【作者】 李彬；

【导师】 季晓平；

【作者基本信息】 山东大学 ， 内科学， 2014， 博士

【摘要】 研究背景随着人们生活方式改变和人口老龄化,目前,缺血性心脏病(ischemic heart disease, IHD)已经成为威胁人类生命健康的主要原因之一。我国现有缺血性心脏病患者超过2000万人,且每年新增患者100万人左右。急性冠状动脉闭塞所致的急性心肌梗死是缺血性心脏病中非常严重的一类疾病,及时开通梗死相关动脉、恢复血流供应是挽救濒死心肌的关键；但是,研究发现心肌持续一段时间缺血后重新恢复血流灌注会给心脏组织带来新的损伤,出现心肌顿抑、心功能降低、恶性心律失常发作等,即心脏缺血/再灌注损伤(ischemia/reperfusion injury, I/RI)。缺血/再灌注损伤的概念是在1960年由Jennings等人首次提出的。研究发现,心脏缺血/再灌注损伤与氧自由基释放、细胞内钙超载、心肌能量代谢障碍、中性粒细胞侵润、血管内皮细胞损伤和细胞凋亡等有关。研究心脏缺血/再灌注损伤的发生机制,探讨在尽早恢复缺血组织血流灌注的同时,减少甚至消除缺血/再灌注损伤的发生成为临床治疗中一个新的挑战。MicroRNA (miRNA)是一类由21～23个碱基组成的、内源性的非编码的单链小分子RNA,通过对mRNA的降解或者翻译抑制在转录后水平负调控靶基因的表达。越来越多的研究发现miRNA在心血管系统中表达丰富,参与了心脏发育、心肌重塑、心肌肥厚、心肌细胞凋亡、心律失常、心力衰竭等病理生理过程。近年来,miRNA在心脏缺血/再灌注损伤中的调控作用越来越受到人们的重视。van Rooij等研究发现,在非热休克导致的心脏缺血/再灌注损伤的小鼠体内注入miR-1、miR-21和miR-24可以减少心肌梗死范围。作为心肌特异性表达的miRNA, miR-1在心脏缺血/再灌注损伤过程中起着重要作用。Ren等研究发现,miR-320在心脏缺血/再灌注损伤中表达明显降低,敲除miR-320可以使热休克蛋白20(heat-shock protein20, Hsp20)表达增加,从而减少心脏缺血/再灌注损伤诱导的心肌细胞凋亡。Ren等在研究心脏缺血/再灌注损伤中miR-320的作用时,通过基因芯片发现miR-7表达升高。近年来有研究发现miR-7是一种肿瘤抑制因子,在多种肿瘤,如肺癌、舌鳞状细胞癌及恶性成神经细胞瘤中抑制肿瘤细胞增殖、促进肿瘤细胞凋亡；但是,关于miR-7在心脏缺血/再灌注损伤中表达的变化,以及miR-7在心脏缺血/再灌注损伤中的作用及其机制尚不清楚。近年来,有一种与细胞损伤有关的酶——多聚ADP核糖聚合酶[poly(ADP-ribose) polymerase, PARP引起人们的重视。PARP是在真核细胞内普遍存在的一类蛋白酶类家族,具有蛋白修饰和核苷酸聚合作用。主要存在于细胞核内,少量存在于细胞浆内。目前发现至少有18个家族成员,包括PARP-1、 PARP-2、PARP-3、PARP-4/VPARP、Tankyrase1, Tankyrase2等。PARP在DNA的损伤修复、基因表达、维持染色质的稳定、细胞的增殖、分化、凋亡和坏死以及基因转录等多种生理、病理过程中起重要作用。细胞在受到外源性有害因子刺激时DNA损伤,PARP在DNA损伤断裂时被激活并参与DNA的损伤修复。当细胞严重受损时,为防止NAD+和ATP的大量消耗,激活的Caspase-3裂解PARP,从而使PARP失活导致细胞凋亡。研究表明PARP在心脏缺血/再灌注损伤、心肌梗死、心力衰竭、脑血管疾病、糖尿病和肿瘤等多种疾病的发生发展过程中起着重要作用。尽管miRNA和PARP在心脏缺血/再灌注损伤中的重要作用均有研究,但其中发生机制却远未清楚。例如miR-7在心脏缺血/再灌注损伤中表达是否发生变化?它又是如何变化的?miR-7是否通过PARP对心脏缺血/再灌注损伤产生影响?二者又是怎样的关系?具体作用如何?这些问题构成了本研究的课题内容。在本研究中,我们拟通过建立心肌细胞模拟缺血/再灌注(缺氧/复氧)损伤模型和大鼠心脏缺血/再灌注损伤模型,从体外实验和体内实验两方面进行深入研究,探讨miR-7在心脏缺血/再灌注损伤中的作用及其作用机制,这可能为心脏缺血/再灌注损伤的临床治疗提供新的治疗靶点。本研究共分三部分进行：第一部分体外试验：MiRNA-7a/b在心肌细胞缺血/再灌注损伤中的表达变化及其对缺血/再灌注损伤介导的细胞凋亡的影响；第二部分体外试验：MiRNA-7a/b通过抑制PARP减少缺血/再灌注损伤诱导的心肌细胞凋亡；第三部分体内试验：MiRNA-7a/b减少大鼠心脏缺血/再灌注诱导的心肌损伤。第一部分体外试验：MiRNA-7a/b在心肌细胞缺血/再灌注损伤中的表达变化及其对缺血/再灌注损伤介导的细胞凋亡的影响研究目的本研究拟培养Wistar大鼠原代心肌细胞和H9C2心肌细胞,体外建立心肌细胞缺氧/复氧模型,测定miR-7a/b的表达；并通过在H9C2细胞中转染miR-7a/b mimic或miR-7a/b inhibitor来调控miR-7a/b的表达,测定心肌细胞凋亡率和乳酸脱氢酶(Lactate dehydrogenase, LDH)水平,明确miR-7a/b在心肌细胞缺氧/复氧中的表达变化及其对缺氧/复氧介导的细胞凋亡的影响。研究方法1.原代心肌细胞的提取：取2日龄Wistar大鼠乳鼠,开胸取心脏,将心脏剪碎成约1mm3大小,应用胰蛋白酶和Ⅱ型胶原酶多次消化,收集细胞,差速贴壁纯化心肌细胞后种板,在37℃培养箱中培养。2.心肌细胞模拟缺血/再灌注损伤模型的建立和分组：取不含血清、不含糖的DMEM培养基做为缺氧液置换培养基,置于37℃密闭的低氧培养箱中(95%N2和5%CO2)培养,其中95%N2和5%CO2混合气体以2L/min的流速通气置换空气。缺氧后置换新鲜培养基继续培养建立心肌细胞模拟缺血/再灌注损伤模型。原代心肌细胞实验分以下2组：对照组(Control组)和模拟缺血/再灌注组(SI/R组)；H9C2心肌细胞实验分以下10组：Control组、Control+miR-7a mimic组、Control+miR-7b mimic组、Control+miR-7a inhibitor组、Control+miR-7b inhibitor组、SI/R组、SI/R+miR-7a mimic组、SI/R+miR-7b mimic组、SI/R+miR-7ainhibitor组、SI/R+miR-7b inhibitor组。3. Real-time PCR检测miR-7a和miR-7b的表达：使用mirVanaTM miRNA提取试剂盒提取心肌细胞中的miRNAs,并用Taqman miRNA反转录试剂盒进行反转录,最后使用’Taqman miRNA引物和TaqMan Universal PCR Master Mix检测心肌细胞中miR-7a和miR-7b的表达,以U6作为内参照。4. miR-7a/b mimic和miR-7a/b inhibitor的转染：使用Lipofectamine2000分别将miR-7a/b mimic和miR-7a/b inhibitor转染入H9C2心肌细胞。5.心肌细胞凋亡的检测：使用流式细胞技术检测心肌细胞的凋亡。6.细胞LDH的测定：采用LDH检测试剂盒测定心肌细胞的LDH水平。研究结果：1.心肌细胞缺氧/复氧后miR-7a和miR-7b升高：在原代心肌细胞和H9C2心肌细胞缺氧/复氧模型中,miR-7a和miR-7b的表达均明显升高；说明miR-7a和miR-7b参与了心脏缺血/再灌注损伤；2.在缺氧/复氧后,心肌细胞凋亡率增加,LDH水平升高：H9C2心肌细胞在缺氧10小时、复氧2小时后细胞凋亡率明显增加,LDH水平显著升高；3. miR-7a和miR-7b在缺氧/复氧条件下对心肌细胞损伤的影响：在H9C2心肌细胞缺氧/复氧模型中,与SI/R组相比,转染miR-7a/b mimic可显著减少心肌细胞的凋亡率和LDH的释放；相反,miR-7a/b inhibitor增加心肌细胞凋亡率和LDH水平；以上结果表明,miR-7a/b在心肌细胞缺氧/复氧中发挥重要作用,过表达miR-7a/b可显著降低缺血/再灌注导致的心肌细胞损伤。结论：1.体外建立心肌细胞缺氧/复氧模型中,miR-7a/b表达明显升高；2.体外建立心肌细胞缺氧/复氧模型中,miR-7a/b mimic可减少心肌细胞凋亡率、降低LDH水平；相反,miR-7a/b inhibitor增加心肌细胞凋亡率、升高LDH水平。第二部分体外试验：MiRNA-7a/b通过抑制PARP减少缺血/再灌注损伤诱导的心肌细胞凋亡研究目的通过miRNA靶基因预测软件分析miRNA有可能发挥作用的靶点,应用双荧光素酶报告基因检测试剂盒及Western Blot技术验证PARP是否是miR-7a/b的靶基因；体外建立心肌细胞缺氧/复氧模型,并通过miR-7a/b mimic或miR-7a/b inhibitor转染H9C2心肌细胞,应用Western Blot技术测定靶基因的表达及其对凋亡的影响,并探讨miR-7a/b在心肌细胞缺血/再灌注损伤中的作用机制。研究方法1.心肌细胞缺氧/复氧模型的建立和分组：取不含血清、不含糖的DMEM培养基做为缺氧液置换培养基,置于37℃密闭的低氧培养箱中(95%N2和5%CO2)培养,其中95%N2和5%C02混合气体以2L/min的流速通气置换空气。缺氧后置换新鲜培养基继续培养建立心肌细胞模拟缺血/再灌注损伤模型。H9C2心肌细胞实验分以下10组：Control组、Control+miR-7a mimic组、Control+miR-7b mimic组、Control+miR-7a inhibitor组、Control+miR-7b inhibitor组、SI/R组、SI/R+miR-7a mimic组、SI/R+miR-7b mimic组、SI/R+miR-7a inhibitor组、SI/R+miR-7b inhibitor组。2. miR-7a/b mimic和miR-7a/b inhibitor的转染：使用Lipofectamine2000分别将miR-7a/b mimic和miR-7a/b inhibitor转染入H9C2心肌细胞,并以scramble control miRNA作为阴性对照。3.双荧光素酶报告基因检测：为检测miR-7a/b是否能与PARP的3’非翻译区(3’-untranslated region,3’-UTR)的mRNA结合,我们采用双荧光素酶报告基因检测的方法。构建GV126-PARP3’-UTR野生型(GV126-PARP3’-UTR-WT)和突变型(GV126-PARP3’-UTR-MU)报告基因载体,我们将miR-7a/b mimic和这些报告基因载体及海肾素表达质粒(pRL-TK vector)转染入HEK293细胞后,用双荧光素酶报告基因检测试剂盒检测并分析其荧光素酶活性,确定miRNA的作用靶点。HEK293细胞实验分以下5组：MiR control+GV126+pRL-TK组、Mimic-7a+GV126-3’ UTR-WT+pRL-TK组、Mimic-7b+GV126-3’UTR-WT+pRL-TK组、Mimic-7a+GV126-3’ UTR-MU+pRL-TK组、Mimic-7b+GV126-3’UTR-MU+pRL-TK组；4. Western Blot检测表达变化的miRNA对靶基因表达的影响及其在心肌细胞缺血/再灌注损伤中对凋亡的影响：将miR-7a/b mimic或miR-7a/b inhibitor转染H9C2心肌细胞后,应用Western Blot技术检测靶基因表达的变化,确定miRNA对靶基因的影响；心肌细胞转染miR-7a/b mimic或1miR-7a/b inhibitor后制做缺氧/复氧损伤模型,Western Blot检测PARP和Caspase-3及其裂解片段的表达,明确其对心肌细胞凋亡的影响。研究结果1.在H9C2心肌细胞中,PARP是miR-7a/b的靶基因：双荧光素酶报告基因检测显示,与对照组相比,转染miR-7a/b mimic后,携带有PARP野生型报告基因载体组的荧光素酶信号强度明显下降,而携带有PARP突变型报告基因载体组荧光素酶信号强度没有明显变化；Western Blot结果也显示,过表达miR-7a/b可使PARP的表达降低,相反,抑制miR-7a/b的表达后,PARP的表达明显升高；以上结果充分证明了在H9C2心肌细胞中,miR-7a/b可以调控PARP蛋白的表达,说明PARP是miR-7a/b的靶基因。2.在心肌细胞缺氧/复氧损伤中,miR-7a/b的表达变化对细胞凋亡产生影响：Western Blot结果显示,与对照组相比,在心肌细胞缺氧/复氧损伤中,PARP表达显著减少,但PARP和Caspase-3裂解片段的表达升高,提示心肌细胞发生了细胞凋亡；过表达miR-7a/b可以降低裂解片段的表达,减少心肌细胞凋亡；相反,抑制miR-7a/b使裂解片段表达明显增加,加重了心肌细胞凋亡；以上结果表明,miR-7a/b可以通过抑制PARP的表达减少在缺血/再灌注损伤中的心肌细胞凋亡,从而保护心肌。结论1.在H9C2心肌细胞中,PARP是miR-7a/b的靶基因；2.体外建立心肌细胞缺氧/复氧模型中,miR-7a/b通过与PARP的3’-UTR相互作用,从而抑制其蛋白的表达,减少心肌细胞凋亡。第三部分体内试验：MiRNA-7a/b减少大鼠心脏缺血/再灌注诱导的心肌损伤研究目的本研究拟制作Wistar大鼠心脏缺血/再灌注损伤模型,测定心肌组织miR-7a/b的表达；并通过心肌内直接注射慢病毒转染miR-7a/b mimic或miR-7a/b inhibitor,测定PARP的表达及心肌梗死范围、心肌细胞凋亡和血清LDH水平的变化,探讨miR-7a/b在心脏缺血/再灌注损伤中的表达变化、miR-7a/b在心脏缺血/再灌注中对心肌损伤的影响及其作用机制。进一步验证体外实验得出的结论。研究方法1. Wistar大鼠心脏缺血/再灌注(ischemia/reperfusion, I/R)损伤模型的建立及分组：采用开胸结扎和松开冠状动脉的方法建立大鼠心脏I/R模型。实验一：miR-7a/b在大鼠心脏I/R损伤中表达变化的研究。实验分为以下2组：sham组和I/R组。每组8只大鼠；实验二：验证PARP是否是miR-7a/b的靶基因。实验分为以下5组：NC组、miR-7a mimic组、miR-7b mimic组、miR-7a inhibitor组、miR-7b inhibitor组。每组8只大鼠；实验三：miR-7a/b在大鼠心脏I/R损伤中作用的研究。实验分为以下7组：sham组、I/R组、I/R+NC组、I/R+miR-7a mimic组、I/R+miR-7b mimic组、I/R+miR-7a inhibitor、I/R+miR-7b inhibitor。每组16只大鼠。2.慢病毒转染：采用心肌内直接注射的方法将携带有ImiR-7a/b mimic或miR-7a/b inhibitor的慢病毒转染大鼠心肌调控miR-7a/b的表达,并以转染空病毒组作为阴性对照(NC)。3. Real-time PCR检测miR-7a和miR-7b的表达：使用TRIzol提取心肌组织中的总RNA,并用Taqman miRNA反转录试剂盒进行反转录,最后使用Taqman miRNA引物和TaqMan Universal PCR Master Mix检测心肌中miR-7a和1miR-7b的表达,以U6作为内参照。4. Real-time PCR检测PARP的表达：利用TRIzol提取心肌组织中的总RNA,使用日本TaKaRa公司的cDNA合成试剂盒将样本总RNA反转录为cDNA,然后使用TaKaRa公司的实时定量PCR试剂盒进行PCR反应,检测心肌中PARP的表达,以β-actin作为内参照。PCR引物由上海博尚生物公司合成。5. Western Blot检测PARP的表达：将携带有miR-7a/b mimic或miR-7a/b inhibitor的慢病毒转染心肌7天后,Western Blot检测PARP的表达,在体内实验中验证PARP是否是miR-7a/b的靶基因。6.心肌梗死范围测定：采用Evans蓝染色和氯化三苯基四氮唑(triphenyl tetrazolium chloride, TTC)染色来鉴别非缺血心肌、缺血心肌和梗死心肌：呈蓝色的部分为非缺血心肌,呈红色部分为缺血心肌,呈白色部分为梗死心肌。以称重法计算缺血心肌和梗死心肌的重量,心肌梗死范围的大小以梗死心肌占缺血心肌重量的百分比表示。7.心肌细胞凋亡的检测：采用末端脱氧核苷酸转移酶介导的末端标记技术(TUNEL)试剂盒检测各组大鼠心肌细胞的凋亡。8.血清LDH测定：采用LDH检测试剂盒检测各组大鼠血清LDH水平。研究结果1. Wistar大鼠在心脏缺血/再灌注损伤后miR-7a和miR-7b升高：与对照组相比,在大鼠心脏缺血/再灌注损伤模型中,niR-7a和miR-7b的表达均明显升高；体内实验也证明了miR-7a和miR-7b参与了心脏缺血/再灌注损伤；2.通过心肌内直接注射,可成功转染慢病毒：采用心肌内直接注射的方法将携带有miR-7a/b mimic或miR-7a/b inhibitor的慢病毒转染大鼠心肌来调控miR-7a/b的表达；慢病毒转染7天后,荧光显微镜下观察大鼠心肌组织中有绿色荧光蛋白(Green Fluoreseent Protein, GFP)的表达,并且阳性率均达到80%以上；同时,采用Real-time PCR方法检测了心肌组织中miR-7a和miR-7b的表达,结果发现,与对照组相比,转染miR-7a/b mimic慢病毒后大鼠心肌组织中miR-7a和miR-7b的表达水平均明显升高,而转染携带有miR-7a/b inhibitor的慢病毒后大鼠心肌组织中miR-7a和miR-7b的表达均显著降低；以上结果证明了在大鼠心肌中成功转染了慢病毒。3. mir-7a和mir-7b抑制PARP表达：大鼠心肌中转染miR-7a/b mimic或miR-7a/b inhibitor慢病毒7天后应用Western Blot的方法检测心肌组织PARP的蛋白表达水平。结果发现,miR-7a/b mimic抑制PARP的蛋白表达,miR-7a/b inhibitor使PARP表达升高；与体外实验一致,在体内实验中证明了PARP是miR-7a/b的靶基因。4.在心脏缺血／再灌注损伤中,miR-7a/b负性调控靶基因：应用Real-timePCR技术测定PARP的表达,与I/R+NC组相比,成功转染含有miR-7a/b mimic的慢病毒后,心肌组织中PARP的表达降低,相反,转染携带有miR-7a/b inhibitor的慢病毒可以增加PARP的表达；以上实验结果表明,在大鼠心脏缺血/再灌注损伤模型中PARP是miR-7a/b的靶基因。5.在大鼠心脏缺血／再灌注损伤模型中,miR-7a/b的表达变化对心肌损伤产生影响：在大鼠心脏缺血／再灌注损伤模型中,与I/R+NC组相比,转染携带有miR-7a/b mimic的慢病毒可显著减少心肌梗死范围、心肌细胞凋亡率及LDH水平；相反,转染携带有miR-7a/b inhibitor的慢病毒增加心肌梗死范围、心肌细胞凋亡率及LDH释放；以上结果表明,体内实验再次证实了miR-7a/b在心脏缺血/再灌注损伤中发挥重要作用,过表达miR-7a/b可保护缺血/再灌注诱导的心肌损伤。结论：1.在大鼠心脏缺血/再灌注损伤模型中,miR-7a/b的表达明显升高；2.在大鼠心肌中,PARP是miR-7a/b的靶基因。3.在大鼠心脏缺血/再灌注损伤模型中,miR-7a/b通过抑制PARP的表达,具有心脏保护作用。创新性及局限性1.创新性(1)本研究首次证实了在大鼠心脏缺血/再灌注损伤中, miR-7a/b的表达升高；(2)本研究首次证实了PARP是miR-7a/b的靶基因,并且在体内体外实验中证明了miR-7a/b通过调节PARP的蛋白表达,减少缺血/再灌注导致的心肌损伤；(3)本研究结果提示miR-7a/b将可能成为临床上治疗心脏缺血/再灌注损伤的潜在靶点。2.局限性(1)本课题没有对miR-7a/b在心功能等方面的作用进行体内实验研究,将在以后的实验中对此做进一步探讨；(2) miRNA是一个复杂的网络,本研究仅对PARP做了研究,对发挥功能作用的miR-7a/b其他靶基因尚待进一步的研究。更多还原

【Abstract】 BackgroundAs the changes of lifestyle and the ageing of population, ischemic heart disease has been one of the major reasons for the threat to the life and health of human. Now, in our country, the amount of patients with ischemic heart disease is more than20million, and there are about1million new patients with ischemic heart disease each year. In ischemic heart disease, acute myocardial infarction which is caused by coronary artery occlusion is a very serious disease, and opening of infarction related artery timely to make the return of blood supply as soon as possible is the key to save the dying myocardium. But, it was found that the return of blood supply may product additional myocardial injury, such as myocardial stunning, diminished cardiac function and malignant arrhythmia, which is the myocardial ischemia/reperfusion injury (I/RI). The concept of ischemia/reperfusion injury was put forward in1960by Jennings for the first time. Studies found that myocardial ischemia/reperfusion injury was related to the release of oxygen free radical, overload of intracellular calcium, metabolic disorders of myocardial energy, invasion of neutrophils, injury of vascular endothelial cell and apoptosis and so on. It is a new challenge to study the pathogenetic mechanism of myocardial ischemia/reperfusion injury and to investigate how to reduce or eliminate the ischemia/reperfusion injury.MicroRNA (miRNA) is a class of endogenous non-coding single and small RNA which is composed with21-23bases that negatively regulating the gene expression at post-transcriptional level by degrading target mRNA or translation repression. Studies found that the expression of miRNA was rich in the cardiovascular system, involved in heart development, myocardial remodeling, myocardial hypertrophy, myocardial cell apoptosis, arrhythmia, heart failure and other pathological and physiological processes. In recent years, the function of miRNA in myocardial ischemia/reperfusion injury was paid to more attention. Van Rooij etc. found that injection of miRNA-1, miRNA-21or miRNA-24into mice can reduce myocardial infarction area in myocardial ischemia/reperfusion injury. miRNA-1, as a kind of myocardial specificity miRNA, plays an important role in myocardial ischemia/reperfusion injury. Ren etc. found that the expression of miRNA-320was significantly decreased in myocardial ischemia/reperfusion injury and knockout of miRNA-320increased the expression of HSP20, reducing myocardial cell apoptosis induced by myocardial ischemia/reperfusion.Ren etc. found that the expression of miRNA-7was increased in myocardial ischemia/reperfusion injury with gene chip. In recent years, studies found that miRNA-7was a kind of tumor suppressor which inhibited tumor cell proliferation and promoted tumor cell apoptosis in various tumors, such as lung cancer, tongue squamous cell carcinoma and malignant neuroblastomas. But, the expression change, the role and mechanism of miRNA-7in myocardial ischemia/reperfusion injury are not clear.Recently, more attention was paid to the poly(ADP-ribose) polymerases (PARP), a kind of enzymes related to cellular injury. PARP is a family of protease existing in eukaryotes which plays a part in protein modification and nucleotide polymerization. PARP mainly exists in the nucleus and there are18superfamily, including PARP-1、PARP-2、PARP-3、PARP-4/VPARP、Tankyrase1, Tankyrase2, and so on. PARP is involved in many physiological and pathological processes, such as the repair of DNA damage, gene expression, the stability of chromatin, cellular proliferation, differentiation, apoptosis, necrosis and gene transcription. PARP is activated and participated in DNA repair when DNA is damaged. PARP is cleaved by activated Caspase-3to avoid exceeding consumption of the NAD+and ATP, leading to cell apoptosis when cells are damaged severely. Studies have shown that PARP plays an important role in the development of some diseases, such as myocardial ischemia/reperfusion injury, myocardial infarction, heart failure, cerebrovascular disease, diabetes and cancer and so on.Though the functions of miRNA and PARP in myocardial ischemia/reperfusion injury were studied, the mechanism is not clear. Is the expression of miRNA-7changed in myocardial ischemia/reperfusion injury? Does miRNA-7have an effect on myocardial ischemia/reperfusion injury by regulating PARP? In our studies, we established myocardial cell ischemia/reperfusion injury model and the rat heart ischemia/reperfusion injury model, investigated the role and the mechanism of miRNA-7in myocardial ischemia/reperfusion injury, so that we can provide a new therapeutic target for myocardial ischemia/reperfusion injury.This study includes three parts:Part I In vitro:The change of expression of microRNA-7a/b and the effect of microRNA-7a/b on cell apoptosis in ischemia/reperfusion injuryPart II In vitro:MicroRNA-7a/b reduces myocardial cell apoptosis in ischemia/reperfusion injury by inhibiting PARPPart III In vivo:MicroRNA-7a/b reduces myocardial injury induced by myocardial ischemia/reperfusion in ratsPart I In vitro:The change of expression of microRNA-7a/b and the effect of microRNA-7a/b on cell apoptosis in ischemia/reperfusion injuryObjectiveTo investigate the change of expression of microRNA-7a/b and the effect on myocardial cell apoptosis in myocardial ischemia/reperfusion injury.Methods1. The extraction of primary myocardial cells:obtain the heart of neonatal Wistar rat, shred to lmm3, and then digest with trypsin and collagenase Ⅱ. After differential adhesion, the myocardial cells are put in incubator at37℃.2. Establish the hyoxia-reoxygenation injury model of myocardial cell.The following experimental groups were studied in primary myocardial cells:(1) control group;(2) SI/R group;The following experimental groups were studied in H9C2myocardial cells:(1) control group;(2) control+mir-7a mimic group;(3) control+mir-7b mimic group;(4) control+mir-7a inhibitor group;(5) control+mir-7b inhibitor group;(6) SI/R group;(7) SI/R+mir-7a mimic group;(8) SI/R+mir-7b mimic group;(9) SI/R+mir-7a inhibitor group;(10) SI/R+mir-7b inhibitor group.3. Real-time quantitative PCR for miR-7a and miR-7b:myocardial cells were harvested and miRNAs were extracted by use of mirVanaTM miRNA extraction kit. The miR-7a/b expression level was determined by real-time quantitative PCR using Taqman miRNA reverse transcription kit and TaqMan Universal PCR Master Mix according to the manufactures’ instruction. The primers of miR-7a/b and U6were obtained from Ambion, and U6as an internal reference for miR-7a/b expression.4. Transfection of miR-7a/b mimic and miR-7a/b inhibitor:For miR-7a/b over-expression or inhibition, miR-7a/b mimic or inhibitor was transfected into H9C2myocardial cells with Lipofectamine2000according to the manufactures’ instruction.5. Detection of apoptosis of myocardial cells:Flow cytometry was used.6. Detection of LDH in myocardial cells:LDH detection kit was used.Results1. The expression of miR-7a/b was increased after myocardial ischemia/reperfusion injury:as compared with controls, the expression of miR-7a/b was significantly increased after myocardial ischemia/reperfusion injury. This indicates that miR-7a/b is involved in the myocardial ischemia/reperfusion injury;2. After myocardial ischemia/reperfusion injury, myocardial cell apoptosis rate was increased and the level of LDH was increased:compared with the control group, the cell apoptosis rate was increased and the level of LDH was increased significantly after H9C2myocardial cell was in hypoxia for10hours and reoxygenation for2hours;3. The effects of mir-7a/b on the damage of myocardial cell in myocardial ischemia/reperfusion injury:compared with the SI/R group, the transfection of mir-7a/b mimic significantly reduced the apoptosis rate of myocardial cells and the release of LDH; On the contrary, the transfection of mir-7a/b inhibitor increased the apoptosis rate of myocardial cells and the release of LDH;The results show that mir-7a/b plays an important role in myocardial ischemia/reperfusion injury and overexpression of mir-7a/b can significantly reduce the myocardial cell injury caused by ischemia/reperfusion.Conclusion1. The expression of miR-7a/b was increased significantly in myocardial stimulated ischemia/reperfusion model;2. In myocardial stimulated ischemia/reperfusion, transfection of miR-7a/b mimic reduced the myocardial cell apoptosis rate and the level of LDH, on the contrary, transfection of miR-7a/b inhibitor increased the myocardial cell apoptosis rate and the level of LDH.Part II In vitro:MicroRNA-7a/b reduces myocardial cell apoptosis in ischemia/reperfusion injury by inhibiting PARPObjectiveTo investigate the target gene of mir-7a/b and the regulatory mechanism of mir-7a/b in myocardial ischemia/reperfusion injury.Methods1. Establish the hyoxia-reoxygenation injury model of myocardial cell.The following experimental groups were studied in H9C2myocardial cells.(1) control group;(2) control+mir-7a mimic group;(3) control+mir-7b mimic group;(4) control+mir-7a inhibitor group;(5) control+mir-7b inhibitor group;(6) SI/R group;(7) SI/R+mir-7a mimic group;(8) SI/R+mir-7b mimic group;(9) SI/R+mir-7a inhibitor group;(10) SI/R+mir-7b inhibitor group.2. Transfection of miR-7a/b mimic and miR-7a/b inhibitor:For miR-7a/b over-expression or inhibition, miR-7a/b mimic or inhibitor was transfected into H9C2myocardial cells with Lipofectamine2000according to the manufactures’ instruction. Scramble control miRNA was used as the negative control. miR-7a/b mimic, miR-7a/b inhibitor and scramble control miRNA were purchased from GenePharm.3. Dual-luciferase reporter assay:To confirm that miR-7a/b can directly bind to the3’-untranslated region (3’-UTR) of PARP, the dual-luciferase reporter assay was performed. After transfection of miR-7a/b mimic and GV126-PARP-3’-UTR-WT (wild type)/MU (mutant type) vectors into HEK293cells, firefly and renilla luciferase activities were measured using the dual-luciferase reporter assay system according to the manufactures’ instruction.The following experimental groups were studied in HEK293cells.(1) MiR control+GV126+pRL-TK;(2) Mimic-7a+GV126-3’UTR-WT+pRL-TK;(3) Mimic-7b+GV126-3’ UTR-WT+pRL-TK;(4) Mimic-7a+GV126-3’ UTR-MU+pRL-TK;(5) Mimic-7b+GV126-3’UTR-MU+pRL-TK.4. Investigating the effect of miR-7a/b on the target gene and the role of miR-7a/b in myocardial ischemia/reperfusion injury:After miR-7a/b mimic or miR-7a/b inhibitor was transfected into H9C2cells, the expression of the target gene was measured by western blot analysis. After transfection of mir-7a/b mimic or miR-7a/b inhibitor, we made myocardial ischemia/reperfusion injury model and detected the expression of PARP, Caspase-3, cleaved PARP and Caspase-3using western blot analysis.Results1. miR-7a/b directly targeted PARP in H9C2cells:The result of dual-luciferase reporter assay showed that compared with the control group, transfection with the miR-7a/b mimic strongly inhibited luciferase activity of the GV126-PARP3’-UTR-WT, but not the GV126-PARP3’-UTR-MU. The expression of PARP was down-regulated with miR-7a/b mimic and up-regulated with miR-7a/b inhibitor by use of western blot analysis.These results demonstrate that miR-7a/b can regulate the expression of PARP directly and PARP is the target gene of miR-7a/b in H9C2cells.2. The effect of miR-7a/b on myocardial cell apoptosis in myocardial hypoxia-reoxygenation injury:PARP was reduced and cleaved PARP and Caspase-3were increased during the hypoxia-reoxygenation injury model. Overexpression of miR-7a/b significantly reduced the levels of cleaved PARP and Caspase-3as compared with SI/R group. In contrast, miR-7a/b inhibitor increased the expression of cleaved PARP and Caspase-3as compared with SI/R group.This indicates that miR-7a/b reduces myocardial cell apoptosis in hypoxia-reoxygenation injury.Conclusion1. PARP is the target gene of miR-7a/b in H9c2cells.2. Mir-7a/b inhibits the expression of PARP via interacting with its3’-UTR and reduces the apoptosis of myocardial cells during the myocardial hypoxia-reoxygenation injury model. Part Ⅲ In vivo:MicroRNA-7a/b reduces myocardial injury induced by myocardial ischemia/reperfusion in ratsObjectiveTo investigate the change of microRNA-7a/b and explore the effect of microRNA-7a/b in rat myocardial ischemia/reperfusion injury.Methods1. Establish the rat model of cardiac I/R.The rat heart I/R model was induced with a4-0silk suture ligating left anterior descending artery (LAD) to block blood flow. After30min of ischemia, the knot was relaxed to establish rat heart I/R model.The following experimental groups for the experiment one were studied (n=8):(1) sham group;(2) I/R group.The following experimental groups for the experiment two were studied (n=8):(1) NC group;(2) mir-7a mimic group;(3) miR-7b mimic group;(4) miR-7a inhibitor group;(5) miR-7b inhibitor group.The following experimental groups for the experiment three were studied (n=16):(1) sham group;(2) I/R group;(3) I/R+NC group;(4) I/R+mir-7a mimic group;(5) I/R+miR-7b mimic group;(6) I/R+miR-7a inhibitor group;(7) I/R+miR-7b inhibitor group.2. Transfection of lentivirus:Lentivirus vector with miR-7a/b mimic or miR-7a/b inhibitor was transfected into the rat myocardium by direct intramyocardial injection. Transfection of vacancy lentivirus was used as a negative control (NC).3. Real-time quantitative PCR for miR-7a and miR-7b:The total RNA was extracted with TRIzol. The miR-7a/b expression level was determined by real-time quantitative PCR using Taqman miRNA reverse transcription kit and TaqMan Universal PCR Master Mix according to the manufactures’ instruction. The primers of miR-7a/b and U6were obtained from Ambion, and U6as an internal reference for miR-7a/b expression.4. Real-time quantitative PCR for PARP:The total RNA was extracted with TRIzol. The expression of mRNA was detected by cDNA synthesis kit and PCR kit of TaKaRa according to the manufactures’ instruction.β-actin was a reference for PARP expression.5. Detect the expression of PARP:After injection of lentivirus into the rat myocardium for7days, western blot analysis was performed to evaluate the expression of PARP.6. Determination of myocardial infarct size:Evans blue and triphenyl tetrazolium chloride (TTC) were used to evaluate the infarcted and noninfarcted areas. Infarct size was expressed as the percentage of the area at risk (AAR).7. Detection of apoptosis of myocardial cells:The TUNEL technique was used.8. Detect the level of LDH in blood serum:LDH detection kit was used.Results1. The expression of miR-7a/b was increased after I/R:As compared with controls, the expression of miR-7a/b was significantly increased after I/R. This shows that miR-7a/b is involved in I/R injury;2. Transfection of lentivirus successfully by direct intramyocardial injection: Lentivirus with miR-7a/b mimic or miR-7a/b inhibitor was transfected into the rat myocardium by direct intramyocardial injection to up-regulate or down-regulate the expression of miR-7a/b;3. Mir-7a/b inhibits the protein expression of PARP:After transfection of lentivirus with mir-7a/b mimic or mir-7a/b inhibitor into rat myocardium for7days, the protein expression level of PARP was detected using western blot method. The protein expression of PARP was inhibited by miR-7a/b mimic, but, increased by miR-7a/b inhibitor. These results demonstrate that PARP is the target gene of miR-7a/b;4. Mir-7a/b negatively regulates the target gene in I/R:As compared with I/R+NC, the expression of PARP was down-regulated with miR-7a/b mimic and up-regulated with miR-7a/b inhibitor in I/R. This indicates that PARP is the target gene of miR-7a/b in I/R.5. The effects of mir-7a/b on the damage of myocardial cell in I/R:As compared with I/R+NC, overexpression of miR-7a/b significantly reduced the infarct size, myocardial cell apoptosis rate and the level of LDH; Conversely, inhibition of miR-7a/b increased the infarct size, myocardial cell apoptosis rate and the level of LDH. This demonstrates that miR-7a/b plays an important role in I/R and overexpression of miR-7a/b protects myocardial cells from I/R injury. Conclusion1. In rat I/R model, the expression of miR-7a/b was increased;2. In rat myocardium, PARP is the target gene of miR-7a/b;3. In rat I/R model, mir-7a/b inhibits the expression of PARP and protects myocardial cells.更多还原