miR-27a-3p调控Ras/Raf/MEK/ERK信号通路抑制肾透明细胞癌细胞增殖的研究

目的 探讨miR-27a-3 p对肾透明细胞癌细胞Ras/Raf/MEK/ERK信号通路的作用,观察其对肾透明细胞癌细胞增殖的影响.方法 使用脂质体试剂lipofectamine 2000将miR-27a-3p模拟物或miR-NC转染至肾透明细胞癌细胞株Caki-1和A-498;qRT-PCR和Western blot检测miR-27a-3p的靶基因KRAS及下游通路蛋白的表达;流式细胞仪检测细胞周期;噻唑蓝(MTT)检测细胞活力;集落形成实验检测细胞增殖能力.结果 KRAS基因和下游通路蛋白的表达降低;转染miR-27a-3p后的肾透明细胞癌细胞周期明显被抑制,G0/G1期的细胞比例上升(P<0.01);肾透明细胞癌细胞活力和增殖能力降低(P<0.05).结论 过表达miR-27a-3p在体外可以显著降低肾透明细胞癌细胞Ras/Raf/MEK/ERK蛋白的表达,进而显著抑制其增殖能力.     

miR-30a-5p在肾癌中的表达分析及临床意义研究

目的:本研究通过检测32对肾细胞癌组织标本及相应癌旁正常组织标本中miR-30a-5p的表达水平,然后分析miR-30a-5p的表达量与患者临床特征之间的关系,为更深一步研究miR-30a-5p在肾细胞癌的发病机制的作用和功能的研究打下基础。方法:收集32对经手术切除的肾细胞癌及相应的癌旁组织标本,提取标本的总RNA,经反转录获得cDNA,并通过实时荧光定量PCR(qRT-PCR)方法检测miR-30a-5p的表达水平,利用统计学方法分析其表达量与患者临床特征之间有无相关性。结果:32对组织标本中有27例(84.38%)肾细胞癌组织的miR-30a-5p表达水平下降(P0.01),且统计学分析表明其下降和患者的年龄、性别、组织学分型及临床分期等临床特征无明显相关性(P0.05)。结论:miR-30a-5p在肾癌组织中表达明显下调,提示其可能与肾癌的发生发展有一定的相关性。     

miR-195-5p通过靶向ROCK1抑制肾癌细胞迁移、侵袭和上皮-间质转化

目的探讨miR-195-5p对肾癌细胞迁移、侵袭和上皮-间质转化的影响。方法通过转染miR-195-5p mimics或inhibitors分别过表达或抑制肾癌细胞中miR-195-5p的表达,转染靶向Rho相关螺旋蛋白激酶1(ROCK1)的小干扰RNA敲低肾癌细胞中ROCK1的表达量,利用细胞划痕实验和Transwell小室实验分别检测肾癌细胞的迁移和侵袭能力。通过双荧光素酶报告实验验证miR-195-5p对ROCK1的靶向调控作用,利用免疫印迹试验检测ROCK1及上皮-间质转化相关蛋白的表达水平。结果过表达miR-195-5p可显著抑制肾癌细胞的迁移、侵袭和上皮-间质转化,而抑制miR-195-5p的表达可明显促进肾癌细胞的迁移、侵袭和上皮-间质转化(P<0.05)。miR-195-5p可通过靶向ROCK1调控其在肾癌细胞中表达。敲低ROCK1后可部分抵消miR-195-5p inhibitors对肾癌细胞迁移、侵袭和上皮-间质转化的影响。结论miR-195-5p可通过靶向ROCK1抑制肾癌细胞的迁移、侵袭和上皮-间质转化。     

miR-195-3p在肾癌中的表达及其临床意义研究

目的 研究miR-195-3p在肾癌与癌旁组织及肾癌细胞与正常细胞系中的表达差异,分析差异表达与患者临床病理特征间的关系,探究其临床意义. 方法 提取27例配对标本(组织及细胞系)的总RNA,经过逆转录、实时荧光定量PCR (qRT-PCR)检测标本中miR-195-3p标本中的表达量,统计分析miR-195-3p在不同组织、细胞系间的表达差异及其与临床病理特征间的相关性.通过转染上调和下调miR-195-3p,并进行细胞增殖实验(MTT),评估miR-195-3p对肾癌细胞增殖的影响. 结果 miR-195-3p在肾癌组织(18例,66.67％)及肾癌细胞系中的表达较癌旁组织及正常细胞系明显上调(P＜0.05),miR-195-3p在肾癌中的表达量与患者的年龄、性别、癌组织类型、TNM分期、AJCC分期无明显相关性(P＞0.05).miR-195-3p在肾癌细胞系(ACHN、786-O)表达上调后促进肾癌细胞增殖,表达下调后抑制肾癌细胞增殖. 结论 miR-195-3p在肾癌组织中明显升高,并且促进肾癌细胞增殖,提示其可能与肾癌的发生发展有一定关系.     

miR-19b-1在肾癌中的表达及临床意义的研究

目的检测肾癌细胞(ACHN、786O、769P)与人胚肾细胞(HEK-293T)、肾癌及对应癌旁组织的miR-19b-1的表达水平,并分析其与临床病理特征之间的关系。方法利用qPCR技术检测miR-19b-1在肾癌细胞与人胚肾细胞、肾癌组织与配对组织中的表达量,运用统计学方法分析其与临床病理之间有无相关性。结果 miR-19b-1在肾癌细胞中的表达量比正常细胞高,在肾癌组织中的表达量是对应癌旁组织的2.2倍,且其差异具有统计学意义(P0.05),与患者的年龄、性别及病理分期无关(P0.05)。结论肾癌组织及肾癌细胞中miR-19b-1表达上调,提示肾癌的发生可能与miR-19b-1上调有关。     

miR-518a-5p靶向HDAC2调控甲状腺鳞癌细胞SW579凋亡的实验研究

目的:探讨miR-518a-5p调控甲状腺鳞癌细胞SW579凋亡的潜在机制.方法:通过实时荧光定量PCR分析33例癌组织和癌旁组织中miR-518a-5p的表达水平.使用miR-518a-5p mimics和miR-518a-5p inhibitor甲状腺鳞癌细胞SW579中过表达或敲低miR-518a-5p,检测细胞...     

MicroRNA-133a-5p调控c-met表达对胆管癌细胞迁移和侵袭的影响

目的:探讨MicroRNA-133(miR-133)在胆管癌细胞系中的表达,研究miR-133基因表达在胆管癌细胞迁移和侵袭过程中的作用。方法:利用实时荧光定量PCR(RT-PCR)技术测定分析miR-133及其不同成熟体(miR-133b、miR-133a-3p、miR-133a-5p)在胆管癌QBC939细胞系中的表达。将胆管癌QBC939细胞系经转染后分为三组:自然生长组(空白对照组),细胞转染无关序列组(阴性对照组)以及细胞转染Anti-miR-133a-5p组(干扰组),利用RT-PCR检测各组胆管癌QBC939细胞系中miR-133a-5p、c-met的表达。然后利用Transwell小室法分别检测分析干扰miR-133a-5p基因表达对胆管癌QBC939细胞系迁移和侵袭能力的影响。结果:RT-PCR检测miR-133不同成熟体miR-133b(0.74±0.07)、miR-133a-3p(0.99±0.12)、miR-133a-5p(1.00±0.06)中的表达,其中miR-133a-5p在胆管癌QBC939细胞系中表达量最高(P0.05)。RT-PCR实验证实干扰组QBC939细胞系中miR-133a-5p(0.70±0.08)表达显著低于对照组(1.43±0.34);干扰组QBC939细胞系中c-met(0.09±0.02)表达也显著低于对照组(1.01±0.07),差异有统计学意义(P0.01)。Transwell小室实验证实干扰组中胆管癌QBC939细胞系迁移(69.5±8.3)和侵袭(19.3±3.3)能力明显低于对照组,差异均有统计学意义(P0.01)。结论:MicroRNA-133a-5p的低表达可以明显减弱c-met的表达,同时可以抑制胆管癌QBC939细胞系的迁移和侵袭能力,miR-133a-5p有可能成为胆管癌基因表达调控的新靶点。     

miR-520a-3p对肾癌细胞增殖、迁移、侵袭和凋亡的影响

目的 探讨miR-520a-3p在肾癌细胞系中的表达水平及其对肾癌细胞增殖、迁移、侵袭和凋亡的影响.方法 通过实时荧光定量聚合酶链式反应检测miR-520a-3p在正常肾小管上皮细胞HK2和肾癌细胞系786-O、A498、OS-RC-2及ACHN中的表达水平.选取miR-520a-3p表达水平最低的肾癌细胞系786-O...     

miR-18a-3p在肝癌中的表达及其调控ADCY1表达对肝癌细胞侵袭及增殖的影响

背景与目的 研究表明多种microRNA（miRNA）可能在肝癌的发生发展中发挥重要作用,其作用机制仍值得进一步研究和探讨。因此,本研究从已报道的肝癌差异表达miRNA中进一步筛选关键miRNA,并验证和探讨其作用机制。方法 从已发表的研究中筛选出肝癌组织及肝癌患者血清/血浆中与正常肝组织及正常血清/血浆中共同的差异表达miRNA;用qRT-PCR在正常肝细胞与肝癌细胞中对筛选出的目标miRNA表达情况进行验证;用过表达和抑制的方法观察目标miRNA对肝癌细胞侵袭能力（Transwell实验）与增殖能力（MTT实验）的影响,以及在30例临床标本中检测目标miRNA的表达并通过KM plotter网站分析其对肝癌患者生存的影响;通过miRDB和GEPIA数据库预测和分析目标miRNA的靶基因,并用逆转实验和双荧光素酶报告实验进一步验证。结果 在肝癌组织（vs.正常肝组织）及肝癌患者血清/血浆（vs.正常人血清/血浆）中共同高表达的miRNA有4个（miR-18a-3p、miR-221-3p、miR-222-3p、miR-224-3p）,共同低表达的miRNA有2个（miR-26a-3p、miR-125b-3p）。qRT-PCR实验证实,与正常肝细胞比较,miR-18a在肝癌细胞中高表达,miR-26a在肝癌细胞中低表达（均P<0.05）。过表达/抑制miR-18a-3p表达能促进/降低肝癌细胞的侵袭及生长能力（均P<0.05）,而过表达/抑制miR-26a-3p对肝癌细胞的侵袭及生长能力影响无不法确定。分析结果显示,ADCY1是miR-18a-3p的靶基因,过表达ADCY1能部分逆转miR-18a-3p对肝癌细胞的上述作用,同时,表达上调的miR-18a-3p能通过结合到ADCY1 mRNA 3''UTR抑制ADCY1的表达。结论 miR-18a-3p可能在肝癌的发生发展中起了关键作用,其在肝癌中表达上调,并能通过抑制下游靶基因ADCY1的表达增强进肝癌细胞的侵袭和增殖能力。     

瘢痕疙瘩microRNA表达谱的筛选及miR-199a-5p生物功能的初步研究

目的 探讨并筛选出瘢痕疙瘩相关微小RNAs(miRNAs),检测其对瘢痕疙瘩成纤维细胞(keloid fibroblasts,KF)增殖的影响.方法 收集手术切除瘢痕疙瘩及正常皮肤组织标本各8例,采用基因芯片检测瘢痕疙瘩与正常皮肤组织差异表达的miRNA,并采用qRT-PCR验证,在瘢痕疙瘩成纤维细胞株中转染miRNA模拟物,模拟细胞中成熟miRNA的高表达,用EdU检测方法检测其对瘢痕疙瘩成纤维细胞增殖的影响.结果 ①通过芯片检测发现包括miR-199a-5p在内的17个差异表达的miRNAs.②qRT-PCR验证结果显示miR-199a-5p表达下调,与芯片检测结果相一致.③miR-199a-5p模拟物转染组与阴性对照组EdU的阳性率分别为(20.72±2.50)％和(27.68±4.92)％,miR-199a-5p模拟物转染组瘢痕疙瘩成纤维细胞的增殖率下降(t=2.183,P=0.047).同时,细胞的生长周期分布也发生改变,MiR-199a-5p模拟物转染组S期与G2/M期细胞百分比分别为(33.93±1.30)％和(10.87±0.80)％,阴性对照组分别为(31.39±0.79)％和(9.27±0.46)％,差异有统计学意义(P＜0.05).结论 ①瘢痕疙瘩中miRNA的差异表达有组织特异性;②miR-199a-5p在瘢痕疙瘩中的显著低表达,可影响瘢痕疙瘩成纤维细胞周期分布,抑制瘢痕疙瘩成纤维细胞增殖,提示miR-199a-5p可能参与了瘢痕疙瘩成纤维细胞增殖的调控.     

Circ_0000069 contributes to the growth,metastasis and glutamine metabolism in renal cell carcinoma (RCC) via regulating miR-125a-5p-dependent SLC1A5 expression

BackgroundCircular RNAs (circRNAs) have emerged as critical mediators in various cancers, including renal cell carcinoma (RCC). In the present research, the functions of circ_0000069 in RCC were explored.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) assay, western blot assay and immunohistochemistry (IHC) assay were performed for the expression of circ_0000069, microRNA-125a-5p (miR-125a-5p) and solute carrier family 1 member 5 (SLC1A5). Cell Counting Kit-8 (CCK-8) assay and 5′-ethynyl-2′-deoxyuridine (EdU) assay were performed for cell proliferation. Flow cytometry assay was manipulated for cell apoptosis. Transwell assay and wound-healing assay were utilized for cell invasion and migration. Glutamine metabolism level was evaluated by examining glutamine consumption, α-ketoglutarate production and glutamate production. Dual-luciferase reporter assay was used to analyze the relationships of circ_0000069, miR-125a-5p and SLC1A5. Murine xenograft model assay was conducted to analyze the function of circ_0000069 in vivo.ResultsCirc_0000069 level was abnormally upregulated in RCC tissues and cells. Knockdown of circ_0000069 inhibited the proliferation, invasion, migration and glutamine metabolism and promoted the apoptosis in RCC cells in vitro and restrained tumor growth in vivo. Circ_0000069 served as the sponge for miR-125a-5p. MiR-125a-5p inhibition ameliorated the effects of circ_0000069 knockdown on RCC cell malignant behaviors. SLC1A5 was identified as the target gene of miR-125a-5p. Moreover, miR-125a-5p overexpression repressed the progression of RCC cells, while SLC1A5 elevation abrogated the effect.ConclusionCirc_0000069 knockdown inhibited the carcinogenesis of RCC by regulating miR-125a-5p/SLC1A5 axis.     

上调lncRNA SNHG12与miR-199a-5p/FZD6轴对肝细胞癌细胞增殖、侵袭和上皮-间质转化的影响

背景与目的:肝细胞癌(HCC)是临床上常见的恶性肿瘤之一,侵袭、转移和术后复发是导致HCC患者死亡的主要原因.目前认为长链非编码RNA (lncRNA)的失调可能与各类癌症的发生和转移有关.有研究显示lncRNA SNHG12在HCC组织表达明显上调,但其具体功能尚不清楚.故本研究探讨lncRNA SNHG12在HCC...     

miR-135a-5p抑制胆囊癌细胞增殖的研究

目的:探讨miR-135a-5p对胆囊癌的作用及其机制。方法:采用实时PCR检测miR-135a-5p和VLDLR在23对胆囊癌与癌旁组织的表达。采用miR-135a-5p模拟物转染胆囊癌细胞,通过细胞增殖(CCK-8)曲线、单克隆集落形成实验和细胞周期实验(流式细胞仪),检测miR-135a-5p对胆囊癌细胞增殖的影响。用Western印迹和Luciferase双荧光素酶报告系统验证和VLDLR受miR-135a-5p的调控。结果:在23对胆囊癌与癌旁组织中,癌组织的miR-135a-5p表达水平低于癌旁组织。体外实验证实miR-135a-5p通过G1期阻滞抑制胆囊癌细胞的增殖,且可通过作用于VLDLR mRNA降低其蛋白质水平。结论:miR-135a-5p抑制胆囊癌的增殖,可能是胆囊癌治疗靶点。     

Benzodiazepines safeguards nerve cells from the toxicity of lidocaine via miR-133a-3p/EGFR pathway

BackgroundLidocaine was an anesthetic commonly used for analgesia, but the neurotoxicity could not be ignored. However, benzodiazepines could alleviate the toxicity when combined with other drugs.PurposeTo explore the molecular mechanism of benzodiazepines in protecting nerve cells after the induction of lidocaine.MethodsPC12 cells were induced by lidocaine (0 mM, 0.1 mM, 0.5 mM and 1 mM) first and then treated by benzodiazepines (0 μM–200 μM). RT-qPCR assays measured RNA expressions of epidermal growth factor receptor (EGFR) and microRNA-133a-3p (miR-133a-3p) in PC12 cell line, respectively. Western blot was for protein detections of EGFR and caspase-3. Flow cytometry assay assessed apoptosis and cellular viability was validated via Cell Counting Kit-8 (CCK-8) test. Bioinformatics analysis predicted the potential link between miR-133a-3p and EGFR and the binding was verified using the Dual luciferase reporter experiment.ResultsBenzodiazepines increased cellular viability of PC12 cells up to 100 μM while suppressed viability between 100 and 200 μM. Benzodiazepines (0 μM, 10 μM, 50 μM and 100 μM) did not regulate PC12 cell viability but promoted the viability of lidocaine-treated PC12 cells. Lidocaine downregulated miR-133a-3p RNA expression but facilitated EGFR mRNA expression, which was reversed after treated by benzodiazepines. MiR-133a-3p targeted and negatively regulated EGFR expressions in mRNA and protein levels. Furthermore, miR-133a-3p inhibitor and overexpressed EGFR transfection both restrained the decreased PC12 cell viability and prompted cell apoptosis caused by benzodiazepines.ConclusionBenzodiazepines restrained lidocaine-induced toxicity in PC12 cells which secured viability and reduced apoptosis via miR-133a-3p/EGFR pathway.     

反义miR-30a-5p对人脑胶质瘤细胞U251生长的抑制作用

目的 观察敲低microRNA(miR)-30a-5p表达对人脑胶质瘤U251细胞生物学特征的影响.方法 用脂质体介导转染寡聚核苷酸抑制物(miR-30a-5p inhibitor)于人脑胶质瘤U251细胞.采用实时聚合酶链反应(real-time PCR)检测转染后U251细胞的miR-30a-5p表达水平以鉴定抑制效果;噻唑蓝(MTT)比色法评价miR-30a-5p inhibitor抑制U251细胞生长的作用;Transwell实验检测细胞侵袭能力变化;流式细胞术检测细胞周期变化;Annexin V法检测细胞早期凋亡.结果 real-time PCR检测结果 显示转染miR-30a-5p inhibitor后,肿瘤细胞miR-30a-5p表达下降,细胞增殖活性降低,细胞侵袭能力明显受到抑制,细胞周期阻滞在G0/G1期及早期凋亡增加.结论 miR-30a-5p可能是癌微RNA(oncomiR)之一,有望成为人脑胶质瘤基因治疗的候选靶点.

Abstract：

Objective To investigate the effect of knock-down of miR-30a-5p on the biological characteristics of U251 glioblastoma cells. Methods miR-30a-5p inhibitor, mediated by Lipofectamine 2000, was transfected to U251 cells for knocking down miR-30a-5p. Real-time polymerase chain reaction (PCR) was conducted to detect the expression of miR-30a-5p in transfected cells. The cell proliferation rate was detected by methyl thiazol tetrazolium (MTT) assay, and cell cycle kinetics was examined by flow cytometry. The cell invasive ability was evaluated by Transwell assay and apoptosis detected by Annexin V assay. Results The expression of miR-30a-5p in the miR-30a-5p inhibitor group was significantly down-regulated. The cell proliferation activity and invasive ability were reduced. Cells were arrested in G0/G1 phase, and apoptosis was induced in cells transfected with miR-30a-5p inhibitor as compared to those of the cells transfected with scramble siRNA and control cells, so suppression of miR-30a-5p expression rendered the glioma cells harboring less aggressive phenotype. Conclusion miR-30a-5p is one of oncomiRs. It may be a candidate target miRNA for gene therapy of gliomas.     

SIX4和miR-103a-3p在胆囊癌组织中的表达及其对胆囊癌细胞增殖和侵袭的影响

目的 探究SIX同源盒蛋白4(SIX4)、微小RNA(miR)-103a-3p在胆囊癌组织中的表达情况,以及二者对胆囊癌细胞增殖、侵袭的影响。方法 胆囊癌组织及癌旁组织取自永州市中心医院2019年10月至2021年9月32例接受手术治疗的胆囊癌患者,并将体外培养的胆囊癌细胞系GBC-SD分为对照组、mimic NC组、miR-103a-3p mimic组、miR-103a-3p mimic+pc DNA3.1组、miR-103a-3p mimic+SIX4组。实时荧光定量PCR(qRT-PCR)检测SIX4 mRNA、miR-103a-3p水平;蛋白印迹法检测SIX4、增殖细胞核相关抗原Ki-67、增殖细胞核抗原(PCNA)以及基质金属蛋白酶(MMP)2、MMP9蛋白水平;CCK-8法检测细胞增殖情况;Transwell检测细胞侵袭情况。双荧光素酶验证SIX4 mRNA 3’UTR与miR-103a-3p的结合作用。结果 与癌旁组织相比,胆囊癌组织中SIX4 mRNA水平升高,miR-103a-3p水平降低(P<0.05)。miR-103a-3p靶向负调控SIX4。与对照组、mi...