The Research On Long Noncoding RNA Related To Atherosclerosis

Objective: Human aortic smooth muscle cells stimulated by oxidized low density lipoprotein were used as models of atherosclerosis in vitro.Lnc RNA with differentially up-regulated expression were screened by high-throughput sequencing.After q PCR validation,sh RNA lentvirus infected cells inhibited the expression of related lnc RNA.The proliferation,migration and cholesterol levels of human aortic smooth muscle cells were detected to provide new targets and ideas for exploring the etiology and treatment of atherosclerosisMethods: 1.In the first stage,HA-VSMC was randomly divided into two groups: oxLDL group and non-OX-LDL group.The former was stimulated with 100ug/ml oxidized low density lipoprotein for 24 h,while the latter was not specially treated.After passing the quality inspection,total RNA was extracted by Trizol method for high-throughput sequencing to screen the differentially up-regulated lnc RNA that met the criteria.Differential lnc RNA were verified by q PCR.2.In the second stage,HA-VSMC was randomly divided into three groups.Normal aortic smooth muscle cells infected with sh RNA lentivirus for 72 hours were treated as experimental group,unloaded virus infection was treated as sh Ctrl group,normal HAVSMC was treated without any treatment as control group(CON group).Fluorescence and infection efficiency were observed under microscope.q PCR was used to detect the expression levels of target genes in the three groups to verify the effectiveness of gene interference in the experimental group.3.The third stage is divided into three groups.Group 1: normal aortic smooth muscle cells were not treated at all Group 2: normal HA-VSMC stimulated by oxidized low density lipoprotein(LDL)only 100ug/ml were added.The third group: the successful cells infected with the above sh RNA lectin virus were ox-LDL+ sh BOLA3-AS1 group,ox-LDL+ sh POC1B-AS1 group and ox-LDL+sh GRXCR1-4 group,respectively.The second group was used as control group,and the cell proliferation activity of the three groups was detected by CCK8 method.transwell cell migration ability and intracellular cholesterol concentration were detected by ELISA.Results: 1.Compared with normal cells treated with ox-LDL,three kinds of long-chain non-coding RNA BOLA3-AS1,POC1B-AS1,and GRXCR1-4 were significantly differentially expressed in Ox-LDL treated human arterial smooth muscle cells.2.It showed that the cell infection efficiency of the experimental group in this stage reached more than 80%,and the cell morphology was normal,indicating successful infection.q PCR detection at this stage compared with the control group,the expression levels of target lnc RNA were significantly decreased(P<0.001),the difference was statistically significant.3.Compared with the control group,the proliferative activity of aortic vascular smooth muscle cells in ox-LDL+sh BOLA3-AS1 group,ox-LDL+sh POC1B-AS1 group and oxLDL+sh GRXCR1-4 group was significantly decreased(P<0.001),and the difference was statistically significant.The cell migration numbers of ox-LDL+ sh BOLA3-AS1 group,ox-LDL+ sh GRXCR1-41 group and ox-LDL+sh POC1B-AS1 group were 52±10.07,58±5.57,43±8.62,respectively,and 120±10.41 in the control group.Compared with the control group,the number of cell migration in the lentivirus infection group was significantly decreased(P<0.01),and the difference was statistically significant.The linear regression equation y=0.4126x+0.0548(R2=0.9943)was obtained according to the concentration and OD value corresponding to the standard substance.Compared with the control group,the cholesterol content in the normal group was significantly decreased(P<0.001),and the cholesterol concentration in the lentivirus infection groups was significantly decreased(P<0.001),and the difference was statistically significant.Conclusions: 1.High throughput sequencing technology can screen lnc RNA in atherosclerosis.2.Lnc RNA may be involved in regulating the progression of atherosclerosis.3.Highly expressed lnc RNA(BOLA3-AS1,POC1B-AS1,GRXCR1-4)have been proven to be associated with atherosclerosis.4.Lnc RNA(BOLA3-AS1,POC1B-AS1,GRXCR1-4)may be a new intervention target,contributing to the etiological research and drug target treatment of atherosclerotic diseases.