简介:Paralysisfollowingspinalcordinjury(SCI)isduetofailureofaxonalregeneration.Itisbelievedthatthecapacitiesofneuronstoregrowtheiraxonsareduepartlytotheirintrinsiccharacteristics,whichinturnaregreatlyinfluencedbyseveraltypesofinhibitorymoleculesthatarepresent,orevenincreasedintheextracellularenvironmentoftheinjuredspinalcord.Manyoftheseinhibitorymoleculeshavebeenstudiedextensivelyinrecentyears.IthasbeensuggestedthatthesmallGTPaseRhoAisanintracellularconvergencepointforsignalingbytheseextracellularinhibitorymolecules,butduetothecomplexityofthecentralnervoussystem(CNS)inmammals,andthelimitationofpharmacologicaltools,thespecificrolesofRhoAareunclear.ByexploitingtheanatomicalandtechnicaladvantagesofthelampreyCNS,werecentlydemonstratedthatRhoAknockdownpromotestrueaxonregenerationthroughthelesionsiteafterSCI.Inaddition,wefoundthatRhoAknockdownprotectsthelarge,identifiedreticulospinalneuronsfromapoptosisaftertheiraxonswereaxotomizedinspinalcord.Therefore,manipulationoftheRhoAsignalingpathwaymaybeanimportantapproachinthedevelopmentoftreatmentsthatarebothneuroprotectiveandaxonregeneration-promoting,toenhancefunctionalrecoveryafterSCI.
简介:摘要目的探讨RhoA在胃癌患者中的表达及与临床病理的关系。方法选择2016年3月—2018年2月收治的58例胃癌患者为研究对象,分别取其胃癌组织和癌旁组织,采用免疫组化测定取获的病灶组织标本中RhoA表达阳性率,同时查阅患者病理,分析不同病理因素下胃癌患者RhoA表达情况。结果胃癌组织中RhoA表达阳性率显著高于癌旁组织,差异有统计学意义(P<0.05);免疫组化结果显示RhoA在胃癌组织中高表达,且主要定位于胞质中,但在癌旁组织中亦有表达;胃癌组织中RhoA表达阳性率与性别、年龄差异无统计学意义(P>0.05),与肿瘤大小、淋巴结转移、肿瘤分期及分化程度有统计学意义(P<0.05)。结论RhoA蛋白在胃癌患者中呈高表达,且表达阳性率与临床病理存在紧密的联系,加强RhoA测定有助于评估患者预后,为临床诊疗提供依据和参考。
简介:目的构建RhoA—siRNA表达载体,研究其对肝癌HepG2细胞肿瘤生物学行为的影响。方法利用pGenesil-1质粒构建RhoA—siRNA表达载体,以脂质体法转染至肝癌HepG2细胞中建立稳定细胞系,并分为3组。转染pGenesil-1-RhoA—siRNA载体者为HepG2/RhoA—siRNA组,转染随机对照载体者为HepG2/control组,未转染的肝癌HepG2细胞作为HepG2组。Westernblot检测RhoA—siRNA对其蛋白表达的抑制情况。分别采用MTT法、细胞划痕损伤和平板克隆形成实验检测转染细胞的增殖、迁移和生长潜能,流式细胞仪检测细胞周期变化。采用单因素方差分析、)f2检验比较各组差异。结果3组细胞蛋白表达水平比较,HepG2/RhoA—siRNA组RhoA蛋白的表达明显下凋(F=178.19,P〈0.05)。HepG2/control组和HepG2组细胞划痕损伤在48h内愈合,而HepG2/RhoA—siRNA组则不能愈合。HepG2/RhoA—siRNA组克隆形成率低于HepG2组和HepG2/control组,分别为39%±3%、67%±5%、70%±6%,其差异有统计学意义(x^2=33.34,38.69,P〈0.05)。RhoA基因沉默显著抑制肝癌HepG2细胞的增殖,细胞周期中G0/G1期细胞数量增多而S期细胞数量减少(F=70.46,76.57,P〈0.05)。结论RhoA—siRNA表达载体能抑制肝癌HepG2细胞的增殖和迁移,可为肝癌的基因治疗提供新的方法。
简介:摘要目的探讨双氢睾酮(DHT)对人外周血早期内皮祖细胞(PB-EPCs)增殖、迁移功能的影响及RhoA/ROCK信号通路在其中的作用。方法取健康成人外周血分离、培养出早期EPCs并鉴定。分别以不同浓度DHT(1、10、100 nmol/L)干预,得出最佳浓度和最佳作用时间后用于后续干预实验。分组:对照组、DHT、RhoA抑制剂C3 exoenzyme+DHT组、ROCK抑制剂Y-27632+DHT组。检测各组EPCs的增殖、迁移能力。ELISA法检测各组细胞上清液液中VEGF蛋白含量。结果DHT在一定范围内呈浓度-时间依赖性促进EPCs增值、迁移功能,分别在浓度为10 nmol/L和24 h达到最大作用效果。与单纯10 nmol/L的DHT刺激组相比,C3 exoenzyme[(0.22±0.02) vs (0.26±0.05),P>0.05]和Y-27632[(0.21±0.04) vs (0.26±0.05), P>0.05]能够减弱DHT诱导的EPCs增殖,但差异无统计学意义。DHT对EPCs迁移能力的促进作用可被C3 exoenzyme[(35.26±4.27) vs (46.92±5.46), P<0.05]和Y-27632[(33.61±5.33) vs (46.92±5.46), P<0.01]抑制。DHT对EPCs分泌VEGF能力的促进作用可被C3 exoenzyme[(116.75±7.42) vs (156.80±21.74), P<0.05]和Y-27632[(121.73±5.33) vs (156.80 ±21.74), P<0.01]抑制。结论DHT呈一定的浓度-时间依赖性促进EPCs的增殖、迁移以及VEGF的表达,其中RhoA/ROCK信号通路参与调控了此过程。
简介:本文对吴茱萸次碱的收缩血管效应及其机制进行了研究。结果表明,吴茱萸次碱体外可明显的引起大鼠胸主动脉血管平滑肌收缩。与血管平滑肌收缩相关的信号蛋白Rho激酶(RhoA)和IP3受体(IP3R)的抑制剂可以抑制吴茱萸次碱的缩血管效应。血管平滑肌细胞A7r5的实验发现,吴茱萸次碱(300μg/L)可以明显升高胞内Ca2+浓度,促进IP3R的mRNA表达,而后者与胞内Ca2+浓度升高有关。预先使用RhoA抑制剂H-1152,吴茱萸次碱仍能使RhoAmRNA表达升高。此外,吴茱萸次碱能够促进肌球蛋白轻链磷酸酶(MLCP)以及肌球蛋白轻链(MLC)的磷酸化。提示吴茱萸次碱的缩血管效应与RhoA/MLCP-MLC信号转导通路有关。本工作对于深入认识吴茱萸次碱的药理活性具有重要的意义。
简介:摘要目的探讨法舒地尔对脓毒症小鼠急性肺损伤的保护作用。方法4~6周龄雄性C57BL小鼠45只随机(随机数字法)分为对照组(Control组)、脂多糖组(LPS组)、法舒地尔干预组(FAS+LPS组),每组15只。LPS溶液腹腔注射及气道内滴注建立脓毒症小鼠急性肺损伤模型。法舒地尔干预组分别于腹腔注射LPS前30 min和气道滴注LPS后1 h,腹腔内注射盐酸法舒地尔溶液(10 mg/kg)。造模后4 h处死小鼠取肺组织。HE染色观察病理形态学变化;测定肺组织湿重/干重比(W/D);TBA比色法测定MDA含量及MPO活性;IHC测定caspase-3表达水平;Western Blot法检测肺组织中RhoA、ROCK1、eNOS及p-eNOS的蛋白表达水平。结果FAS预处理后肺组织中炎性细胞浸润和红细胞渗出显著减少,间质水肿及肺泡结构破坏显著减轻。与LPS组相比,FAS+LPS组的W/D值、MDA含量及MPO活性较LPS组均显著降低(P<0.01)。FAS+LPS组的Caspase-3表达较LPS组显著下降(P<0.01)。与Control组比,LPS组的RhoA、ROCK1的蛋白表达水平增高(P<0.05),p-eNOS的蛋白表达水平下降(P<0.05);与LPS组比,FAS+LPS组的RhoA和ROCK1的蛋白表达下调(P<0.05),但p-eNOS的蛋白表达上调(P<0.05);三组的eNOS总蛋白表达水平差异无统计学意义(P>0.05)。结论法舒地尔可减轻脓毒症小鼠肺组织炎性细胞浸润程度,下调肺组织细胞凋亡,抑制RhoA/ROCK1信号通路活性并促进eNOS的磷酸化表达。
简介:摘要目的探讨RhoA/Rho激酶1(ROCK1)下调抑制肌球蛋白轻链(MLC)磷酸化在主动脉夹层(AD)形成过程中的作用。方法检测正常(NA)和AD主动脉原代平滑肌细胞(SMC)中RhoA/ROCK1的表达与MLC的磷酸化程度。免疫荧光观察SMC中MLC磷酸化程度和结构。β-氨基丙腈(BAPN)联合应用ROCK1抑制剂Fasudil,验证RhoA/ROCK1下调在AD形成中的作用。组间比较采用t检验或χ2检验,Kaplan-Meier生存曲线采用非参数检验。结果NA组和AD组年龄(t=-1.439,P>0.05)、性别(χ2=0.900,P>0.05)差异无统计学意义,AD组高血压患者多于正常组(χ2=5.562,P<0.05)。AD主动脉SMC中RhoA(NA:42 782±31 339,AD:6 975±3 130,t=2.585,P<0.05)和ROCK1表达下调(NA:64 626±38 822,AD:13 851±961,t=2.977,P<0.05),MLC磷酸化减少(NA:230 193±27 749,AD:51 142±48 151,t=6.561,P<0.01)。免疫荧光显示AD组SMC和Fasudil处理的SMC中MLC磷酸化降低,结构受损。Fasudil联合BAPN的夹层形成率(100%)高于BAPN的夹层形成率(50%,χ2=22.780,P<0.01)。结论RhoA/ROCK1下调抑制MLC磷酸化促进了AD的发生。
简介:AbstractBackground:Dysuria is one of the main symptoms of genitourinary syndrome of menopause, which causes serious disruption to the normal life of peri-menopausal women. Studies have shown that it is related to decrease of detrusor contractile function, but the exact mechanism is still poorly understood. Previous results have suggested that the sphingosine-1-phosphate (S1P) pathway can regulate detrusor contraction, and this pathway is affected by estrogen in various tissues. However, how estrogen affects this pathway in the detrusor has not been investigated. In this study, we detected changes of the S1P/RhoA/Rho associated kinases (ROCK)/myosin light chain (MLC) pathway in the detrusor of ovariectomized rats in order to explore the underlying mechanism of dysuria during peri-menopause.Methods:Thirty-six female Sprague-Dawley rats were randomly divided into SHAM (sham operation), OVX (ovariectomy), and E groups (ovariectomy + estrogen), with 12 rats in each group. We obtained bladder detrusor tissues from each group and examined the mRNA and protein levels of the major components of the S1P/RhoA/ROCK/MLC pathway using quantitative real-time polymerase chain reaction and Western blotting, respectively. We also quantified the content of S1P in the detrusor using an enzyme linked immunosorbent assay. Finally, we compared results between the groups with one-way analysis of variance.Results:The components of the S1P pathway and the RhoA/ROCK/MLC pathway of the OVX group were significantly decreased, as compared with SHAM group. The percent decreases of the components in the S1P pathway were as follows: sphingosine kinase 1 (mRNA: 39%, protein: 45%) (both P < 0.05), S1P (21.73 ± 1.09 nmol/g vs. 18.86 ± 0.69 nmol/g) (P < 0.05), and S1P receptor 2/3 (S1PR2/3) (mRNA: 25%, 27%, respectively) (P < 0.05). However, the protein expression levels of S1PR2/3 and the protein and mRNA levels of SphK2 and S1PR1 did not show significant differences between groups (P > 0.05). The percent decreases of the components in the RhoA/ROCK/MLC pathway were as follows: ROCK2 (protein: 41%, mRNA: 36%) (both P < 0.05), p-MYPT1 (protein: 54%) (P < 0.05), and p-MLC20 (protein: 47%) (P < 0.05), but there were no significant differences in the mRNA and protein levels of RhoA, ROCK1, MYPT1, and MLC20 (all P > 0.05). In addition, all of the above-mentioned decreases could be reversed after estrogen supplementation (E group vs. SHAM group) (all P > 0.05).Conclusion:In this study, we confirmed that ovariectomy is closely associated with the down-regulation of the S1P/RhoA/ROCK/MLC pathway in the rat detrusor, which may be one mechanism of dysuria caused by decreased contractile function of the female detrusor during peri-menopause.
简介:目的探讨乳腺癌细胞MDA-MB-231中癌基因RhoA对VEGF蛋白表达和胞外分泌的影响及可能的分子机制。方法将RhoA过表达质粒pcDNA3.0-V14RhoA、对照质粒pcDNA3.0、RhoA沉默质粒pcDNA3.0-shRhoA转染到MDA-MB-231细胞中,通过Westernblot和ELISA实验分别检测细胞内外VEGF蛋白的表达,通过Westernblot和实时PCR方法分别检测RhoA对p53表达的影响及对VEGF的调控作用。均数比较用t检验;计量资料用x^-±s表示,采用方差分析。结果MDA-MB-231细胞中上调RhoA表达后,胞内VEGF的蛋白表达水平增加;胞外分泌水平显著增加,与对照组相比差异具有统计学意义(F=4.020,P=0.032);MDA-MB-231细胞中沉默RhoA表达后,胞内VEGF的蛋白表达水平降低;胞外分泌水平显著降低,与对照组相比差异具有统计学意义(F=5.131,P=0.001);并且与0h相比,在MDA-MB-231细胞中上调RhoA可以抑制p53的表达(48h:F=3.231,P=0.043),而p53表达的降低可以增加VEGF的表达水平(48h:F=3.226,P=0.015),均差异具有统计学意义。结论乳腺癌细胞MDA-MB-231中RhoA表达的变化可以引起胞内外VEGF水平的变化,并且RhoA可能是通过抑制p53的表达从而增加VEGF表达。
简介:目的探讨温针灸对类风湿性关节炎模型大鼠RhoA(Rashomologgenefamily,memberA)蛋白表达影响的实验研究。方法清洁级健康3月龄SD大鼠60只(雌雄各半),随机分为正常组、模型组和温针灸组三组,每组20只,采用皮内注射牛II型胶原接种诱发制备大鼠类风湿性关节炎模型。温针灸组于造模第1天取足三里和肾俞穴温针灸治疗,采用手针,刺激量以大鼠耐受为度,1次/d,共21d。所有大鼠于相应处理结束后,采用排水法测大鼠膝关节部体积,放免法检测血清白介素含量,免疫印迹法检测膝关节滑膜组织RhoA蛋白的表达。结果与正常组比较,模型组大鼠膝关节肿胀明显,血清中白介素含量较高,膝关节滑膜组织RhoA蛋白表达增加,差异具有统计学意义(P〈0.01);与模型组比较,温针灸组大鼠膝关节肿胀改善明显,白介素含量减少,RhoA蛋白表达均降低,差异具有统计学意义(P〈0.05)。结论温针灸能有效调节RA大鼠关节炎性损伤状况,降低RA大鼠血清白介素和骨关节滑膜组织RhoA蛋白含量,这可能是温针灸通过调节RhoA蛋白改善RA的调节机制之一。
简介:摘要目的探究LncRNA BBOX1-AS1在卵巢癌(ovarian cancer,OC)放疗敏感性中的作用及潜在机制。方法qRT-PCR检测OC组织和细胞中BBOX1-AS1、miR-185-5p的表达。双荧光素酶报告实验确认BBOX1-AS1、miR-185-5p、RHOA之间的相互作用。MTT及流式细胞术观测OC细胞的增殖和凋亡。结果BBOX1-AS1在OC组织和细胞中上调,相对于si-NC组在2、3、4天的细胞增殖活力(0.89±0.07)(1.48±0.13)(1.69±0.15),si-BBOX1-AS1组的增殖活力(0.59±0.06)(0.97±0.09)(1.21±0.10)显著下降(均P<0.05)。相对于si-NC组(6.24±0.28),敲减BBOX1-AS1能诱导OC细胞的凋亡(12.07±1.33)(均P<0.05)。miR-185-5p在OC组织和细胞中表达下调,BBOX1-AS1与miR-185-5p、RHOA与miR-185-5p的靶向关系被证明。敲低miR-185-5p能逆转BBOX1-AS1对OC细胞的影响(均P<0.05)。结论LncRNA BBOX1-AS1通过调控miR-185-5p/RHOA轴抑制OC细胞放疗敏感性。
简介:摘要目的评价Rho亚家族蛋白A(RhoA)/Rho相关的卷曲连接蛋白激酶2(ROCK2)信号通路在多次七氟烷麻醉致新生大鼠远期认知功能障碍中的作用。方法SPF级健康新生SD大鼠60只,雌雄不拘,6日龄,体重12~20 g,采用随机数字表法分为3组(n=20):对照组(C组)、多次七氟烷麻醉组(S组)和RhoA/ROCK2信号通路抑制剂Y-27632组(Y组)。S组和Y组于出生后6、7和8 d时吸入3%七氟烷麻醉2 h;Y组于吸入七氟烷麻醉前腹腔注射Y-27632 5 mg/kg。于出生后35 d时行旷场实验评估自发活动能力;于出生后36 d时行Morris水迷宫实验评估认知功能。水迷宫实验结束后处死大鼠分离海马组织,采用流式细胞术检测神经元凋亡率和胞浆钙离子浓度([Ca2+]i),采用Western blot法测定磷酸化RhoA(p-RhoA)、ROCK2和裂解的caspase-3(cleaved-caspase-3)的表达水平,透射电镜下观察神经元超微结构。结果3组旷场实验运动速度、路程及旷场中心停留时间比较差异无统计学意义(P>0.05)。与C组比较,S组逃避潜伏期延长,穿越原平台次数减少,海马神经元凋亡率和[Ca2+]i升高,p-RhoA、ROCK2和cleaved-caspase-3表达上调(P<0.05),海马神经元发生病理学损伤。与S组比较,Y组逃避潜伏期缩短,穿越原平台次数增加,海马神经元凋亡率和[Ca2+]i降低,p-RhoA、ROCK2和cleaved-caspase-3表达下调(P<0.05),海马神经元病理学损伤减轻。结论多次七氟烷麻醉诱发新生大鼠远期认知功能障碍的机制与激活RhoA/ROCK2信号通路,诱发海马神经元凋亡有关。
简介:摘要目的探索微小RNA(miR)-340-5p在胰腺导管腺癌细胞(PDAC)中表达及影响其生物学行为的机制。方法实时定量反转录聚合酶链反应(RT-qPCR)检测40例胰腺导管腺癌与正常细胞中miR-340-5p的表达,研究其表达水平与临床特征之间的联系。检测不同胰腺导管腺癌细胞及人胰腺导管上皮细胞中miR-340-5p水平。miR-340-5p模拟物转染PDAC细胞株PANC-1;甲基噻唑蓝(MTT)比色法检测细胞增殖活性;划痕实验、Transwell侵袭实验检测细胞侵袭迁移能力。RT-qPCR及蛋白质印迹法(Western blot)检测N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、E-钙黏蛋白(E-cadherin)、基质金属蛋白酶-9(MMP-9)的表达。双荧光素酶报告基因分析实验检测miR-340-5p对RhoA的调控功能。结果PDAC组织中miR-340-5p水平低于正常胰腺组织(0.362 4±0.083 7、0.965 2±0.129 9,t=21.272,P<0.01);PDAC中miR-340-5p表达降低与胰腺癌淋巴结转移(χ2=7.782,P<0.01)、肝转移(χ2=17.109,P<0.01)有相关。双荧光素酶报告基因分析结果表明,野生型RhoA的荧光素酶活性在转染miR-340-5p模拟物后低于对照组(0.992 5±0.099 8比0.177 5±0.019 0,t=18.496,P<0.01)。转染后,转染组细胞增殖活性降低(0.505 2±0.077 1比1.031 0±0.069 1、1.029 5±0.113 5,t=38.012、7.637,P<0.01、迁移距离减少(0.753 3±0.050 1、0.725 0±0.071 5比0.338 3±0.087 7,t=11.098、7.031,P<0.01)、迁移细胞数减少(57.000 0±4.290 0、52.666 7±5.785 0比19.833 3±2.994 0,P<0.01)。Western blot及RT-qPCR结果显示,与空白对照组(NC)及阴性对照组(Scramble)比较,miR-340-5p模拟物转染组N-cadherin(1.048 3±0.076 9、1.040 7±0.079 2比0.457 3±0.074 5,t=11.800、11.585,P<0.01)、Vimentin(1.045 3±0.062 8、0.993 3±0.068 6比0.521 7±0.057 9,t=10.982、9.585,P<0.05)、MMP-9(1.047 0±0.069 6、1.154 3±0.101 1比0.560 3±0.457 1,t=32.803、18.559,P<0.01)表达显著降低,E-cadherin(1.053 3±0.100 3、0.987 7±0.083 4比1.976 0±0.154 2,t=-29.502、-24.203,P<0.01)表达升高。结论胰腺导管腺癌中miR-340-5p表达降低,过表达miR-340-5p后能明显抑制PANC-1细胞的体外增殖和转移。其对RhoA信号通路介导的上皮-间充质转化(EMT)进程具有潜在抑制作用。
简介:AIM:ToinvestigatetheroleofRho-associatedproteinkinase(ROCK)inhibitor,Y27632,inmediatingtheproductionofextracellularmatrix(ECM)componentsincludingfibronectin,matrixmetallo-proteinase-2(MMP-2)andtypeIcollagenasinducedbyconnectivetissuegrowthfactor(CTGF)ortransforminggrowthfactor-β(TGF-β)inahumanretinalpigmentepithelialcellline,ARPE-19.METHODS:TheeffectofY27632ontheCTGForTGF-βinducedphenotypeinARPE-19cellswasmeasuredwithimmunocytochemistryasthechangeinF-actin.ARPE-19cellsweretreatedwithCTGF(1,10,100ng/mL)andTGF-β(10ng/mL)inserumfreemedia,andanalyzedforfibronectin,laminin,andMMP-2andtypeIcollagenbyRT-qPCRandimmunocytochemistry.CellswerealsopretreatedwithanROCKinhibitor,Y27632,toanalyzethesignalingcontributingtoECMproduction.·RESULTS:TreatmentofARPE-19cellsinculturewithTGF-βorCTGFinducedanECMchangefromacobblestonemorphologytoamoreelongatedswirlpatternindicatingamesenchymalphenotype.RT-qPCRanalysisanddifferentgeneexpressionanalysisdemonstratedanupregulationinexpressionofgenesassociatedwithcytoskeletalstructureandmotility.CTGForTGF-βsignificantlyincreasedexpressionoffibronectinmRNA(P=0.006,P=0.003respectively),lamininmRNA(P=0.006,P=0.005),MMP-2mRNA(P=0.006,P=0.001),COL1A1mRNA(P=0.001,P=0.001),COL1A2mRNA(P=0.001,P=0.001).PreincubationofARPE-19withY27632(10mmol/L)significantlypreventedCTGForTGF-βinducedfibronectin(P=0.005,P=0.003respectively),MMP-2(P=0.003,P=0.002),COL1A1(P=0.006,P=0.003),andCOL1A2(P=0.006,P=0.004)geneexpression,butnotlaminin(P=0.375,P=0.516).CONCLUSION:OurstudydemonstratedthatbothTGF-βandCTGFupregulatetheexpressionofECMcomponentsincludingfibronectin,laminin,MMP-2andtypeIcollagenbyactivatingtheRhoA/ROCKsignalingpathway.Duringthisprocess,ARPE-19cellswereshowntochangefromanepithelialtoamesenchymalphenotypeinvi
简介:AbstractBackground:Microribose nucleic acids (miRNAs) are implicated in the progression of lung adenocarcinoma. MicroRNA-345-5p (miR-345-5p) is a recently identified anti-oncogene in some human cancers, but its functional role and possible molecular mechanism in lung adenocarcinoma remain unknown. This study aimed to identify the biological function and underlying mechanism of miR-345-5p in lung adenocarcinoma cells.Methods:In this study, lung adenocarcinoma tissues and adjacent tissues were collected in the First Affiliated Hospital of Anhui Medical University between April 2016 and February 2017. The expression of miR-345-5p and ras homolog family member A (RhoA) in lung adenocarcinoma tissues and human lung adenocarcinoma cell lines (A549, H1650, PC-9, and H441) was detected by reverse transcription quantitative polymerase chain reaction analysis. Functional assays including colony formation, flow cytometry analysis, wound healing, and transwell assays were performed to assess the proliferation, apoptosis, migration, and invasion of lung adenocarcinoma cells. In addition, RNA pulldown and luciferase reporter assays were conducted to evaluate the relationship between miR-345-5p and RhoA. Difference between the two groups was analyzed with Student’s t test, while that among multiple groups was analyzed with one-way analysis of variance.Results:MiR-345-5p expression displayed lower level in lung adenocarcinoma tissues (0.241 ± 0.095 vs.1.000 ± 0.233, t = 19.247, P < 0.001) and cell lines (F = 56.992, P < 0.001) than control tissues and cells. Functional experiments demonstrated that upregulation of miR-345-5p inhibited the malignant phenotypes of lung adenocarcinoma cells via suppressing cell proliferation, migration, invasion, and facilitating cell apoptosis. Additionally, RhoA was verified to be the downstream target of miR-345-5p. Expression of RhoA was downregulated by overexpression of miR-345-5p in PC-9 (0.321 ± 0.047 vs. 1.000 ± 0.127, t = 8.536, P < 0.001) and H1650 (0.398 ± 0.054 vs. 1.000 ± 0.156, t = 4.429, P = 0.011) cells. Rescue assays revealed that overexpression of RhoA rescued the suppressive effects of miR-345-5p upregulation on proliferation, migration, and invasion of lung adenocarcinoma cells. Further, miR-345-5p was found to regulate the Rho/Rho-associated protein kinase (ROCK) signaling pathway by downregulation of RhoA in lung adenocarcinoma cells.Conclusions:MiR-345-5p plays a tumor suppressor role in lung adenocarcinoma cells by downregulating RhoA to inactivate the Rho/ROCK pathway.
简介:摘要目的探讨RhoA/Rho激酶1(ROCK1)参与细胞外基质(ECM)沉积对慢性阻塞性肺疾病(COPD)患者肺小静脉、肺小动脉及小气道平滑肌重塑的病理生理意义。方法本研究为病例对照研究。采用非随机抽样法,分析2019年10月至2020年12月宁夏医科大学总医院收治的因原发性肺肿瘤而接受肺切除术患者的肺组织,结合患者肺功能及临床病史分为COPD组(n=13),对照组(n=12)。使用α-平滑肌肌动蛋白(α-SMA)抗体鉴定平滑肌细胞,选取高倍视野下结构完整的肺小静脉及肺小动脉(血管横断面外径<500 μm)、小气道(气道横断面外径<2 000 μm),使用Image-Pro Plus测量肺小静脉、肺小动脉血管外径,小气道基底膜周长,计算肺小静脉、肺小动脉及小气道平滑肌层面积占管腔横断面面积百分比(WA%)作为评估平滑肌重塑的指标。采用免疫组织化学及蛋白免疫印迹法检测目的蛋白的表达。分析形态学和临床资料之间的相关性。结果COPD组肺小静脉、肺小动脉、小气道WA%均较对照组增高(P值均<0.05)。免疫组织化学检测显示COPD组肺小静脉、肺小动脉、小气道平滑肌细胞α-SMA、纤维连接蛋白(FN)、ROCK1表达中位平均光密度(AOD)值均较对照组增高(P值均<0.05)。蛋白免疫印迹法显示,COPD组肺组织α-SMA、FN、ROCK1蛋白表达灰度值分别为[(1.31±1.07)、(0.55±0.23)、(0.64±0.20)],均较对照组增高,分别为[(0.59±0.37)、(0.26±0.16)、(0.33±0.18),t值分别为2.30、3.86、4.06,P值均<0.05]。相关性分析显示,第1秒用力呼气容积/用力肺活量、第1秒用力呼气容积占预计之百分比与肺小静脉、肺小动脉、小气道WA%均呈负相关,与肺小静脉、肺小动脉、小气道平滑肌细胞ROCK1表达AOD值均呈负相关(P值均<0.05)。COPD组患者外周血中性粒细胞绝对值及超敏C反应蛋白较对照组升高(P值均<0.05)。结论轻中度稳定期COPD患者的肺小静脉、肺小动脉、小气道存在以平滑肌层增厚及ECM蛋白沉积为表现的重塑。ROCK1参与COPD患者肺小静脉、肺小动脉、小气道平滑肌重塑。炎症反应参与稳定期轻中度COPD发病。肺小静脉、肺小动脉、小气道平滑肌重塑及ROCK1蛋白表达与气流阻塞程度相关。