| Objective:Atherosclerosis（AS）is one of the most common pathological diseases of arteries,which is the important pathophysiological basis of coronary heart disease（CHD）.AS is a vicious circulatory system disease caused by the long-term interaction of multiple factors,although many theories have been formed after years of research and exploration,the mechanism of AS has not been fully elucidated until now.HGP（human genome project）found that more than 98%of mammalian genes were transcribed into non-coding RNAs while less than 2%genes were transcribed into proteins.Long non-coding RNAs（lncRNAs）is a kind of regulatory RNA with a length of more than 200 nt and no protein encoding function.lncRNA taurine up-regulated gene 1（TUG1）is located on chromosome 22q12.2,7.1 kb in length,which was initially detected in taurine-treated mouse retinal cells.Recent studies showed that lncRNA TUG1 could participate in the progression of cardiovascular diseases.Nevertheless,the molecular mechanism by which lncRNA TUG1 regulates the progression of AS is yet to known,especially by far still no report on whether it can regulate autophagy and inflammation response to play the role of anti-AS.Thus,to know more about the pathogenesis of AS,we take the peripheral blood of patients with CHD,human umbilical vein endothelial cells（HUVECs）and AS rats as models in the research,to study the effects and molecular mechanism of lncRNA TUG1 in AS.Method:Part of autophagy:Firstly,quantitative real time-PCR（q RT-PCR）was used to detect the expression of TUG1 in the blood of thirty-five patients with CHD and thirty-eight controls without CHD.After cells were transfected with small interfering of TUG1（si-TUG1）,Cell Counting Kit-8（CCK-8）,5-Ethynyl-2’-deoxyuridine（Ed U）,and wound healing assay were performed to detect the proliferation and migration ability of HUVECs cells;m RFP-GFP-LC3 and Western blot were used to detect the expression of autophagosomes,and the protein levels of p62 and LC3,respectivelly.q RT-PCR was used to detect TUG1 expression levels after cells were incubated with metformin.Then,TUG1 overexpression plasmid was transfected into cells to observe its rescue effect of metformin on cells,AMPK inhibitor（Compound C）was used to observe the rescue effect of si-TUG1 on cells,then analyze the molecular mechanism of TUG1and the therapeutic effect of metformin on cells.Wistar rats were used to induce AS.After 30 days,40μl of TUG1(1.1×1013V.G/ml)adeno-associated virus or empty vector was injected into the corresponding rats through sublingual vein.In addition,metformin group was given metformin of100 mg/kg/day,positive control group was given 2.1 mg/kg/day atorvastatin,the control group（without AS）and AS model group were given the same volume of normal saline,all for 30 days.Rats were then sacrificed for the following analysis:HE staining was performed to calculate the lesion area;q RT-PCR was used to detect the expression of TUG1 in each group;immunohistochemistry was then performed to measure the levels of p62 and LC3;Western blot was used to measure the protein levels of AMPK/mTOR signaling pathway.Part of inflammation:q RT-PCR was performed to detect the m RNA levels of TUG1 in cells incubated with different concentrations of ox-LDL,the levels of miR-140-3p and IL-8 in the peripheral blood of patients with CHD.q RT-PCR and Western blot were used to detect the levels of IL-8,MMPs,and miR-140-3p in cells incubated with ox-LDL or transfected with si-TUG1;and the levels of IL-8,MMPs in cells transfected with miR-140-3p-inhibitor or corresponding-mimics.The luciferase reporter assay was used to detect the binding site between TUG1 and miR-140-3p,miR-140-3p and IL-8.To make clear the effect of TUG1 on inflammatory response via miR-140-3p,and miR-140-3p via IL-8,rescue experiments were performed.Western blot and ELISA were used to detect the protein expression of IL-8,MMPs in cells and supernatant,respectively.The arterial tissues in the control group（no AS）,AS model group,sh-NC group,sh-TUG1 group were collected for the following experiments:immunohistochemistry was used to detect the expression of IL-8,MMPs;fluorescence in situ hybridization was used to detect the expression of miR-140-3p.Results:Part of autophagy:The results of q RT-PCR showed that the expression level of TUG1 in the peripheral blood of patients with CHD was significantly higher than that of the controls.After transfected with si-TUG1,the proliferation and migration ability of HUVECs decreased,while the autophagy ability of HUVECs increased.After metformin administration,the expression of TUG1 in HUVECs decreased in a time-dependent manner.Besides,after metformin incubation,the proliferation and migration abilities of HUVECs decreased,while the autophagy ability of HUVECs increased.The results of rescue experiment indicated that silencing of lncRNA TUG1promotes autophagy via AMPK/mTOR signaling pathway,and metformin could activeate AMPK/mTOR pathway via lncRNA TUG1.HE staining suggested that compared with the control group,the lesion area in the AS model group and the sh-NC group were obviously increased,indicating the success of AS model induction.The lesion area in the metformin group and the sh-TUG1 group were significantly reduced compared with that of AS model group or the sh-NC group,suggesting that metformin and sh-TUG1 have protective effects on high-fat-diet induced AS injury.Through q RT-PCR,we demonstrated that compared with the control group,the level of TUG1 in the AS model group and the sh-NC group was upregulated;besides,compared with the AS model group or the sh-NC group,the levels of TUG1 decreased in the metformin group,sh-TUG1 group and metformin+sh-TUG1 group.The results of immunohistochemistry and Western blot indicated that compared with the control group,the AS model group has some certain of autophagy defects;compared with the AS model group or sh-NC group,metformin and sh-TUG1 could promote autophagy via activating the AMPK/mTOR signaling pathway.Part of inflammation:The results of q RT-PCR and Western blot showed that:after ox-LDL incubation,the levels of TUG1 in HUVECs cells upregulated in a concentration-dependent manner;the levels of IL-8,MMP-2,and MMP-9 upregulated,while the level of miR-140-3p downregulated obviously.After cells were transfected with si-TUG1,the results were contray to the results of ox-LDL incubation.Furthermore,the m RNA levels of IL-8、MMP-2 and MMP-9 upregulated in cells transfected with miR-140-3p-inhibitor.Besides,the results of q RT-PCR showed that compared with the control group,the expression of miR-140-3p in the peripheral blood of patients with CHD was much lower while IL-8 was higher.The results of luciferase report indicating direct binding site between TUG1 and miR-140-3p.Also,there was a direct binding site between IL-8 and miR-140-3p.The results of Western blot and ELISA showed that the changes of si-TUG1 on protein levels were reversed by si-TUG1 and miR-140-3p inhibitor transfected simultaneousl,co-transfected with IL-8 overexpression plasmid and miR-140-3p mimics could partially reverse the protein level changes caused by miR-140-3p mimics alone.Results of immunohistochemistry and fluorescence in situ hybridization showed that the levels of IL-8,MMP-2,and MMP-9 in the AS model group upregulated,while miR-140-3p downregulated compared with that of the control group;besides,compared with the sh-NC group,the levels of IL-8,MMP-2,and MMP-9 in the sh-TUG1 group downregulated while miR-140-3p increased.Conclusion:1.The expression of lncRNA TUG1 and IL-8 were significantly increased in the peripheral blood of patients with CHD and in AS rats,while expression of miR-140-3p decreased.2.Both metformin and si-TUG1 could reduce the proliferation,migration abilities and activate autophagy of HUVECs,coz metformin could active the AMPK/mTOR signaling pathway to play the role of anti-atherosclerosis through targetting lncRNA TUG1.3.Si-TUG1 could increase the expression of miR-140-3p while reduce that of IL-8,coz si-TUG1 could inhibit inflammation response through miR-140-3p/IL-8axis,thus providing a new theoretical basis and a possible new target for the clinical treatment of AS. |
