肿瘤坏死因子相关凋亡诱导配体受体在肾癌中的分布及其意义

目的：研究肿瘤坏死因子相关诱导凋亡配体（TRAIL）受体在肾癌组织中的分布及其意义。方法：采用RT－PCR及NorthernBlot的方法检测TRAIL受体在肾癌组织及正常肾组织,肾癌细胞系GRC－I和正常肾小管细胞系HK－2中的表达。结果：死亡受体DR4、DT4在肾癌组织及正常肾组织、肾癌细胞系GRC－I和正常肾小管细胞系HK－2中强表达;假受体DcR-1在正常肾组织和正常肾小管细胞系HK－2中强表达;假受体DcR-2未见表达。结论TRAIL基因在肾癌肿瘤细胞的凋亡机制中可能发挥重要的作用。     

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...     

T细胞因子4在肾癌中的表达及其意义

目的　研究Wnt/Frizzled信号通路在核内的转导及其主要信号分子T细胞因子 4(TCF4)在肾癌发生发展中的作用。　方法　采用RT PCR及NorthernBlot的方法检测了TCF4基因在肾癌组织及正常肾组织 ,肾癌细胞系GRC I和正常肾小管上皮细胞系HK 2中的表达。　结果　TCF4在肾癌组织及细胞系中强表达 ,且存在剪接性突变 ,形成了 30 0bp左右的突变基因。　结论　TCF4作为Wnt/Frizzled信号通路的核内信号分子 ,在肾癌发生和发展中具有重要作用 ,其致病机理与TCF4的剪接性突变有一定关系。     

EZH2在肾癌组织和肾癌细胞系中的表达及意义

目的：探讨EZH2蛋白在肾癌和肾癌细胞系中的表达情况及其与肾癌发生发展的关系。方法：应用Western blot检测12例肾癌和正常肾组织及两种细胞系ACHN和786—0中EZH2蛋白的表达,采用免疫组化法检测64例肾癌和12例正常肾组织中EZH2蛋白的表达情况,并分析与肾癌临床病理特征的关系。结果：West—ernblot检测结果显示,EZH2蛋白在正常肾小管上皮细胞系HK-2的表达极低,而肾癌细胞系786-0和ACHN的表达水平较高;正常肾组织EZH2蛋白表达阴性,肾癌组织均有不同程度的表达,差异有统计学意义（P〈0．05）。免疫组化结果示,肾癌和正常肾组织EZH2蛋白表达率分别为78．1％和16．7％,差异有统计学意义（P〈0．01）。EZH2蛋白表达率在局限性和局部进展性肾癌分别为63．9％和92．90A,差异有统计学意义（P〈0．01）;EZH2蛋白表达率在肾癌不同病理分级组和是否淋巴结转移组间差异有统计学意义（P％0．05）。结论：EZH2异常表达与肾癌的分期、分级和淋巴结转移有关,与肾癌的临床进展关系密切。     

透明质酸合成酶2在肾癌中的表达及临床意义

目的：研究透明质酸合成酶2在肾癌中的表达,并探讨其潜在的临床意义。方法：运用实时定量聚合酶链反应（qPCR）方法检测五种肾癌细胞（ACHN、Caki-1、OS-RC-2、786-O、SN12PM6）透明质酸合成酶三种亚型（HAs1、HAS2、HAs3）mRNA的表达,运用Westernblot方法进一步检测mRNA表达含量高的HAS亚型在五种肾癌细胞系及肾透明细胞癌（ccRCC）组织中的蛋白表达。结果：在五种肾癌细胞系中HAS2mRNA表达水平均明显高于正常肾小管上皮细胞（HK-2）,其中在。肾癌SN12PM6细胞系中表达水平最高（均P〈0．05）,HAS1mRNA的表达水平均明显低于正常肾小管上皮细胞（均P〉0．05）,而HAS3mRNA表达水平除肾癌Caki-1细胞系外均低于正常肾小管上皮细胞（P〉0．05）。在五种肾癌细胞系中HAS2蛋白均明显表达,而正常‘肾小管上皮细胞无明显表达。在肾透明细胞癌组织中HAs2蛋白表达也明显高于相应癌旁组织。结论：透明质酸合成酶2在多种肾癌细胞系和肾透明细胞癌组织中均明显表达,提示透明质酸合成酶2可能在肾癌尤其在肾透明细胞癌发生发展过程中起着至关重要的某种作用,并可能成为新的肾透明细胞癌分子标志物。     

OIP5基因在肾癌中的表达及作用

目的:探讨OIP5基因与肾癌发生发展的相关性,及下调OIP5基因对肾癌细胞增值活力、侵袭力及成瘤能力的影响。方法:利用RT-PCR及Real-time PCR在4对肾癌组织及癌旁正常组织中检测OIP5的表达情况,利用Real-time PCR在肾正常细胞系HK-2及肾癌细胞系786-O中检测OIP5基因的表达情况。转染shRNA下调OIP5基因后,检测肾癌细胞的增殖活力、侵袭能力及成瘤能力等。进一步利用免疫组化的方法在裸鼠成瘤的组织中检测OIP5蛋白的表达。结果:我们发现OIP5基因在肾癌组织及肾癌细胞系中都明显的高表达。降低OIP5基因的表达后,肾癌细胞的增殖能力、侵袭能力及成瘤能力都明显的减弱了。结论:OIP5基因与肾癌的发生发展有关,OIP5可能是肾癌基因治疗的一个新靶点。     

肾癌相关新基因GYLZ-RCC18反义寡核苷酸转染对肾癌细胞系GRC-1的作用

目的：探讨肾癌相关新基因GYLZ－RCC18在肾癌发生发展中的作用机制。方法：采用逆转录PCR方法检测GYLZ－RCC18在肾癌和正常肾细胞系中的表达,将第一编码区起始处的20个碱基的反义和正义寡核苷酸导入肾癌细胞系GRC－1,连续观察对肾癌细胞生长、增殖凋亡、死亡率、形态学的影响。结果：GYLZ－RCC18在肾癌细胞系中表达明显高于正常肾细胞系;导入GYLZ－RCC18反义寡核苷酸后,GRC－1细胞的核分裂可明显减少,导致GRC－1细胞死亡,抑制癌细胞的生长和增殖活性,并可特异诱导GRC－1连续凋亡。结论：GYLZ－RCC18是肾癌发生发展密切相关的癌基因,其表达可促进肾癌细胞的生长和增殖,并通过抗癌细胞死亡和凋亡机制对癌细胞起保护作用。     

miR-1303靶向抑制LPAR3对肾癌786-O细胞增殖和迁移的影响及机制研究

目的:探讨微小RNA(miRNA)-1303通过靶向调控溶血磷脂酸受体3(LPAR3)的表达抑制肾癌786-O细胞增殖和迁移的机制。方法:采用实时荧光定量聚合酶链反应(qRT-PCR)检测肾癌细胞株(A498、ACHN、786-O、OS-RC-2)及正常肾小管上皮细胞HK-2中miR-1303的相对表达量。将miR-1...     

T细胞因子4显性负调节基因△NTCF4对肾癌细胞GRC—Ⅰ生物学性状的影响

目的观察T细胞因子4（TCF4）显性负调节基因△NTCF4对肾癌细胞GRC—Ⅰ生长、增殖等生物学行为的影响。方法将缺失N末端的TCF4显忡负调节基因直核表达质粒pCDNA3-△NTCF4和pCDNA3空载体分别转染肾癌细胞GRC—Ⅰ，建立稳定表达△NTCF4基因的肾癌细胞系GRC—Ⅰ／△NTCF4和空载体细胞系GRC-1／Mock，光镜下观察细胞生长状态，绘制生长曲线，MTT法检测细胞增殖活性，免疫细胞化学和Western blot检测Wnt信号通路下游靶基因C-Myc、Cox-2蛋白表达水平。结果稳定表达△NTCF4基因的GRC—Ⅰ／△NTCF4细胞形态由圆形变为长条状，细胞生长速度变慢，核分裂相减少，出现向正常肾小管细胞逆转的趋势；GRC-Ⅰ／△NTCF4细胞增殖活性受到明混抑制，与GRC—Ⅰ对照细胞相比抑制率达11．2％～35．5％（P〈0．05），而GRC—Ⅰ／Mock与对照细胞相比差异无统计学意义；表达显性负调节基因的肾癌细胞系GRC—Ⅰ／△NTCF4中Cox-2蛋白表达水平显著降低。结论转染T细胞因子4显性负调节基因△NTCF4能部分抑制肾煽细胞GRC—Ⅰ生长，降低Wnt通路靶基因C—Mye、Cox-2表达，为通过阻断Wnt信号通路对肾痛进行细胞信号治疗提供了实验依据。     

小干扰RNA对肾癌细胞Ki67基因表达及其增殖的抑制作用

目的探讨小干扰RNA(siRNA)对人肾癌细胞增殖基因Ki67表达及其增殖、凋亡的影响。方法将Ki67 siRNA(100 nmol/L)转染人肾癌786-0细胞。采用RT-PCR、免疫印迹、免疫组化技术检测Ki67 mRNA及蛋白表达,MTT法检测细胞增殖,免疫组化TUNEL法检测细胞凋亡。结果Ki67 siRNA处理组786-0细胞Ki67 mRNA表达(37．6±1．9)%、Ki67蛋白表达(46．4±0．9)%、免疫组化Ki67表达吸光度(A)值52．5±2．3,阴性siRNA对照组分别为(97．3±0．9)%、(95．3±0．9)%、114．5±4．9,2组比较差异均有统计学意义(P＜0．01)。Ki67 siRNA处理组细胞增殖抑制率(63．6±1．6)%、凋亡细胞阳性率(41．7±0．6)%,阴性siRNA对照组分别为(2．8±0．2)%、(10．3±1．4)%,2组比较差异有统计学意义(P＜0．01)。结论肿瘤增殖基因Ki67 siRNA能抑制人肾癌786-0细胞Ki67基因表达,进而抑制其增殖,促进其凋亡,有望成为肾癌基因治疗的有效工具。     

肾小管上皮细胞内质网应激时NGAL表达改变的调控机制研究

目的:探讨人肾小管上皮细胞(HK-2细胞)发生内质网应激时,NGAL表达增加的上游调控机制。方法:将HK-2细胞分为对照组(正常HK-2细胞),TG(毒胡萝卜素,thapsigargin)组(5μmol/L TG处理8 h),单纯转染组(siRNAATF4试剂转染24 h),转染+TG组(siRNA-ATF4试剂转染24 h后,5μmol/L TG处理8 h),阴性对照组(siRNA-阴性对照物转染24 h),DMSO组(5μmol/L DMSO处理8 h)。采用Western blot检测各组细胞内质网源性转录因子(CHOP)、内质网分子伴侣葡萄糖调节蛋白78(GRP78)、中性粒细胞明胶酶相关性载脂蛋白(NGAL)、激活转录因子4(ATF4)的表达,采用Real-time PCR方法测得ATF4mRNA、NGALmRNA表达量。结果:与对照组相比,TG组细胞NGAL、ATF4、ATF4mRNA、NGALmRNA表达量显著提高(P <0. 05),而转染+TG组、单纯转染组、阴性对照组、DMSO组中ATF4及NGAL差异无统计学意义(P> 0. 05)。与TG组相比,转染+TG组ATF4、NGAL、ATF4mRNA及NGALmRNA表达量呈显著降低趋势(P <0. 05)。在TG组与转染+TG组细胞中,CHOP和GRP78呈过表达状态(P <0. 05),而转染+TG组细胞CHOP和GRP78提升趋势明显低于TG组细胞(P <0. 05)。结论:(1) TG可诱导人肾小管上皮HK-2细胞发生内质网应激反应。(2) HK-2细胞发生内质网应激反应时,抑制ATF4表达会引起NGAL降低,提示ATF4是NGAL表达的上游调控因子。(3) HK-2细胞发生内质网应激反应时,抑制ATF4不能阻止CHOP和GRP78发生过表达,但可降低其升高程度,提示ATF4及NGAL降低可能对内质网应激反应介导HK-2细胞损伤起到一定的缓解作用。     

激活转录因子5的克隆及其在真核细胞中的表达定位

目的克隆激活转录因子(ATF)5,构建其真核表达载体,观察其在细胞中的表达定位。方法根据人ATF5的cDNA序列设计引物,通过巢式PCR扩增ATF5全长及其N端(ATF5/N)和C端(ATF5/C),构建重组表达质粒pCS2MT-ATF5、pCS2MT-ATF5/N和pCS2MT-ATF5/C及融合绿色荧光蛋白(GFP)基因的质粒pEGFP-ATF5。将表达质粒转染293T和Cos7细胞,Westernblot检测其表达情况,荧光显微镜观察ATF5在293T和Hela细胞的定位。结果巢式PCR产物与预期大小一致,重组质粒pCS2MT-ATF5、pCS2MT-ATF5/N和pCS2MT-ATF5/C能表达携带Myc标签的蛋白Myc-ATF5、Myc-ATF5/N和Myc-ATF5/C,分子量分别为52、35和43×103。荧光显微镜观察发现融合了GFP的ATF5蛋白定位在细胞核。结论成功的克隆和表达ATF5基因为进一步研究其在肾肿瘤发生、发展过程中的作用奠定了基础。     

bcl-2高表达提高肾小管上皮细胞抗凋亡能力的研究

目的 观察凋亡相关基因bcl-2高表达抑制过氧化氢诱导肾小管上皮细胞凋亡的作用.方法 构建含有人bcl-2 cDNA的逆转录病毒真核表达载体PLXSN,脂质体法将重组质粒转染PA317细胞,G418筛选阳性克隆,鉴定后浓缩收集病毒上清,浓缩病毒液,感染人肾小管上皮细胞株HK-2,Western blot检测bcl-2 mRNA表达.5 mmol/L过氧化氢(HO2)诱导细胞凋亡后,流式细胞仪检测细胞凋亡发生变化.结果 流式细胞仪分析显示5 mmol/L H2O2:成功诱导肾小管上皮细胞凋亡,bel-2逆转录病毒感染肾小管上皮细胞后,bcl-2蛋白水平呈高表达,HK bcl-2组细胞凋亡数量(12.41±3.46)较HK-2组(19.62±4.20)显著减少(P<0.05).而转染空载体的对照组无明显变化(19.62±4.20,P<0.05).结论 bel-2蛋白高表达显著抑制氧化剂诱导的肾小管上皮细胞凋亡.     

P-GLYCOPROTEIN IN HK-2 PROXIMAL TUBULE CELL LINE

P-glycoprotein (PGP) is an efflux pump physiologically expressed in the apical membrane of the proximal tubular cells. PGP may play a role in the elimination of exogenous substances such as chemotherapeutic drugs, calcium channel blockers and immunosuppressors. The involvement of renal PGP in the transport of endogenous substrates is under investigation. HK-2 is an immortalized proximal tubule cell line from normal adult human kidney, reported to retain a phenotype indicative of a well-differentiated state. No data regarding expression and/or activity of PGP in this cell line are available. The aim of this study was to ascertain the usefulness of HK-2 cell line to investigate the properties and roles of PGP in proximal tubular cells. PGP expression in HK-2 cells was determined by immunoblotting analysis using the monoclonal antibody C219. The activity of PGP was assessed by measuring the transport of the fluorescent probe Rhodamine 123 (R-123) in intact cell monostrates. The interactions of putative PGP modulators, including verapamil and cyclosporin A were also evaluated. Western blot revealed a C219 immunoreactive band of about 150kDa consistent with the presence of PGP. HK-2 cells preloaded with R-123 rapidly effluxed the dye, the efflux being inhibited by verapamil. Verapamil and, to a major extent cyclosporin A, significantly increased R-123 intracellular accumulation. PGP immunoblottable amount was increased when cells were cultured in the presence of either cyclosporin A or dexamethasone. The results suggest that the HK-2 cells, among the various differentiation features of proximal tubules, retain also the expression of a functional PGP in their membranes and that both PGP activity and expression may be modulated by drugs. Therefore, HK-2 line appears a suitable and promising tool for the study in vitro of renal transport processes dependent on PGP.     

P-glycoprotein in HK-2 proximal tubule cell line

P-glycoprotein (PGP) is an efflux pump physiologically expressed in the apical membrane of the proximal tubular cells. PGP may play a role in the elimination of exogenous substances such as chemotherapeutic drugs, calcium channel blockers and immunosuppressors. The involvement of renal PGP in the transport of endogenous substrates is under investigation. HK-2 is an immortalized proximal tubule cell line from normal adult human kidney, reported to retain a phenotype indicative of a well-differentiated state. No data regarding expression and/or activity of PGP in this cell line are available. The aim of this study was to ascertain the usefulness of HK-2 cell line to investigate the properties and roles of PGP in proximal tubular cells. PGP expression in HK-2 cells was determined by immunoblotting analysis using the monoclonal antibody C219. The activity of PGP was assessed by measuring the transport of the fluorescent probe Rhodamine 123 (R-123) in intact cell monostrates. The interactions of putative PGP modulators, including verapamil and cyclosporin A were also evaluated. Western blot revealed a C219 immunoreactive band of about 150 kDa consistent with the presence of PGP. HK-2 cells preloaded with R-123 rapidly effluxed the dye, the efflux being inhibited by verapamil. Verapamil and, to a major extent cyclosporin A, significantly increased R-123 intracellular accumulation. PGP immunoblottable amount was increased when cells were cultured in the presence of either cyclosporin A or dexamethasone. The results suggest that the HK-2 cells, among the various differentiation features of proximal tubules, retain also the expression of a functional PGP in their membranes and that both PGP activity and expression may be modulated by drugs. Therefore, HK-2 line appears a suitable and promising tool for the study in vitro of renal transport processes dependent on PGP.     

长链非编码RNA MALAT-1通过EZH2-β-catenin信号通路调控肾癌细胞的增殖、凋亡及侵袭

目的探究长链非编码RNA肺腺癌转移相关转录因子1(LncRNA MALAT-1)在肾癌组织及肾细胞癌(RCC)细胞株中的表达情况及其在调控RCC细胞增殖、凋亡及侵袭过程中的作用机制。方法利用实时荧光定量聚合酶链反应(qRT-PCR)实验检测正常和RCC组织以及HK-2、786-O、ACHN及Caki-1细胞中MALAT-1的表达情况;将786-O细胞随机分为3组,分别转染MALAT-1-siRNA沉默载体(si-MALAT-1组)及MALAT-1-siRNA阴性表达载体(si-NC组),空白对照组加入PBS(Blank组)。采用CCK-8法、流式细胞术法、Transwell法检测细胞增殖、凋亡及侵袭能力;利用Western blot法检测Zeste基因同源物增强子2(EZH2)及β-catenin的蛋白表达水平。结果与正常组织及细胞株HK-2相比,肾癌组织及细胞株786-O、ACHN及Caki-1中MALAT-1的表达水平明显增加(P<0.05);与Blank组和si-NC组相比,si-MALAT-1组RCC细胞活性和侵袭能力明显降低、细胞凋亡率明显增加、EZH2及β-catenin的蛋白表达水平明显升高(P<0.05)。结论LncRNA MALAT-1在肾癌组织及RCC细胞中表达上调;抑制MALAT-1的表达能够抑制RCC细胞的增殖和侵袭,促进凋亡,其机制可能与下调EZH2-β-catenin信号通路的表达有关。     

Downregulation of miR-574-5p inhibits HK-2 cell viability and predicts the onset of acute kidney injury in sepsis patients

BackgroundIncreased levels of microRNA-574-5p (miR-574-5p) have been found to be associated with increased survival of septic patients, indicating the potential role of miR-574-5p in protecting against septic progression and complications. Acute kidney injury (AKI) is one of the most common and serious complications of sepsis. Therefore, the aim of this study was to test these hypotheses: (1) in a renal cell culture line (HK-2), upregulated expression of miR-574-5p increases, and downregulated expression of miR-574-5p decreases cell viability, and (2) serum levels of miR-574-5p from patients with sepsis and AKI are lower than those of patients with sepsis but no AKI.MethodsThe expression of miR-574-5p was regulated by cell transfection in HK-2 cells, and HK-2 cell viability was measured using the Cell Counting Kit-8. Serum miR-574-5p expression was analyzed using qRT-PCR. The predictive value of miR-574-5p for AKI onset was evaluated using the receiver operating characteristic curve and logistic regression analysis.ResultsThe overexpression of miR-574-5p promoted HK-2 cell viability. Fifty-eight sepsis patients developed AKI, who had significantly lower miR-574-5p expression. miR-574-5p expression was decreased with AKI stage increase and correlated with kidney injury biomarker and had relatively high accuracy to predict AKI occurrence from sepsis patients.ConclusionOverexpression of miR-574-5p in cultured HK-2 cells increases cell viability and knocked-down expression of miR-574-5p decreases cell viability. Consistently, septic patients with AKI were found to have less upregulation of miR-574-5p expression compared to septic patients without AKI. Thus, serum miR-574-5p may provide a novel biomarker for septic AKI.     

p53 inhibition attenuates cisplatin-induced acute kidney injury through microRNA-142-5p regulating SIRT7/NF-κB

Renal tubular epithelial cell apoptosis is the main mechanism of cisplatin-induced acute kidney injury. The role of microRNAs (miRNAs) in the apoptosis of renal tubular epithelial cells has been suggested, but the underlying mechanism has not been fully elucidated. We used microarray analysis to identify miR-142-5p involved in cisplatin-induced acute kidney injury. miR-142-5p was down-regulated in human renal tubular epithelial (HK-2) cells with cisplatin treatment. Notably, the overexpression of miR-142-5p attenuated the cisplatin-induced HK-2 cell apoptosis and inhibition of miR-142-5p aggravated cisplatin-induced HK-2 cell apoptosis. During cisplatin treatment, p53 was activated. The inhibition of p53 by pifithrin-α attenuated the cisplatin-induced kidney injury and up-regulated miR-142-5p expression. We also identified the Sirtuin7 (SIRT7) as a target of miR-142-5p. Furthermore, we demonstrated that the inhibition of SIRT7 prevented cisplatin-induced HK-2 cell apoptosis and decreased the expression of nuclear factor kappa B (NF-κB). Our data revealed that p53 inhibition could attenuate cisplatin-induced acute kidney injury by up-regulating miR-142-5p to repress SIRT7/NF-κB. These findings may provide a novel therapeutic target of cisplatin-induced acute kidney injury.