The Study On Pathological Changes And Roles Of MiR-29b-3p In Unstable Angina

Objective: Unstable angina (UA) is a set of clinical symptoms betweenstable angina and acute myocardial infarction. Due to its fast onset andprogressive aggravation, UA is easy to develop for myocardial infarctionwithout timely diagnosis and treatment. In the past, risk factors for UA weremanaged from the epidemiology perspective, however, its incidence is stillsurprisingly high. This indicates that there are still unknown factors thatinfluence the onset of UA, and it is critical to explore the possible mechanismand to find new molecular biomarkers for UA. MicroRNAs (miRNAs), smallregulatory RNAs, could complementarily bind with the target messenger RNA(mRNA), degrade or inhibit its translation, and furture regulate the expressionof gene at the posttranscriptional level. Studies found that miRNAs involve inthe regulation of many physiological and pathological processes in body, suchas growth, development, differentiation, apoptosis and tumor formation.Recently, it has been reported that miRNAs could stably existed in the tissuefluid, such as plasma, refered to circulating miRNAs. There are reportsshowed that expression profile of plasma miRNAs could be correlated withcertain pathological changes, which revealed that circulating miRNAsmolecules can be used as kind of new markers for disease diagnosis andprognosis. MiR-29b-3p is a member of miR-29family, which has been shownto inhibit myocardial fibrosis, calcification of vascular smooth muscle andparticipate in the regulation of cardiovascular disease, whereras the role ofmiR-29b-3p in UA has not been reported.In this research, Real-time quantitative PCR (qRT-PCR) technique wasused to detect the levels of miR-29b-3p in plasma of UA patients and controlsubjects respectively. The difference of circulating levels of miR-29b-3pbetween these two groups was analyzed and ROC was caculated. In vitro, miR-29b-3p mimics were transfected into HepG2cells using transienttransfection technique. We detected the mRNA and protein levels of liver Xreceptor α/β (LXRα/β) and ATP-binding cassette Transporter A1(ABCA1)with qRT-PCR and Western blotting technique after transfection. Theexpression level of DNA methyltransferase3a (Dnmt3a), a target protein ofmiR-29b-3p, was also investgated. We aim to elusidate the functionalmechanism of miR-29b-3p to UA from the prospective of HDL-C regulationby the posible Dnmt3a-ABCA1pathway.Methods:137UA patients were recruited according to inclusion criteria as experi-mental group from Tangshan Gongren Hospital cardiovascular departmentfrom Mar2012to Mar2013, including19males and18females, average age55±10. We also selected age-and sex-matched37healthy subjects from Centerof Health Examination of Tangshan Gongren Hospital during the same time,including19males and18females, average age54±5.2-3ml venous bloodwas collected from each case of UA group (before clinical interference) andhealthy control. After that, plasma was collected from the centrifuged bloodand saved in-80℃refrigerator.2Total RNAs were extracted from each plasma sample of UA group andhealthy control. After reverse transcription, the levels of plasma miR-29b-3pwere detected by qRT-PCR technique.3HepG2cells were cultured under the condition of37℃,5.0%CO2andsaturated humidity. miR-29b-3p mimics were tranfected into HepG2cells, thisgroup was termed transfection group (T29). Meanwhile, another group, withthe same culture condition except transfection miR-29b-3p mimics, was set asnegative control group (Control).4Total RNAs were extracted from HepG2cells of both T29group andControl group with Trizol respectively. The expression levels of LXRα/β,ABCA1mRNA of the two groups were detected by qRT-PCR technique.5Total protein were extracted from cells of T29group and Control groupwith PIPA respectively, then the expression levels of Dnmt3a, LXRα/β, and ABCA1of the two groups were detected with Western blotting technique.6The experimental data was analyzed by SPSS13.0statistical software,and showed as mean±SD or median (quartile). t test or Mann-Whitney U testwas used to analyze the differences of two independent groups, P<0.05wasregarded as statistical significance.Results:1Plasma qRT-PCR results: The levels of plasma miR-29b-3p in UAgroup and healthy control were0.000076(0.000026,0.004863) and0.006109(0.003429,0.011532), circulating miR-29b-3p level in UA group was higherthan that in healthy control (Z=-4.33, P<0.001), the difference was statisticallysignificant. The ROC curve was used to analyzed the diagnostic value ofmiR-29b-3p for UA, the area under the ROC curve was0.793(P<0.0001).2Cells experiment qRT-PCR results: The expression level of ABCA1mRNA in T29group was higher than that in control, the difference wasstatistically significant (P=0.004).There was no statistical difference of theexpression of LXRα/β mRNA in T29group compared with control group (Pvalue was0.956and0.965).3Cells experiment Western blot results: The protein level of Dnmt3a inT29group was lower than that in control (P=0.020), the difference wasstatistically significant. The protein level of ABCA1was higher than that incontrol, the difference was statistically significant (P=0.011). As for LXRαand LXRβ protein there was no statistical difference between them (P valuewas0.813and0.816).Conclusion:1Compare to the healthy contral, the plasma levels of miR-29b-3p wassignificantly decreased in UA patients, suggests that miR-29b-3p is a potentialbiomarker for the diagnosis of UA and miR-29b-3p was involved in theregulation of UA.2After the transfection of miR-29b-3p into HepG2cells, the protein levelof Dnmt3a was significantly down-regulated, which suggests that miR-29b-3pcan inhibit the expression of Dnmt3a by target inhibitory regulation. 3After the transfection of miR-29b-3p into HepG2cells, the expressionof LXRα/β did not change at the mRNA and protein level, but the expressionof ABCA1was significantly upregulated at both levels. These results indicatethat LXRα/β is not involved in miR-29b-3p-mediated ABCA1upregulation.4miR-29b-3p upregulate the expression of ABCA1at transcriptional andtranslational level in HepG2cells, which are correlated with the down-regulation of Dnmt3a. Taken together, our findings suggest that miR-29b-3p-mediated ABCA1upregulation may be related with the epigenetic regulationof ABCA1induced by Dnmt3a.