, and 3BDO as the powerful tool for studying TGFB2-OT1 functions. Furthermore,3BDO could inhibit promotion of nuclear protein, transcriptional regulator,1 (NUPR1) levels and vascular endothelial cells (VECs) injury induced by lipopolysaccharides (LPS). As the important transcriptional regulator, we speculated NUPRl might participate in TIA1 expression regulation, and then affecting TGFB2-OT1 production. In this dissertation, we would prove this speculation.Whereas the degree of TIA1 phosphorylation was the key to control TGFB-OT1 expression, so identifying the factors directly affecting TIA1 phosphorylation was another way to regulate TGFB2-OT1 expression, but there is no report on the inhibitor of TIA1 phosophorylation. In this dissertation, we would solve this important issue.In previous study, utilizing yeast two-hybrid and co-immunoprecipitation techniques, we identified annexin A7 (ANXA7) interacted with TIA1. Simultaneously, we found a chemical small molecule 6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (ABO) could target ANXA7, inhibit ANXA7 GTPase activity and phosphorylation of its interacting proteins in vascular endothelial cells. These findings indicated ANXA7 might be the regulator of TIA1 phosphorylation. Therefore, in this study, we would prove our speculation using chemical small molecules 3BD0 and ABO as the tools.Vascular endothelial cells (VECs) are closely contact with blood in our body and comprise the morphological basis of cardiovascular system. VECs dysfunction is tightly related with the occurrence and progression of various cardiovascular diseases, and inflammatory factors stimulation could induce VECs dysfunction. Activation of the endothelium by inflammatory mediators leads to the recruitment of inflammatory cells, which drives atherosclerotic plaque formation and progression. Therefore, identified new factors regulating VECs inflammatory response has an important significance in cardiovascular diseases therapy.The involvement of lncRNAs in VECs biology is just beginning to be explored, and the research of lncRNAs in inflammation is focused on other cell types, such as macrophages. Therefore, so far the role of lncRNAs in regulating VECs inflammation is unclear. Our results found that TGFB2-OT1 could act as competing endogenous RNAs (ceRNAs), binding with MIR4459, and promoted autophagy by increasing ATG13 levels. Further studies showed that 3BDO could inhibit the production of inflammatory factors induced by oxidized low-density lipoprotein (oxLDL) in vitro and in vivo. Autophagy and inflammation are closely related processes. Autophagy could modulate inflammatory responses and inflammatory related factors could regulate autophagy. Thus, we speculated TGFB2-OT1 participated in inflammatory response in VECs. In this study, to prove this speculation, we profoundly studied-the role and molecular mechamism of TGFB2-OT1 in inflammation and autophagy of VECs.In conclusions, this study aimed to investigate the key factors in regulating lncRNA TGFB2-OT1 expression and its biological functions in VECs. By using the chemical small molecules, we explored the expression regulation mechanism and the role of TGFB2-OT1 in autophagy and inflammation of VECs, and identified the newly key factors and signal pathway participated in this process, thus provided new targets and effective tools for clinical treatment of cardivascular diseases.STUDY CONTENTS1. To determine the expression regulation mechanism of TGFB2-OT1 by NUPR1 and TIA1.2. To investigate the interaction and mechanism between ANXA7 and TIA1 in regulating TGFB2-OT1 expression.3. To investigate the role of lncRNA TGFB2-OT1 in inflammatory response of VECs.4. To study the molecular mechanism of TGFB2-OT1 in regulating autophagy and inflammatory response of VECs.METHODS1. We used human umbilical vein endothelial cells (HUVECs), human embryonic kidney 293 cells (HEK293 cells) as the cell model, apoE-/-mice as the animal model, and used chemical small molecule 3BDO and ABO as the tools, to do the related study.2. Detection the expression regulation of TGFB2-OT1 by NUPR1 and TIA1:2.1 In situ hybrization and quantitative real-time PCR analysis the effect of LPS treatment on TGFB2-OT1 expression with or without 3BDO in HUVECs.2.2 Western blot analysis the effect of LPS on TIA1 protein level, and the effect of oxLDL on NUPR1 and TTA1 protein levels.2.3 RNA interference, Western blot and quantitative real-time PCR analysis the relationship between NUPR1 and TIA1 in regulating TGFB2-OT1 expression.3. Detection the interaction and modulation between ANXA7 and TIA1:3.1 We treated HUVECs with ABO in a time course, and extracted the cellular protein. Co-immunoprecipitation assay analyzed the effect of ABO on the interaction between ANXA7 and TIA1.3.2 We treated HUVECs with ABO in a time course, and TIA1 siRNA treatment to confirm the specificity of the p-Ser band. Immunoprecipitation assay determinined the effect of ABO on TIA1 phosphorylation level.3.3 We transfected HUVECs with ANXA7 siRNA, and then treated with ABO. Immunoprecipitation assay determinined the effect of ANXA7 on TIA1 phosphorylation level.4. Detection the expression regulation of TGFB2-OT1 by ANXA7 and TIA1:4.1 Real-time PCR analysis the level of TGFB2-OT1 by ABO treatment in HUVECs.4.2 Western blot analysis of ATG13 protein level by ABO treatment in HUVECs.4.3 RNA interference, Western blot and immunofluorescence assay analyzed the autophagy levels induced by ABO when TIA1 knockdown.4.4 In HUVECs, Western blot and immunofluorescence assay analyzed the LC3-II protein level and localization in cells to determine the autophagy level by co-treatment with 3BD0 and ABO.5. Confirmation ANXA7 and TIA1 action model in vivo:5.1 ApoE"’" mice were fed with high-fat diet for 14 weeks, and to build the atherosclerosis plaque model, and then intraperitoneal injections of with ABO (50 mg/kg/day) for 8 weeks, equal DMSO as control.5.2 The frozen tissue sections of thoracic aorta were used in double-immunofluorescence assay to determine the co-localization between ANXA7 and TIA1 in CD31 positive endothelium.5.3 The frozen tissue sections of thoracic aorta were used in immunofluorescence assay with ATG13 antibody in CD31 positive endothelium.6. Identification new miRNAs binding with TGFB2-OT1:6.1 MiRNA array analysis of miRNA regulated by TGFB2-OT1.6.2 Bioinformatics prediction of miRNA could bind with TGFB2-OT1.6.3 Compare miRNA array and prediction results, identify new miRNA, and quantitative real-time PCR confirm the results.6.4 We re-combined the TGFB2-OT1 cDNA and mutational cDNA with the presumed MIR3960 or MIR4488 recognition sequences deleted downstream of the iuciferase reporter gene. Transfection miRNA mimics with TGFB2-OT1 wild type or mutational plasmids into HEK293 cells, and detect the luciferase activity to confirm the direct binding between MIR3960 or MIR4488 and TGFB2-OT1.7. Confirmation of MIR3960 and MIR4488 target genes:7.1 Bioinformatics prediction the target genes of MIR3960 and MIR4488.7.2 Transfection MIR3960 or MIR4488 mimics and inhibitor into HUVECs, and determine the effect on the mRNA and protein levels of target genes.7.3 We cloned the 3’UTRs of CERS1 and NAT8L within the predicted binding sites of MIR3960 or MIR4488 into the luciferase reporter vector and transfected the vectors into HEK293 cells with the corresponding MIR3960 or MIR4488 mimics. The mearsurement of luciferase activity confirmed the direct binding of CERS1 and NAT8L with MIR3960 and MIR4488.7.4 Western blot and overexpression assay analysis to detect the effect of TGFB2-OT1 on the target genes CERS1 and NAT8L.8. Detection the role of MIR4459 target LARP1 in VECs inflammation:8.1 MIR4459 mimics transfection with HUVECs, and Western blot analysis the protein level of LARP1.8.2 Western blot and overexpression assay analysis to detect the effect of TGFB2-OT1 on LARP1.8.3 Western blot analysis the protein level of SQSTM1 with MIR4459 mimics transfection.8.4 Overexpression technology, Western blot and quantitative real-time PCR analysis of the protein level of cleaved Caspase land ILIB mRNA level.9. Detection the role of non-related lncRNA AF007131 in inflammation:9.1 Overexpression technology, Western blot and immunofluorescence analysis the expression of SQSTM1 with AF007131 overexpression.9.2 Overexpression technology and immunofluorescence analysis of NFKB RELA localization in HUVECs with AF007131 overexpression.RESULTS1. NUPR1 induced TGFB2-OT1 expression by promoting TIA1 protein level.1.1 In situ hybrization and quantitative real-time PCR results showed that LPS treatment could induce TGFB2-OT1 levels in a time and dose course,3BDO inhibited the promotion of TGFB2-OT1 levels induced by LPS.1.2 In HUVECs, LPS and oxLDL treatment increased the protein levels of NUPR1 and TIA1, which was inhibited by 3BD0.1.3 In HUVECs, NUPR1 or TIA1 knockdown significantly inhibited the TGFB2-OT1 levels promotion induced by LPS.1.4 When NUPR1 was knocked down, LPS did not increase the TIA1 level in HUVECs.2. ABO promoted the interaction of ANXA7 and TIA1, and inhibited TIA1 phosphorylation.2.1 ABO treatment for 2 h and 3 h could greatly promote the interaction between ANXA7 and TIA1 in HUVECs.2.2 ABO treatment for 3 h significantly decreased TIA1 phosphorylation at serine residues. When ANXA7 knockdown, the decline of TIA1 phosphorylation induced by ABO was suppressed.2.3 When TIA1 knockdown, ABO treatment for 24 h could not increase the level of LC3-Ⅱ and the puncta of LC3B, decrease the level of SQSTM1, indicating TIA1 interacting with ANXA7 involved in autophagy induced by ABO.3. The interaction between ANXA7 and TIA1 promoted TGFB2-OT1 production.3.1 ABO treatment for 12 h and 24 h in HUVECs significantly increased TGFB2-OT1 level and ATG13 expression.3.2 In HUVECs,3BDO co-treatment with ABO could inhibit the increase of LC3-Ⅱ protein level and LC3B puncta induced by ABO.4. ABO could increase the co-localization of ANXA7 and TIA1 and ATG13 levels in the endothelium of apoE-/ mice.4.1 The double-immunofluorescent experiment result showed that ABO treatment could increase the co-localization of ANXA7 and TIA1 in the endothelium of apoE-/- mice as compared with controls.4.2 The aortic roots of ABO-treated groups showed significantly increased staining for ATG13 in endothelium as compared with controls.5. TGFB2-OT1 could directly bind with MIR3960 and MIR4488,5.1 We performed miRNA microarray assay to identify miRNA with changed expression when TGFB2-OT1 was up-or downregulated. The bioinformatics analysis revealed that 57 presumed miRNAs could recognize and bind to TGFB2-OT1. We considered both bioinformatics analysis and microarray assay results and identified the top 5 miRNAs. We further chose MIR3960 and MIR4488 for further research by screening expression abundance and miRNA target genes.5.2 We verified the miRNA microarray results by quantitative real-time PCR. TGFB2-OT1 downregulation by 3BDO increased and TGFB2-O1 overexpression decreased the levels of MIR3960 and MIR4488.5.3 With MIR3960 or MIR4488 mimics transfection, luciferase activity was reduced by about 60% and 40% respectively, as compared with control miRNA. When adding mutant substrates for MIR3960 and MIR4488, the decreased luciferase activity was abolished.6. TGFB2-OT1 regulated the level of CERS1 and NAT8L by binding with MIR3960 and MIR4488.6.1 HUVECs were transfected with MIR3960 and MIR4488 mimics or inhibitor. The efficiency of miRNA mimics and inhibitor was determined by quantitative real-time PCR. MiRNA mimics and the inhibitor at 25 and 50 nM effectively increased or decreased the corresponding miRNA level, respectively. We further determined the mRNA level of miRNA targets by transfecting HUVECs with miRNA mimics or inhibitor. MiRNA mimics decreased the corresponding target mRNA levels, and miRNA inhibitor increased the corresponding target mRNA levels. Furthermore, MIR3960 and MIR4488 mimics at 50 nM significantly decreased the protein levels of CERS1 and NAT8L. Moreover, the MR3960 and MIR4488 inhibitor increased the protein levels.6.2 The luciferase activity of Luc-CERS1-3’UTR was significantly decreased by MIR3960 mimics. The luciferase activity of Luc-NAT8L-3’UTR was significantly decreased by MIR4488 mimics.6.3 In HUVECs, TGFB2-OT1 downregulation by 3BDO decreased the mRNA and protein levels of CERS1and NAT8L, whereas TGFB2-OT1 overexpression in HEK293 cells increased the protein levels of CERS1 and NAT8L.7. TGFB2-OT1 regulated protein synthesis and inflammatory response through MIR4459 and LARP1.7.1 In HUVECs, MIR4459 transfection could decrease the protein levels of LARP1 and SQSTM1.7.2 In HUVECs, TGFB2-OT1 downregulation by 3BDO decreased the mRNA and protein levels of LARP1 in dose-dependent course, whereas TGFB2-OTI overexpression in HEK293 cells increased the protein levels of CERS1 and NAT8L.7.3 TGFB2-OTI overexpression could increase cleaved Caspase 1 protein level and IL1B mRNA level.3BDO treatment could inhibit the increase of IL1B mRNA level.CONCLUSIONS1. By using inflammation inducers LPS and oxLDL, NUPR1 could increase TIA1 protein level and then promote TGFB2-OT1 expression, indicating that NUPR1 and TIA1 participated in regulating TGFB2-OT1 expression.2. The interaction of ANXA7 and TIA1 suppressed TIA1 phosphorylation, and then promoted TGFB2-OT1 processing, indicating by affecting TIA1 phosphorylation, ANXA7 participated in TGFB2-OT1 expression regulation with TIA1.3. TGFB2-OT1 acted as competing endogenous RNA, could bind with MIR3960 and MIR4488, increased CERS1 and NAT8L levels, and then promoted autophagy.4. TGFB2-OT1 could bind with MR4459, increased the target LARP1 level, promoted cleaved Caspase 1 and IL1B mRNA level through SQSTM1, and then induced inflammation. |
