肝纤维化进程中miRNA-19b表达变化的研究

目的:观察微小RNA(miRNA)-19b在肝纤维化大鼠肝脏组织的表达,探讨miRNA-19b在肝纤维化进程中的作用.方法:将20只SD大鼠随机分成正常对照组和肝纤维化模型组.采用改良式复合因素法复制大鼠肝纤维化模型,分别在6、7周后,取各组大鼠肝脏组织,用荧光实时定量PCR方法检测miRNA-19b的表达.结果:6周后肝纤维化模型组大鼠肝脏组织miRNA-19b的表达低于正常对照大鼠组(P＜0.05),7周模型组大鼠肝脏组织miRNA-19b的表达低于6周模型组(P＜0.05).结论:miRNA-19b在肝纤维化大鼠肝脏组织中表达降低,可能与纤维化进程相关,且可能成为肝纤维的诊断以及预后的一个指标.     

下调UCA1通过靶向miR-23b及其下游靶基因抑制胃癌细胞的增殖和转移

摘要 目的：探讨长链非编码RNA尿路上皮癌相关基因1（UCA1）调控胃癌细胞增殖和转移的分子机制。方法：将人胃癌细胞株SGC7901分为：对照组、siRNA-NC组、siRNA-UCA1组、inhibitor-NC组和miR-inhibitor组、si-UCA1+inhibitor-NC组和si-UCA1+miR-inhibitor组。对SGC7901细胞分别转染siRNA-UCA1及阴性对照（siRNA-NC）、miR-inhibitor及阴性对照（inhibitor-NC）,未转染的细胞作为对照组。通过RT-qPCR检测细胞中UCA1和miR-23b-3p的水平。通过CCK-8法、伤口愈合实验和Transwell实验评价细胞的增殖、迁移和侵袭能力。通过Western blot分析细胞中IL6R、BCL2和HSP90B1蛋白的表达。使用pcDNA-UCA1/pcDNA-NC与pGL3-miR-23b-3p-WT/pGL3-miR-23b-3p-Mut共转染细胞,通过双荧光素酶报告实验验证UCA1与miR-23b-3p的靶向关系。结果：细胞培养48 h和72 h后,与对照组比较,siRNA-UCA1组的细胞活力分别降低了31.58%和31.40%（P<0.05）。与对照组比较,siRNA-UCA1组的细胞迁移率[(61.46±5.43)% vs (23.16±3.17)%]、侵袭细胞数量（109.17±9.66 vs 50.83±6.96）、IL6R、BCL2和HSP90B1的蛋白相对表达量均显著降低,而miR-23b-3p相对表达量升高（P<0.05）。与pGL3-miR-23b-3p-WT共转染后,与pcDNA-NC组比较,pcDNA-UCA1组的相对荧光酶活性降低了66.12%（P<0.05）。与si-UCA1+inhibitor-NC组比较,si-UCA1+miR-inhibitor组的细胞活力、细胞迁移率、侵袭细胞数量、IL6R、BCL2和HSP90B1的蛋白相对表达量均显著升高（P<0.05）。结论：下调UCA1通过靶向miR-23b-3p及其下游基因IL6R、BCL2和HSP90B1来抑制胃癌细胞的增殖和转移。     

miR-99b在牛肌卫星细胞分化中的表达及靶基因预测

miRNA作为进化保守的非编码RNA,对于细胞的发生、发展和增殖过程发挥重要的功能.miR-99b参与多种细胞的分化及增殖,但其在牛(Bas taurus)肌卫星细胞增殖分化中的表达以及可能靶向的基因尚不明确.本研究首先对牛基因组中miR-99b的基本信息进行详细介绍,采用茎环荧光定量PCR方法检测牛肌卫星细胞增殖及分...     

细胞焦亡与糖尿病肾病肾间质纤维化

肾间质纤维化是糖尿病肾病等慢性肾脏疾病进展至终末期肾病的不可逆性危险因素。细胞焦亡是一种新型程序性细胞死亡,通过诱导炎症反应的发生参与糖尿病肾病。焦亡引起的慢性炎症和纤维化被认为是糖尿病肾病发病的重要因素。因此,明确细胞焦亡与糖尿病肾病肾间质纤维化之间的关系对延缓糖尿病肾病进展至关重要。本文综述了近年来细胞焦亡在糖尿病肾病肾间质纤维化发病机制中的研究进展,以期为临床防治糖尿病肾病提供更多的理论基础。     

肾小球内皮细胞-间质转化在糖尿病肾病肾纤维化中的作用

肾纤维化是糖尿病肾病(diabetic kidney disease,DKD)重要的病理特征。肾小球内皮细胞-间质转化(endothelial-to-mesenchymal transition,EndoMT)可促进肾纤维化,在DKD的发生发展中起重要作用,但其具体机制仍有待研究。本文就EndoMT在DKD肾纤维化中的分子机制以及目前通过抗EndoMT治疗延缓DKD肾纤维化的研究进展作一综述,以期为DKD的临床治疗提供新的理论依据。     

miR-199b-5p在糖尿病肾病大鼠肾脏纤维化中作用机制

[目的]探索miR-199b-5p在糖尿病肾病大鼠肾脏纤维化中的作用机制。[方法]选择92只雄性大鼠,随机分为空白对照组、DN组、慢病毒对照组、miR-199b-5p敲低组,造模结束后第12 w处死大鼠。对比上述4组大鼠在肾组织病理学、肾功能指标,Klotho、Wnt、β-catenin等相关蛋白表达差异。[结果] DN组、慢病毒对照组的miR-199b-5p表达高于空白对照组和miR-199b-5p敲低组,miR-199b-5p敲低组高于空白对照组(1.08±0.24、3.27±0.12、3.22±0.53、1.62±0.15;P<0.05);DN组、慢病毒对照组的Scr、BUN、β-catenin、α-SMA、MMP-7均显著高于空白对照组和miR-199b-5p敲低组,miR-199b-5p敲低组高于空白对照组(78.66±2.12 vs 61.14±3.78;14.04±1.09 vs 9.26±1.73;32.33±2.57 vs 27.23±1.78;0.97±0.02 vs 0.34±0.35;3.49±0.28 vs 1.43±0.23;P<0.05);而D...     

阿魏酸钠联合缬沙坦对糖尿病肾病患者的疗效及对肾脏纤维化的影响

目的:研究阿魏酸钠联合缬沙坦对糖尿病肾病(diabetic kidney disease,DKD)患者的治疗效果及对肾脏纤维化的影响。方法:选择2017年1月～2018年12月山西医科大学附属晋中医院收治的101例糖尿病肾病患者,随机分为两组。两组均低蛋白饮食,口服他汀类药物调脂,口服降糖药和(或)胰岛素控制血糖。对照组加服缬沙坦8 0 mg/d治疗,观察组服缬沙坦联合静脉滴注0.3 g的阿魏酸钠,每天1次。两组均连续治疗1个月。观察两组肾功能指标、血糖指标以及肾脏纤维化指标的变化。结果:治疗后,观察组的治疗总有效率92.00%(46/50),显著高于对照组[68.63%(35/51)](P0.05)。治疗后,两组患者血清的纤维化相关指标Ⅲ型前胶原(procollagen typeⅢ,PCⅢ)和Ⅳ型胶原(typeⅣCollagen,CⅣ)均明显降低(P0.05),且观察组的明显低于对照组(P0.05);治疗后,两组的空腹血糖(fasting plasma glucose,FPG)、餐后2 h血糖(2 h plasma glucose,2 h PG)、血尿素氮(blood urea nitrogen,BUN)、血肌酐(serum creatinine,SCr)、β2-微球蛋白(β2-Microglobulin,β2-MG)、24 h尿白蛋白排泄率(24 h urine microalbumin excretion,24 h UAER)均明显降低(P0.05),且观察组的指标均显著低于对照组(P0.05),两组肾小球滤过率(GFR)增高(P0.05),且观察组指标明显高于对照组(P0.05)。结论:阿魏酸钠联合缬沙坦对DKD患者有较好的疗效,能明显延缓机体肾脏纤维化的进展速度,进而有效改善肾功能。     

TRB3基因敲除减轻糖尿病小鼠中骨骼肌萎缩和纤维化的研究

摘要 目的：探讨TRB3基因敲除对糖尿病小鼠中骨骼肌萎缩和纤维化的影响。方法：选择30只TRB3敲除（TRB3-/-）小鼠和30只C57/BL6J小鼠,随机分为4组,包括TRB3基因敲除糖尿病模型组（A组）、TRB3基因敲除正常对照组（B组）、普通对照组（C组）和糖尿病模型组（D组）。正常对照组小鼠喂养标准大鼠饲料,糖尿病模型小鼠使用腹腔注射小剂量链脲佐菌素的方法建立糖尿病小鼠模型。进行前肢握力测试、悬栅试验、肌肉功能试验,同时测定骨微结构各指标水平、抗I型胶原蛋白和抗III型胶原蛋白水平、萎缩基因MuRF1和Atrogin-1水平。结果：A组、B组、D组小鼠前肢握力显著低于C组,其中D组小鼠前肢握力最低（P<0.05）;A组、B组、D组小鼠悬栅试验倒挂时间显著低于C组,其中D组小鼠悬栅试验倒挂时间最短（P<0.05）;A组、B组、D组小鼠肌肉功能CSA值显著低于C组,其中D组小鼠肌肉功能CSA值最低（P<0.05）;A组、B组、D组小鼠骨微结构指标BMD、BV/TV、Tb.N、Tb.Th值显著小于C组,其中D组数值最低（P<0.05）;而Tb.Sp和SMI值显著大于C组,其中D组数值最高（P<0.05）;A组、B组、D组小鼠抗I型胶原蛋白和抗III型胶原蛋白水平显著高于C组,其中D组抗I型胶原蛋白和抗III型胶原蛋白水平最高（P<0.05）;A组、B组、D组小鼠萎缩基因MuRF1和Atrogin-1水平显著高于C组,其中D组萎缩基因MuRF1和Atrogin-1水平最高（P<0.05）。结论：TRB3基因敲除可显著减轻糖尿病小鼠中骨骼肌萎缩和纤维化,增加骨强度。     

外源性精胺对糖尿病肾病小鼠肾纤维化的影响及其机制

目的: 观察外源性精胺对糖尿病肾病(DN)肾纤维化的保护作用,并探讨其机制。方法: 24 只雄性 C57 小鼠随机分为正常组(Control)、糖尿病组(T1D)和精胺预处理组(T1D+Sp,每组 n=8)。一次性注射 STZ(60 mg/kg)复制 1 型糖尿病小鼠模型,精胺预处理组在 STZ 注射前两周每天腹腔注射精胺(Sp,5 mg/(kg·d)),随后隔天注射精胺,第 12 周处死小鼠,检测血清肌酐、尿素氮判断肾功能变化,HE、PAS 和 Masson 染色观察肾组织损伤和纤维化水平。Western blot 法检测小鼠肾组织中基质金属蛋白酶(MMP-2、MMP-9)、IV型胶原(Coll-IV)蛋白的表达。结果: 与 Control 相比,T1D 组血糖(5.67±0.22 vs 28.40±0.57 mmol/L)、肌酐(14.33±1.22 vs 30.67±4.73 μmol/L)、尿素氮(6.93±4.94 vs 22.00±1.04 mmol/L)明显升高(P＜0.05),肾组织基底膜增厚,胶原含量明显增加,MMP-2、MMP-9 和 Coll-IV 蛋白表达均升高(分别为 0.57±0.07 vs 1.06±0.20、47.00±0.04 vs 1.29±0.09和0.42±0.16 vs 0.95±0.18,P＜0.05),精胺预处理明显减轻上述变化。结论: 外源性精胺预处理通过调节 MMPs 与胶原的平衡减轻 DN 小鼠的肾纤维化。     

microRNA-181b通过靶定MLK2调节白血病细胞HL-60的增殖

microRNA在细胞的转移、凋亡、分化等生命过程中发挥着重要的作用. miR-181b在急髓性白血病组织中高表达, 通过靶定MLK2调节急髓性白血病细胞的增殖. 本结果提示, miR-181b在急髓性白血病的生物学过程发挥着重要的作用, 同时为急髓性白血病的治疗提供了新的解决方案.     

Downregulation of microRNA-23b protects against ischemia-reperfusion injury via p53 signaling pathway by upregulating MDM4 in rats

Total knee arthroplasty is a commonly performed safe procedure and typically executed in severe knee arthritis, but it also triggers ischemia-reperfusion injury (IRI). More recently, microRNAs (miRs) have been reported to play a contributory role in IRI through the key signaling pathway. Hence, the current study aimed to investigate the effect and specific mechanism of microRNA-23b (miR-23b), murine double minute 4 (MDM4), and the p53 signaling pathway in IRI rat models. First, the IRI model was established, and the expression pattern of miR-23b, MDM4, and the p53 signaling pathway-related genes was characterized in cartilaginous tissues. Then, miR-23b mimics or inhibitors were applied for the elevation or the depletion of the miR-23b expression and siRNA-MDM4 for the depletion of the MDM4 expression in the articular chondrocytes. By means of immunohistochemistry, quantitative real-time polymerase chain reaction, and Western blot analysis, IRI rats exhibited increased miR-23b expression, activated p53 signaling pathway, and decreased MDM4 expression. MDM4 was verified as a target gene of miR-23b through. Downregulated miR-23b increased the expression of MDM4, AKT, and Bcl-2, but decreased the expression of p53, p21, and Bax. In addition, a series of cell experiments demonstrated that downregulated miR-23b promoted articular chondrocyte proliferation and cell cycle entry, but inhibited articular chondrocyte apoptosis. The absence of the effects of miR-23b was observed after MDM4 knocked down. Our results indicate that silencing miR-23b could act to attenuate IRI and reduce the apoptosis of articular chondrocytes through inactivation of the p53 signaling pathway by upregulating MDM4, which provide basic therapeutic considerations for a novel target against IRI.     

MiR-874 alleviates renal injury and inflammatory response in diabetic nephropathy through targeting toll-like receptor-4

Diabetic nephropathy (DN) is a kind of diabetic complication with capillary damage, and its pathogenesis remains obscure. Recently, microRNAs have been identified as diagnostic biomarkers in various diseases including DN. Toll-like receptor 4 (TLR4) contributes to inflammation, and it has been implicated in diabetes pathophysiology. This study was designed to investigate the role of miR-874 and TLR4 in a streptozotocin (STZ)-induced DN rat model and glucose-induced mouse podocyte model. In the current study, we reported that miR-874 was markedly downregulated in DN rats and glucose-induced mouse podocytes compared with the corresponding control groups with the activation of TLR4. In addition, we observed that overexpression of miR-874 was able to alleviate renal injury in DN rats. The cell counting kit (CCK-8) assay and 5-Ethynyl-2′-deoxyuridine (EdU) assay demonstrated that glucose simulation significantly inhibited podocyte proliferation and induced cell apoptosis, which can be reversed by miR-874 mimics significantly. Notably, miR-874 overexpression dramatically attenuated the inflammatory response, indicated by the decreased levels of interleukin-6, L-1β, and tumor necrosis factor α (TNF-α). Finally, the binding correlation between miR-874 and TLR4 was confirmed by carrying out dual-luciferase reporter assay in our study. It was found that overexpression of miR-874 depressed TLR4 levels in podocytes. These findings implied for the first time that the overexpression of miR-874 repressed glucose-triggered podocyte injury through targeting TLR4 and suggested that miR-874/TLR4 axis might represent a pathological mechanism of DN.     

血管生成素样蛋白2在糖尿病肾病小鼠肾脏中的表达研究

利用半定量RT-PCR和免疫组化的方法同时从mRNA水平和蛋白质水平对血管生成素样蛋白2在不同病理阶段的2型糖尿病肾病模型小鼠--db/db小鼠肾脏中的表达情况进行了较为系统的分析.结果发现:a.在糖尿病前的db/db小鼠(4周龄的db/db小鼠),血管生成素样蛋白2与作为正常对照的db/m小鼠相比,差异不是很大,随着肥胖的加剧,高血糖、蛋白尿的出现,血管生成素样蛋白2在db/db小鼠肾脏中的表达无论从mRNA水平还是从蛋白质水平均显著升高.b.从免疫组化的分析结果来看,血管生成素样蛋白2主要分布于小鼠肾脏的肾小球部分,主要是沿毛细血管袢呈线性分布,其位置与足细胞的位置重叠,足细胞是小鼠肾脏中血管生成素样蛋白2的主要分泌细胞.c.小鼠肾脏血管生成素样蛋白2的表达水平似乎还与鼠龄相关:虽然变化幅度不是很大,但在周龄较大的小鼠(如20周龄以上),其表达水平相对较高.上述工作不仅印证了先前对2型糖尿病肾病患者肾小球基因表达谱的分析结果,更加明确了血管生成素样蛋白2与糖尿病肾病的相关性,同时揭示了血管生成素样蛋白2在正常小鼠和糖尿病肾病小鼠肾脏中的表达、分布和变化规律,有利于进一步揭示血管生成素样蛋白2的功能及其在糖尿病肾病发生、发展过程中的可能作用,探讨糖尿病肾病的分子机制.     

miR-125b靶向MCL1抑制胃癌 MGC-803细胞增殖

探讨mi R-125b对胃癌MGC-803细胞增殖的影响及机制,为阐明胃癌发病的分子机制提供实验依据.采用q RT-PCR和原位杂交,检测mi R-125b在正常胃黏膜(NGM)和胃癌(GAC)组织中的表达.将mi R-125b导入胃癌MGC-803细胞,观察mi R-125b高表达对MGC-803细胞增殖的影响.利用Targetscan 6.2软件及荧光素酶报告基因检测,分析mi R-125b对MCL1基因的靶向性作用.构建MCL1干扰载体,观察干扰MCL1基因表达对MGC-803细胞增殖的影响.结果发现,mi R-125b在胃癌组织中低表达,其表达与胃癌的分化程度及患者预后呈正相关,与TNM分期、淋巴结转移呈负相关(P0.01).mi R-125b高表达后MGC-803细胞的增殖降低、凋亡率增加、裂解caspase-3与裂解PARP表达增加(P0.01);mi R-125b与MCL1基因的3′UTR(2 613~2 620)结合,抑制MCL1的m RNA及蛋白质表达(P0.01);沉默MCL1基因表达后MGC-803细胞的增殖降低、凋亡率增加、裂解caspase-3与裂解PARP表达增加(P0.01).从而得出结论,mi R-125b在胃癌组织中低表达,其表达与胃癌组织分化程度、TNM分期、淋巴结转移及患者预后密切相关;mi R-125b靶向抑制MCL1基因表达,活化caspase-3信号通路,抑制MGC-803细胞增殖.     

MiR-92d-3p suppresses the progression of diabetic nephropathy renal fibrosis by inhibiting the C3/HMGB1/TGF-β1 pathway

The pathogenesis of diabetic nephropathy (DN) has not been fully elucidated. MicroRNAs (miRNAs) play an important role in the onset and development of DN renal fibrosis. Thus, the present study aimed to investigate the effect of miR-92d-3p on the progression of DN renal fibrosis. We used qRT-PCR to detect the expression levels of miR-92d-3p in the kidneys of patients with DN. Then, after transfecting lentiviruses containing miR-92d-3p into the kidneys of a DN mouse model and HK-2 cell line, we used qRT-PCR to detect the expression levels of miR-92d-3p, C3, HMGB1, TGF-β1, α-SMA, E-cadherin, and Col I. The expression levels of interleukin (IL) 1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in the HK-2 cells were detected through enzyme-linked immunosorbent assay (ELISA), and Western blotting and immunofluorescence were used in detecting the expression levels of fibronectin, α-SMA, E-cadherin, and vimentin. Results showed that the expression levels of miR-92d-3p in the kidney tissues of patients with DN and DN animal model mice decreased, and C3 stimulated HK-2 cells to produce inflammatory cytokines. The C3/HMGB1/TGF-β1 pathway was activated, and epithelial-to-interstitial transition (EMT) was induced in the HK-2 cells after human recombinant C3 and TGF-β1 protein were added. miR-92d-3p inhibited inflammatory factor production by C3 in the HK-2 cells and the activation of the C3/HMGB1/TGF-β1 pathway and EMT by C3 and TGF-β1. miR-92d-3p suppressed the progression of DN renal fibrosis by inhibiting the activation of the C3/HMGB1/TGF-β1 pathway and EMT.     

AL023001基因在2型糖尿病肾病小鼠中的差异表达分析

利用Affymetrix寡核苷酸基因表达谱芯片对2型糖尿病肾病模型动物——db/db小鼠的肾脏基因表达谱进行了研究.在此基础上,利用末端快速扩增法和RT-PCR的方法,对筛选出来的一个糖尿病肾病相关EST进行了cDNA克隆和表达分析.得到了一长为1.7 kb的cDNA片段.序列分析和网上数据库比对发现,此cDNA片段是小鼠表达序列AL023001的一部分.根据AL023001序列设计特异性引物,利用半定量RT-PCR的方法对AL023001在db/db小鼠肾脏、肝脏、脂肪、骨胳肌、脑等组织中的表达情况进行了分析,发现AL023001在db/db小鼠肾脏中的表达情况与基因芯片检测结果吻合,且AL023001在肝脏、脂肪、骨胳肌和脑等糖尿病肾病相关联组织中均有差异表达.这些结果提示：AL023001与db/db小鼠的糖尿病肾病具有相关性.上述工作有利于揭示AL023001基因的功能,探讨糖尿病肾病的分子机制.     

Dysregulation of microRNA-181b and TIMP3 is functionally involved in the pathogenesis of diabetic nephropathy

This study aimed to study the roleof microRNA (miR)-181b and its target TIMP3 in the development of diabetic nephropathy (DMN) via inhibiting the apoptosis of mesangial cells. Real-time polymerase chain reaction (RT-PCR) was adopted to compare the miR-181b expression between subjects with diabetic nephropathy (DN) and normal control. In addition, luciferase assays were utilized to explore the regulatory relationship between TIMP3 and miR-181b. Real-time PCR and densitometry analysis were conducted to measure the levels of TIMP3 mRNA/protein in DMN or in cells treated by miR-181b inhibitors, miR-181b mimics, and TIMP3 siRNA. And the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was adopted to study the effect of miR-181b on cell survival and apoptosis. miR-181b expression was much higher in the DN group, and the results of computational analysis identified TIMP3 as a miR-181b target. The luciferase activity of cells transfected with wild-type TIMP3 and mutant2 TIMP3 was significantly reduced, whereas the luciferase activity of cells transfected with mutant1 TIMP3 was evidently higher. Furthermore, a negative regulatory relationship was established between TIMP3 and miR-181b expression with a correlation efficient of −0.5351. The levels of TIMP3 mRNA/protein expression were apparently increased in the DN group. In addition, the treatment of cells with miR-181b mimics and TIMP3 siRNA remarkably lowered the levels of TIMP3 mRNA/protein, whereas the transfection of cells with miR-181b inhibitors notably elevated the expression of TIMP3 mRNA/protein. miR-181b promoted the survival of cells and inhibited their apoptosis. The miR-181b expression was related to the development of DMN and could be used as a prognosis biomarker of DMN in the patients with DM.