| IntroductionAs one of the most serious diseases threatening human health,atherosclerosis remains the main pathological risk of cardiovascular disease(CAD).Hypercholesterolemia is a significant risk factor leading to development of atherosclerosis and it is meaningful to explore new pathway to prevent atherosclerosis through regulating cholesterol metabolism.long non-coding RNA(lncRNA)is transcription production by RNA polymeraseⅡ and has no protein coding function,which is longer than 200 nucleotides.Researchers have found that IncRNA participated in chromosome inactivation,chromatin modification,transcriptional activation/interference.Recently,some researches showed that IncRNA was involved in regulation of angiopoiesis,endothelial cell migration and inflammation.The role of IncRNA in atherosclerosis has attracted more and more attention at present.HOXC6 is a member of homeobox gene family,which encode proteins sharing a highly conserved 61-amino-acid domain and participate in regulating morphogenesis of multicellular organisms.HOX genes regulated the identity of different regions along the mammalian body axis,and the rules mainly complied with temporal and spatial collinearity.Some HOX genes were reported to participate in regulating development of cardiovascular system.Studies have shown that fat loss caused by bariatric surgery can promote the expression of HOXC6.However,the relationship between HOXC6 and atherosclerosis has not been reported.Materials and Methods1.Cell cultureHuman monocytic THP-1 cells were purchased from American Type Culture Collection(ATCC,Manassas,VA).THP-1 cells were cultured with Roswell Park Memorial Institute(RPMI)1640 medium containing 1%penicillin/streptomycin and 10%fetal calf serum.Cells were incubated at 37℃ in an atmosphere of 5%CO2.THP-1 cells differentiated into macrophages after being incubated with phorbol 12-myristate 13-acetate(PMA)for 24h.2.Sample resourceAll of the specimens in this research were obtained between April 2013 and April 2014 from Nanfang Hospital,Southern Medical University.Fresh,unfixed material was used for extracting protein and RNA.3.RNA Isolation and Real-time Quantitative PCR Analysis(qPCR)Total RNA from cultured cells and human intima tissues was extracted by using TRIzol reagent(Invitrogen,Carlsbad,CA)in accordance with the standard manufacturer’s instructions.The mRNA levels were evaluated by quantitative reverse transcription PCR(qRT-PCR)with SYBR-Green Detection chemistry(TaKaRa Bio,Inc.)by ABI 7500 Fast Real-Time PCR system.The expression of GAPDH was used as the internal control for analysis of mRNA level of HOXC6.The expression of U6 RNA was used as the endogenous control for analysis of level of IncRNA HOXC-AS1 from tissues and cells.△△Ct method was used for determining quantitative measurements.All samples were measured in triplicate and the mean value was considered for comparative analysis.4.Western blot analysesCells and human tissues were harvested and protein extracts were prepared according to standard methods.Proteins were separated by 10%sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to western blot analysis using rabbit polyclonal anti-β-actin antibodies and rabbit anti_HOXC6 antibodies.The proteins were visualized with the chemiluminescence method.5.Lentivirus production and cells infectionPacked empty lentivirus vectors(LV-Mock)and LV-mediated IncRNA HOXC-AS1 overexpression vector(LV-HOXC-AS1)were prepared.The cells were infected with the LV stock at a multiplicity of infection of 100(THP-1 cells)transducing units per cell in the presence of 8μg/ml of polybrene.The cells were washed with fresh complete media after 12h of incubation.6.High-performance liquid chromatographyAbandon the culture medium after the treatment,wash the cells with PBS for 3 times,and then add 200μl cell lysis solution,repeated freezing and thawing for 3 times.Protein concentration was quantified by BCA method,and then adding 7.2%trichloroacetic acid to precipitate the protein.Ordinary centrifuge with 800g speed for 10 min,and draw the supernatant to a new markup EP tube for detection of cholesterol.Stigmasterol is used as internal standard.Take 100 μl supernatant for the detection of total cholesterol,and add 200μl 8.9 mol/L of potassium hydroxide solution to hydrolyze cholesterol ester.The sample is prepared for total cholesterol detection.The absorbance at 216nm was detected,and the data were analyzed with TotalChrom software from PerkinElmer.8,Statistical analysisResults are displayed as means ± standard deviations(S.D).The data was compared by one-way analysis of variance and the Student’s t-test,using the Statistical Package for the Social Sciences(versionl7.0)software(SPSS,Inc.,Chicago,IL,USA).Differences were considered to be significant when p values were less than 0.01.Results1.The levels of IncRNA HOXC-AS1 and HOXC6 were downregulated in atherosclerotic tissues.Microarray analysis was utilized to explore possible changes in RNA levels between atherosclerotic plaque tissues and normal intima tissues.Results showed that expression of IncRNA HOXC-AS 1 was decreased 31.77 times while the expression of HOXC6 was decreased 4.64 times in atherosclerotic tissues.Next,we performed qRT-PCR and western blot analysis to determine the expression of IncRNA HOXC-AS1 and HOXC6 in atherosclerotic plaques and normal arterial intima tissues.The level of IncRNA HOXC-AS1 was decreased to 7.82%while the mRNA level of HOXC6 was reduced to 25.76%in atherosclerotic plaques.Furthermore,protein level of HOXC6 was reduced to 23.12%in atherosclerotic plaques.2.The expression of HOXC6 was promoted by IncRNA HOXC-AS1 in THP-1 macrophages.Bioinformatic analysis revealed that IncRNA HOXC-AS1 and HOXC6 genes were both located on chromosome 12 and have opposite transcription direction.Therefore,we explored whether IncRNA HOXC-AS1 regulates the expression of HOXC6 in THP-1 macrophages.After using LVs to deliver the gene of IncRNA HOXC-AS1 into THP-1 macrophages,the level of IncRNA HOXC-AS1 was enhanced to 4275.21%while the mRNA level of HOXC6 was increased to 901.26%than that of THP-1 macrophages transfected with empty lentiviral vectors(LV-Mock).Next,western blot analysis showed that protein level of HOXC6 was increased to 428.26%by lentivirus-mediated overexpression of IncRNA HOXC-AS1.3.The expression levels of IncRNA HOXC-AS1 and HOXC6 could be suppressed by OX-LDL in THP-1 macrophages.We treated THP-1 cells with 50 pg/ml OX-LDL and found that OX-LDL significantly decreased the expression of IncRNA HOXC-AS1 and HOXC6 in a time-dependent manner in THP-1 macrophages.The inhibition of OX-LDL on HOXC6 was totally compensated after overexpressing of IncRNA HOXC-AS1.4.Cholesterol accumulation induced by OX-LDL could be inhibited by IncRNA HOXC-AS1 in THP-1 macrophages.We further to explore the role of OX-LDL-lncRNA HOXC-AS 1 pathway on cholesterol metabolism in THP-1 cells.Results of High Performance Liquid Chromatography(HPLC)demonstrated that the levels of total cholesterol(TC),free cholesterol(FC),and cholesterol ester(CE)were significant increased after treating THP-1 macrophages with OX-LDL.Moreover,the induction of cholesterol content by OX-LDL could be partly inhibited by lentivirus-mediated overexpression of IncRNA HOXC-AS1. |
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