MicroRNA-199a-3p Enhances Cardiomyocyte Hypertrophy through Targeting Retinoblastoma Transcriptional Corespressor 1 / 中山大学学报(医学科学版)

@#【Objective】To investigate the role and the potential target of miR-199a-3p in mouse cardiac hypertrophy.【Methods】Neonatal mouse ventricular cardiomyocytes（NMVC）were isolated from the hearts of 0- 3- day- old newborn C57BL/6 mice. MiR-199a-3p mimic and retinoblastoma transcriptional corepressor 1（Rb-1）siRNA were transfected in? to NMVC to elevate the level of miR-199a-3p and inhibit Rb-1 expression，respectively. NMVC were stained with FITC- phalloidin solution to determine the size of NMVC. Dual luciferase reporter assay was performed to identify the interaction between miR- 199a- 3p and the 3’UTR of Rb- 1. mRNA and protein expression of cardiac hypertrophy associated genes were determined by RT-qPCR and Western blotting assay，respectively.【Results】（1）Over-expression of miR-199a-3pcould significantly enhance the expression of cardiac hypertrophy-related genes in NMVC ；（2）Dual-luciferase reporter assay results verified that miR- 199a-3p can interact with the 3’UTR of Rb-1. MiR-199a-3p could suppress Rb-1 ex? pression at the post-transcriptional level；（3）Functionally，miR-199a-3p mimic，consistent with Rb-1 siRNA，could increase cell size and the expression of Nppa，Acta1 and Myh7 in NMVC，and promote the nuclear translocation of E2f2 in NMVC.【Conclusions】MiR-199a-3p promotes the entry of E2f2 into the nucleus through inhibiting the expression of Rb-1，contributing to cardiomyocyte hypertrophy.

Effect of impaired glucose tolerance on cardiac dysfunction in a rat model of prediabetes / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The effect of impaired glucose tolerance (IGT) on cardiac function during the chronic prediabetes state is complicated and plays an important role in clinical outcome. However, the molecular mechanisms are not fully understood. This study was designed to observe cardiac dysfunction in prediabetic rats with IGT and to determine whether glucose metabolic abnormalities, inflammation and apoptosis are linked to it.</p><p><b>METHODS</b>The IGT rat models were induced by streptozocin, and the heart functions were assessed by echocardiography. Myocardial glucose metabolism was analyzed by glycogen periodic acid-Schiff staining, and the pro-apoptotic effect of IGT was evaluated by TUNEL staining. Additionally, caspase-3 activation, macrophage migration inhibitory factor (MIF) and G-protein coupled receptor kinase 2 (GRK2) were detected by Western blotting in cardiac tissue lysates.</p><p><b>RESULTS</b>Area-under-the-curve of blood glucose in rats injected with streptozotocin was higher than that in controls, increased by 16.28%, 38.60% and 38.61% at 2, 4 and 6 weeks respectively (F = 15.370, P = 0.003). Abnormal cardiac functions and apoptotic cardiomyocytes were observed in the IGT rats, the ejection fraction (EF) being (68.59 ± 6.62)% in IGT rats vs. (81.07 ± 4.59)% in controls (t = 4.020, P = 0.002). There was more glucose which was converted to glycogen in the myocardial tissues of IGT rats, especially in cardiac perivascular tissues. Compared to controls, the cleaved caspase-3, MIF and GRK2 were expressed at higher levels in the myocardial tissues of IGT rats.</p><p><b>CONCLUSIONS</b>IGT in the prediabetes period resulted in cardiac dysfunction linked to abnormal glycogen storage and apoptosis. Additionally, MIF and GRK2 may be involved in the pathogenesis of cardiac dysfunction in prediabetes and their regulation may contribute to the design of novel diagnostic and therapeutic strategies for those who have potential risks for diabetic cardiovascular complications.</p>

Correlation of angiotensin-converting enzyme 2 gene polymorphisms to essential hypertension and ischemic stroke / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the relationship between angiotensin-converting enzyme 2 (ACE2) gene polymorphisms and the risk factor for essential hypertension (EH) with concurrent ischemic stroke in southern Chinese population.</p><p><b>METHODS</b>The G9570A polymorphism in ACE2 gene were detected in 139 patients with EH and stroke using polymerase chain reaction-restriction fragment length polymorphism. Detailed clinical and biochemistrical data of the patients, including the pulse pressure, high sensitivity C-reactive protein (hsCRP), intima-media thickness (IMT), high-density lipoprotein cholesterol (HDL-C) and uric acid levels, were collected to study the relationship between ACE2 gene and the risk factor of EH and stroke.</p><p><b>RESULTS</b>The levels of hsCRP (OR=1.022), uric acid (OR=1.224), IMT and pulse pressure was positively correlated to the incidence of EH and stroke. The pulse pressure, hsCRP, IMT, and HDL-C levels in male stroke patients carrying A allele was significantly higher than those in patients carrying G allele (P<0.05). In female stroke patients, the pulse pressure, hsCRP, IMT, and HDL-C levels were also significantly different with regard to the genotype of ACE2 gene (P<0.05).</p><p><b>CONCLUSIONS</b>The patients with EH and ischemic stroke carrying the A/AA allele of ACE2 gene have higher risks than those carrying other allele, and can be also more vulnerable to stroke recurrence.</p>

Efficacy comparison between transplanting microenvironmental induced and non-induced bone marrow mesenchymal stem cells in ischemic rat hearts / 中华心血管病杂志

<p><b>OBJECTIVE</b>To compare the efficacy of transplanting bone marrow mesenchymal stem cell (BMSC) or microenvironmental induced BMSC (iBMSC) into the ischemic myocardium of rats with myocardial infarction.</p><p><b>METHODS</b>iBMSC was defined as BMSC co-cultured with myocardial cells for 2 weeks. The stem cells or equal volume PBS were injected into ischemic border zone 1 wk after experimental infarction. Cardiac performance was evaluated at 1, 2, and 4 wk after cell transplantation by echocardiography and analyzed histologically at 4 wk after cell transplantations.</p><p><b>RESULTS</b>Compared with PBS group, both BMSC and iBMSC transplantations reduced infarct size. iBMSC enhanced the beneficial effects of BMSC on improving cardiac function (FS: 28.5% +/- 4.3% in PBS, 29.0% +/- 2.0% in BMSC and 45.1% +/- 3.1% in iBMSC group at 4 weeks post transplantation, iBMSC group vs. PBS group P < 0.05, iBMSC group vs. BMSC group P < 0.05). Immunofluorescence microscopy results revealed co-localization of SPIO-labeled transplanted cells with cardiac markers for cardiomyocytes, indicating regeneration of damaged myocardium.</p><p><b>CONCLUSION</b>Our data suggest that iBMSC implantation is more effective on improving cardiac function than BMSC implantation in this model. iBMSC might serve as a new promising therapeutic cell source for regenerating ischemic myocardium in patients with post-infarction heart failure.</p>

Identification of the interactions between the truncated fragments of macrophage migration inhibitory factor and CD74 using a yeast two-hybrid system / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the interaction domains between macrophage migration inhibitory factors (MIF) and the extracellular segment of type-II trans-membrane protein CD74 using a yeast two-hybrid system.</p><p><b>METHODS</b>By using molecular cloning techniques, the DNA fragments encoding MIF, MIF(50-65) and MIF(1-50/65-115) were introduced into the pGBKT7 vector to construct the corresponding recombinant bait plasmids, and the DNA fragments encoding CD74(73-232), CD74(73-109), CD74(1109-149) and CD74(149-232) into the pGADT7 vector to construct the recombinant activation domain (AD) plasmids. PEG/LiAC method was employed to transform the above 3 recombinant bait plasmids paired with each of the 4 recombinant AD plasmids into the chemical competent yeast AH109 cells. The transformed yeast AH109 cells were screened consecutively on SD/-Trp-Leu and SD/-Trp-Leu-Ade-His/X-alpha-gal nutritional media.</p><p><b>RESULTS</b>The results of restriction endonuclease digestion and DNA sequencing verified the correct construction of all the recombinant plasmids. The yeast AH109 cells transformed with each of the 3 recombinant bait plasmids could grow on SD/-trp nutritional media without autonomous activation effect on the reporter gene MEL1. The cells transformed with each of the 4 recombinant AD plasmids could also grow on SD/-leu nutritional media without activation of the reporter gene MEL1. Only the yeast AH109 cells co-transformed with MIF, MIF(50-65), or MIF(1-50/65-115) plasmid and CD74(73-232) plasmid could grow on SD/-Trp-Leu-Ade-His nutritional media with transcription activation of the reporter gene MEL1.</p><p><b>CONCLUSION</b>MIF interacts with the intact extracellular segment of CD74 (CD74(73-232)) independent of the functional domain of MIF(50-65).</p>

Effects of Panax notoginsenoside on TNF-alpha and MMP-2 expressions in rats with post-myocardial infarction ventricular remodeling and the mechanism / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effects of Panax notoginsenoside (PNS) on tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinases-2 (MMP-2) expressions in rats with post-myocardial infarction ventricular remodeling and explore the mechanism.</p><p><b>METHODS</b>Rat models of acute infarction ventricular (AMI) were established by ligation of the left anterior descending coronary artery. Twenty-four hours after the operation, the rats were randomized into control and experimental groups for intragastric administration of normal saline (control), fosinopril and PNS at the low, medium and high doses for 4 consecutive weeks. The effects of PNS on the cardiac function index including the left ventricular end-diastolic dimension (LVIDd), left ventricular end-systolic diameter (LVIDs), ejection fraction (EF), percentage of left ventricular systole (FS), mitral early diastolic flow velocity mouth (MV), and heart rate (HR) were observed, and the changes in TNF-alpha and MMP-2 expression were detected after post-myocardial infarction ventricular remodeling.</p><p><b>RESULTS</b>Compared with the control group, PNS at the medium and high doses produced significant improvements in the EF, FS and MV of the rats (P<0.01 or 0.05). TNF-alpha and MMP-2 expressions were significantly decreased by PNS treatment at low, medium and high doses (P<0.01).</p><p><b>CONCLUSION</b>PNS can inhibit or reduce the expression of TNF-alpha and MMP-2, thereby enhancing left ventricular systolic and diastolic functions, decreasing peripheral resistance, and improving the cardiac function of rats with post-myocardial infarction left ventricular remodeling.</p>

Human bone marrow mesenchymal stem cells cocultured with semi-permeable membrane separated neonatal rat ventricular myocytes differentiated into cardiomyocyte phenotype / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the ability of human bone marrow mesenchymal stem cells (hBMSCs), cocultured with semi-permeable membrane separated neonatal rat ventricular myocytes, to differentiate into cardiomyocytes.</p><p><b>METHODS</b>hBMSCs were isolated and purified by density gradient centrifugation and adherence screening method. Cells were expanded as undifferentiated cells in culture for more than 3 passages and their phenotypes were identified with flow cytometer. hBMSCs were cocultured with neonatal rat ventricular myocytes in a rate of 1:10 separated by semi-permeable membrane. GATA4 mRNA was detected by RT-PCR; Immunocytochemistry, and Immunostaining were used to detect sarcomeric alpha-actinin, desmin, cTnT, and cTnI protein level.</p><p><b>RESULTS</b>CD29 (98.64% +/- 0.80%) and CD44 (96.70% +/- 1.50%) were the major surface markers of hBMSCs. After coculturing with semi-permeable membrane separated neonatal rat ventricular myocytes, the first contraction of single cells was noted at day 7 and GATA4 expression was detected on these cells by RT-PCR after 1 to 3 weeks coculture. Desmin, sarcomeric alpha-actinin, cTnI and cTnT could be detected by immunocytochemistry and immunostaining on some of these cells.</p><p><b>CONCLUSION</b>hBMSCs possess the potential to differentiate into myocardial cell phenotype in the cardiac microenvironment. Direct contact with cardiomyocytes was not necessary required for hBMSCs differentiation.</p>

Plasmid-mediated miRNA-1-2 specifically inhibits Hand2 protein expression in H9C2 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct an eukaryotic expression vector for miRNA-1-2 that can be expressed in rat H9C2 cardiomyocytes.</p><p><b>METHODS</b>The precursor miRNA (pre-miRNA) DNA template for miRNA-1-2 was designed and generated by PCR amplification. The DNA template was inserted into the hairpin RNA expression vector pSilence-4.1-neo and identified by DNA sequencing analysis. The recombinant plasmid DNA was then transfected into H9C2 cells via Lipofectamine, and the green fluorescence protein expression vector pEGFP-N3 served as the transfection marker. Twenty-four hours after transfection, the total cellular RNA was extracted using TRIzol reagent, and thermoscript reverse transcriptase (RT)-PCR was performed to determine miRNA-1-2 precursor expression.</p><p><b>RESULTS AND CONCLUSION</b>DNA sequencing indicated that the miR-1-2 expression plasmid was correctly constructed. The precursor miRNA-1-2 was successfully expressed in the H9C2 cells, and the expression of Hand2 protein could be efficiently inhibited by miRNA-1.</p>

MicroRNAs can be expressed in cardiomyocyte-like cells differentiated from human mesenchymal stem cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of representative heart-specific primary microRNAs (pri-miRNAs) in the cardiomyocyte-like cells differentiated from human mesenchymal stem cells (hMSCs).</p><p><b>METHODS</b>The phenotype of hMSCs isolated was identified by flow cytometry using monoclonal antibodies against FITC-conjugated CD29, CD34, and CD11b. The third-passage hMSCs were induced to differentiate into cardiomyocyte-like cells by 5-azacytidine and indirect coculture with neonatal rat myocytes, respectively. Immunocytochemical analysis was performed to detect the expression of the cardiac-specific proteins, namely cardiac troponin I (cTnI) and sarcomeric alpha-actinin, in the cardiomyocyte-like cells differentiated from hMSCs. RT-PCR and DNA sequencing were used to identify the expression of the 5 representative heart-specific pri-miRNAs.</p><p><b>RESULTS</b>High hMSC marker CD29 expression rate (98.87%) and low hematopoietic cell markers CD34 (5%) and CD11b (0.4%) expression rates were identified in the hMSCs isolated. cTnI and sarcomeric alpha-actinin expression occurred in the hMSCs following induction with the 2 differentiation-inducing methods. miRNA-143 and -181 expressions were induced in the hMSCs by 5-azacytidine and miRNA-143, -181, -206, and -208 expressions were induced by indirect coculture with neonatal rat myocytes, but pri-miRNA-1-2 expression failed to be induced by these two induction methods.</p><p><b>CONCLUSION</b>Expressions of the representative heart-specific pri-miRNAs in different patterns can be induced in cardiomyocyte-like cells differentiated from hMSCs by 5-azacytidine and indirect coculture with neonatal rat myocytes.</p>

An method for small hairpin RNA expression vector reconstruction for easy single restriction endonuclease identification / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop an effective method for screening recombinant hairpin RNA expression plasmids using single restriction endonuclease analysis.</p><p><b>METHODS</b>The double-strand DNA fragment containing a ClaI site (the flanking sequences of which were not complementary) was annealed and ligated into small hairpin RNA (shRNA) expression vector pSilencer-4.1 that did not contain ClaI site to construct the circular pSilencer-4.1-ClaI vector. With BamHI and HindIII, the pSilencer-4.1-ClaIwas digested and ligated with the DNA template of green fluorescence protein (GFP) shRNA that did not include a ClaI site. The plasmid DNA of the positive clones was extracted and digested with ClaI, and the inserted DNA sequence of the non-linearized plasmid was identified by sequence analysis.</p><p><b>RESULT AND CONCLUSION</b>DNA sequencing showed that pSilencer-4.1-ClaI was correctly constructed and the plasmids resistant to ClaI digestion were all recombinant vectors encoding GFP shRNA. The constructed pSilencer-4.1-ClaI can be used as a universal vector to construct the shRNA expression plasmid, and the incorporated ClaI sites may allow efficient screening of recombinant shRNA expression vectors.</p>

Association of the polymorphism of macrophage migration inhibitory factor gene with coronary heart disease in Chinese population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Inflammation is involved in the process of coronary heart disease (CHD). Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine which can inhibit the random migration of macrophages and concentrate macrophages at the inflammatory site, and is thought to play an important role in cell mediated immunity. The present study is to investigate the association of the -173 G/C polymorphism of MIF gene with the outcome of the CHD.</p><p><b>METHODS</b>One hundred and thirty-eight patients with coronary angiography (CAG) proved CHD were studied, and 163 healthy matched controls in Guangdong were studied. Patients and controls were genotyped for a single nucleotide polymorphism in the 5'-flanking region at position -173 of the MIF gene, using PCR-RFLP analysis, followed by DNA sequencing identification.</p><p><b>RESULTS</b>Only MIF -173G/G and MIF -173G/C genotypes were detected in CHD patients and controls. The MIF -173 G allele was detected in 0.966 of normal controls and 0.917 of patients, while MIF -173 C allele was detected in 0.034 of normal controls and 0.083 of patients. Individuals possessing a MIF-173*C genotype have an increased risk of CHD (16.7% versus 6.8%) (OR: 2.764, 95% CI: 1.295-5.899; P= 0.007).</p><p><b>CONCLUSION</b>These results suggest that MIF -173G /C polymorphism was associated with CHD in Chinese population, the MIF -173C allele might be a risk factor for CHD in Chinese Han nationality.</p>

Relationship Between the Angiotensin-Converting Enzyme 2 Genotype and Effect of Irbesartan on Left Ventricular Structure and Function in Hypertensive Patients / 中华高血压杂志

Background Experimental data have shown tnat polymorpnisms in tne angiotensm-converting en- zyme 2(ACE2)gene are related to echocardiographically determined parameters of left ventricular mass,structure or function in the general population whether ACE2 genotype influences the effect of angi0tensin Ⅱ receptor blocker which improve left ventricular remodeling and function is unknown.Objective To investigate the association be- tween ACE2 gene G9570A polymorphism and the effect of irbesartan on left ventricular structure and function in hy- pertensive patients.Methods Two hundred and five male patients and 190 female patients who were preliminaryly diagnosised with mild and moderate essential hypertension were treated with irbesartan for 48 weeks with initial dose of 150 mg/d and titrated to 300 mg/d to reach the targed BP.Gene polymorphisms of ACE2 G9570A were detected by PCR-RFLP methods.The association between changes in the SBP,DBP,parameters of left ventricular struc- ture and function and genotypes of the ACE2 gene locus were analyzed.Results Irbesartan reducted in blood pres- sure in all patients(P