【作者】 王娜娜；

【导师】 李玲；

【作者基本信息】 中国医科大学 ， 内科学， 2022， 博士

【摘要】 目的:近年来,随着糖尿病发病率在世界范围内的逐年上升,糖尿病及其并发症带来了深远的社会影响。糖尿病肾病(diabetes kidney disease,DKD)是糖尿病重要的并发症之一,目前占据中国终末期肾病病因的第2位和肾病住院病因的第1位。糖尿病肾病是一种以进行性肾纤维化为特征的肾脏疾病,发病机制尚不十分清楚,可能有多种因素参与。目前经典的DKD发病过程中是以肾小球理论为中心提出,但是近期研究发现,糖尿病肾病早期肾小管病变独立于肾小球病变更早的出现,并且严重肾小管病变的占比高于典型肾小球结构改变,这提示肾小管损伤性改变也是DKD的重要病理改变。因此,尽管肾小球在DKD发病过程中发挥着重要的作用,但是管间质才是疾病进程的主要“驱动力”。在肾小管结构改变机理中,的上皮-间质转化(epithelial-mesenchymal transition,EMT)机制已成为当前研究的一个热门课题。研究结果表明,在肾脏上皮细胞中,阻断肾脏上皮细胞的EMT能有效地减少肾脏纤维化,改善肾脏功能,这说明,EMT的发生可能参与肾脏纤维化的发生发展过程,但目前对其分子调节机制的研究甚少。N-myc下游调节基因(N-myc downstream regulated gene,NDRG)家族是新发现的一族新基因,对其研究发现NDRG家族参与了EMT过程,NDRG过表达可以明显抑制细胞EMT的发生。NDRG家族成员NDRG1在肾脏表达丰富,但目前尚无关于NDRG1对糖尿病肾病EMT作用的研究。因此,本研究针对NDRG1在高糖诱导肾病模型过程中的具体调控作用进行深入研究,以期明确NDRG1基因参与糖尿病肾病的具体分子机制,为糖尿病肾病基础理论研究提供新的方向。研究方法:第一部分:全转录组测序分析用含5.6mmol/L葡萄糖的DMEM培养基培养肾小管上皮细胞HK2细胞,高糖组利用含30mmol/L葡萄糖的DMEM培养基培养HK2细胞,构建高糖诱导肾小管上皮细胞EMT模型,培养72小时后,收集细胞,行全转录组测序(transcriptome sequencing,RNA-seq),将经分析得到的差异表达m RNA进一步进行GO分析和KEGG富集分析。根据结果,结合m RNA、mi RNA和circRNA测序结果,进一步分析内源性竞争抑制(Ce RNA)分析,获得候选Ce RNA调控网络。第二部分:NDRG1抑制高糖诱导肾小管上皮细胞的上皮-间质转化过程研究首先,分别在葡萄糖终浓度为5.6mmol/L、30mmol/L和45mmol/L的DMEM培养中培养HK2细胞,构建高糖诱导HK2细胞EMT模型。根据预实验结果,在随后的研究中,最终选择了30mmol/L葡萄糖的DMEM培养基为造模浓度。以30mmol/L葡萄糖的高糖DMEM培养基培养HK2细胞,72小时后收集细胞,行实时荧光定量PCR(Quantitative Real-time PCR,q RT-PCR)、Western-blot、免疫荧光分析NDRG1、信号传导与转录激活因子蛋白3(Signal Transducer And Activator Of Transcription 3,STAT3)、Snail家族转录抑制因子1(Snai1 family transcription suppressor 1,Snai1)、间质细胞标志物α平滑肌激动蛋白(αsmooth muscle actin,α-SMA)和上皮细胞标志物钙黏蛋白E(Epithelial cadherin,E-cadherin)表达量的变化。然后,分别构建NDRG1基因过表达、基因敲除慢病毒质粒p SLenti-SFH-NDRG1、p SLenti-U6-sh RNA-NDRG1及对照质粒p SLenti-SFH-NC和p SLenti-U6-sh RNA-NC,转染HK2细胞,72小时后收集细胞,然后经q RT-PCR测定、Western-blot分析和免疫荧光分析分别测定NDRG1,STAT3,Snai1,α-SMA和E-cadherin的m RNA改变和蛋白质表达量的变化。然后应用STAT3抑制剂JSI-124抑制HK2细胞内STAT3后,检测对NDRG1、STAT3、Snai1、E-cadherin和α-SMA表达量变化的影响。最后,实验中构建db/db 2型糖尿病小鼠自发性糖尿病肾病模型,每2周监测小鼠体重、血糖变化,20周龄后检测24h尿蛋白定量。处死小鼠后取肾脏组织,分别行:观察形态学改变的光镜(HE、Masson染色)和电镜检查;抗NDRG1、E-cadherin和α-SMA的免疫荧光检测;提取总RNA,检测肾组织内NDRG1、STAT3、Snai1、E-cadherin和α-SMA表达量变化情况。对照组选用db/m小鼠和C57BL/6小鼠。实验期间,小鼠给予标准食谱,自由进食水。第三部分:circRNA 1860/hsa-mi R-125b-5p/NDRG1 Ce RNA调控网络对EMT作用研究首先通过全转录组基因测序筛选候选的circRNA和mi RNA,通过q RT-PCR法检测高糖培养下HK2细胞候选circRNA和mi RNA表达量的变化,确定最终的Ce RNA调控网络为circRNA 1860/hsa-mi R-125b-5p/NDRG1。对比分别以convergent引物和divergent引物扩增的RT-PCR产物,以鉴定circRNA1860的环形结构。对比高糖下circRNA 1860和hsa-mi R-125b-5p的亚细胞定位,初预测circRNA1860/hsa-mi R-125b-5p是否通过Ce RNA方式调控NDRG1的表达。分别构建circRNA1860的过表达及敲除载体,转染HK2细胞,72h后收集细胞,q RT-PCR检测circRNA1860、hsa-mi R-125b-5p、NDRG1、α-SMA和E-cadherin的m RNA表达量变化情况;通过Western-blot法检测NDRG1、α-SMA和E-cadherin三种蛋白质的表达量变化。分别应用hsa-mi R-125b-5p的模拟物和hsa-mi R-125b-5p的抑制物,转染HK2细胞,72h后收集细胞,q RT-PCR检测hsa-mi R-125b-5p、NDRG1、E-cadherin和α-SMA;Western-blot法检测NDRG1、E-cadherin和α-SMA的蛋白质表达变化。最后应用AGO2-RIP及双荧光素酶法检测分析hsa-mi R-125b-5p与NDRG1 m RNA、circRNA1860和hsa-mi R-125b-5p的结合。结果:第一部分:肾小管上皮细胞高糖诱导EMT全转录组分析与对照组相比,全转录组高通量测序中共获得66072种基因m RNA的表达情况,高糖组差异表达基因共194个,其中上调131个,下调63个;共获得1050种mi RNA的表达,与对照组相比,高糖组差异表达mi RNA共24个,其中上调10个,下调14个;共获得6245种circRNA的表达情况,经对比,高糖组表达差异性显著的circRNA共39个,其中上调22个,下调17个。实验中分别选取了基因表达上升和下降的差异倍数最明显的前5位m RNA进行初步验证,5种上调m RNA分别为:EIF2S3B、SMIM15、SLC28A3、BASP1、C12orf45;5种下调的m RNA分别为:RIMBP3B、HSPA6、HID1、LAMP3、NDRG1。经q RT-PCR初步验证,确定的上调基因是EIF2S3B、SMIM15、BASP1,确定的下调基因是NDRG1。结合公共数据库查询比对上述蛋白的功能及通路,其中NDRG1与EMT相关。因此,在后续的研究中,实验研究了NDRG1在肾小管上皮细胞EMT的高糖诱导机制。经Targetscan和Miranda数据库的预测,候选的circRNA-mi RNA-m RNA Ce RNA网络共有26个circRNA节点,mi RNA节点共24个,m RNA 677个,组成5870种可能的候选Ce RNA互作网络。筛选其中NDRG1相关节点,获得5个circRNA节点,5个mi RNA节点,形成6种可能的互作网络,用于后续研究。第二部分:NDRG1抑制高糖诱导肾小管上皮细胞的上皮-间质转化过程研究在含30mmol/L葡萄糖的DMEM高糖培养条件下,HK2细胞中E-cadherin m RNA的表达量下降,而α-SMA m RNA的表达量增多。Western-blot分析结果与q RT-PCR结果一致,高糖下,HK2细胞内上皮标记物E-cadherin蛋白表达量下降,α-SMA蛋白表达量增多。通过对细胞形态的观察,发现HK2细胞发生了楔形变,划痕实验及Transwell实验证明其迁移能力增强。这说明,在高糖条件下,HK2细胞发生了EMT。同时,通过q RT-PCR和Western-blot分析,高糖下HK2细胞内NDRG1的m RNA和蛋白表达量均下降,这说明NDRG1可能参与了高糖环境下HK2细胞的EMT过程。HK2细胞转染p SLenti-SFH-NDRG1慢病毒颗粒过表达NDRG1后,q RT-PCR和Western-blot结果显示,HK2细胞内NDRG1表达升高,STAT3,Snai1的表达下降,E-cadherin表达增多,α-SMA表达减少。转染p SLenti-U6-sh RNA-NDRG1敲除NDRG1后,HK2细胞内NDRG1的表达下降,STAT3,Snai1的表达量升高,E-cadherin的表达量下降,而α-SMA表达量增多。上述结果说明,NDRG1能够抑制EMT的发生,并可能与STAT3/Snai1通路有关。应用STAT3抑制剂JSI-124抑制HK2细胞内p STAT3后,Snai1表达下降,E-cadherin表达增多,α-SMA表达下降。并且JSI-124能够抑制过表达NDRG1所造成的EMT。上述结果进一步表明,NDRG1通过STAT3/Snai1通路抑制了高糖下HK2细胞EMT的发生。与对照组相比,db/db糖尿病肾病小鼠模型中,db/db组小鼠体型肥胖,饮水量增多,进食量增多,尿量增多。HE染色镜下可见体积增大的肾小球,肾小囊变小消失,肾小球基底膜增厚。在肾小管基底膜增厚的同时,肾小管结构内可见明显的细胞水肿的病理改变,肾小管上皮细胞内可见颗粒样和空泡样变性。Masson染色显示,肾小球内基底膜基质明显增多,基底膜增厚,肾小管基底膜蓝染的纤维蛋白原明显增多,肾小管基底膜增厚。电镜下,肾病组小鼠肾小球基底膜均质性明显增厚,系膜基质增多。肾小管上皮细胞线粒体数目减少,细胞形态肿胀,线粒体嵴结构紊乱。db/db组小鼠组织内NDRG1和E-cadherin表达量下降,STAT3、Snai1和α-SMA表达量上升。免疫荧光结果显示,肾病小鼠肾脏内E-cadherin表达量明显降低,α-SMA的表达量明显增多,NDRG 1在肾小管内表达量明显降低。并且,NDRG1蛋白只在肾小管内特异性表达,未在肾小球结构内观察到NDRG1的表达。第三部分:circRNA 1860/hsa-mi R-125b-5p/NDRG1 Ce RNA调控网络对EMT作用研究从全转录组高通量分析结果中筛选出可能与NDRG1基因表达有关的5种circRNA和5种mi RNA,经q RT-PCR验证后,最终选择circRNA 1860和hsa-mi R-125b-5p用于后续研究。circRNA 1860的Convergent引物能够使c DNA和g DNA均能扩增出条带,但是Divergent引物只能使c DNA扩增储条带,而g DNA不能扩增出条带。RNase处理后琼脂糖凝胶电泳显示circRNA1860条带仍然存在。测序鉴定结果显示circRNA 1860由FBXL5的2-10外显子反向剪切环化形成。因此,circRNA1860是环状结构。过表达circRNA1860后,与对照组相比,hsa-mi R-125b-5p的表达量明显降低,NDRG1 m RNA表达增多。而敲除circRNA1860后,hsa-mi R-125b-5p的表达量明显升高,NDRG1表达减少。Western-blot结果与q RT-PCR一致。证明circRNA1860可以促进NDRG1基因的表达,可能通过与hsa-mi R-125b-5p的竞争性结合发挥调控作用。应用hsa-mi R-125b-5p模拟物,转染HK2细胞后,q RT-PCR结果证明hsa-mi R-125b-5p表达量明显增加,而NDRG1 m RNA表达量下降;Western-blot结果表明,NDRG1蛋白表达量在hsa-mi R-125b-5p过表达后明显下降。相反地,NDRG1m RNA和蛋白表达水平在抑制hsa-mi R-125b-5p后有明显的升高。原位探针杂交结果显示circRNA 1860和hsa-mi R-125b-5p主要位于细胞质内。高糖下,circRNA 1860表达量明显下降,而hsa-mi R-125b-5p表达量明显上升,提示两者可能是通过竞争性抑制原理调控NDRG1基因的表达。AGO2-RIP结果显示AGO2与circRNA1860及hsa-mi R-125b-5p均有结合。双荧光素酶法检测结果显示NDRG1 m RNA的208-214位UUCAGGG能够与hsa-mi R-125b-5p的15-21位GAGUCCC结合。circRNA 1860的1759-1767位点GGUCUCAGGG碱基能够与hsa-mi R-125b-5p的12-21位点CCAGAGUCCC碱基特异性结合;初步证明了,circRNA 1860和hsa-mi R-125b-5p通过Ce RNA原理调控了NDRG1基因的表达。结论:NDRG1基因在高糖诱导的EMT过程中发挥了重要的调控作用。高糖下,NDRG1表达减少,激活STAT3/Snai1通路使肾小管上皮细胞发生EMT。高糖条件下NDRG1的减少与circRNA1860减少有关。在circRNA1860减少后,与hsa-mi R-125b-5p的竞争结合减少,hsa-mi R-125b-5p抑制了NDRG1的转录,调控了NDRG1的表达,进而引起EMT发生。本研究首次证明了NDRG1参与了高糖下肾小管上皮细胞EMT的发生,为糖尿病肾病的发病机理研究提供了新的理论基础。更多还原

【Abstract】 Objective:In recent years,with the increasing incidence of diabetes mellitus(DM)all over the world,heavy burden has been loaded to patients caused by DM and related complications.Diabetic kidney disease(DKD)is one of the most serious complications of diabetes.In present,it has been the the second cause of end stage renal failure and the first cause of hospital cause of renal disease in China.DKD is a kidney disease characterized by progressive renal fibrosis.The pathogenesis of DKD is not clear.There may be a number of factors involved.At present,the classic pathogenesis of DKD is based on the center of glomerulus theory,but recent studies have found that the pathological changes of tubular cells are separately earlier than glomerular changes at the early statge of DKD,and the proportion of severe tubular lesions was higher than that of typical glomerular structural changes in microalbuminuria DKD,suggesting that tubular injury is also an important pathological change of DKD.Therefore,it has been suggested that although the glomerulus is greatly affected in DKD,the tubulointerstitium is the main initiation of progression.Among mechanisms of tubular changes,epithelial-mesenchymal transformation(EMT)of renal epithelial cells is one of the hot spots.Recent studies have shown blocking EMT of renal epithelial cells can effectively alleviate renal fibrosis and improve renal function,indicating that EMT plays an important role in the process of renal fibrosis.Therefore,it is important to study the mechanism of EMT in diabetic kidney disease.N-MYC downstream regulated gene(NDRG)is a new discovered gene family in recent years.Studies on NDRG family have demonstrated that they are involved in EMT process in tumor disease,and overexpression of NDRG family significantly inhibited EMT process.NDRG1,an important member of NDRG family,is expressed highly in the kidney organ.But,there is no research on NDRG1 against EMT in DKD.Therefore,this study is desiged to explore the effect of NDRG1 on DKD to provide new theory mechanism of DKD.Methods: Part Ⅰ: Transcriptome sequencing analysis We used 30 mmol/L glucose DMEM medium as high glucose group and 5.6 mmol/L glucose DMEM medium as control.72 hours later,cells were collected and subjected to whole transcriptome sequencing(transcriptome sequencing,RNA-seq),the differentially expressed m RNA was analyzed by GO and KEGG enrichment analysis.According to the results,we combined the results of m RNA,miRNA and circRNA sequencing to further analyze the endogenous competition inhibition(Ce RNA)analysis and get the candidate Ce RNA regulatory networks.Part II: Study on inhibition effect of NDRG1 on epithelial-to-mesenchymal transition(EMT)of renal epithelial cells induced by hyperglycemia.Firstly,the renal tubular epithelial cells HK2 were cultured in DMEM medium with different gradients of glucose concentrations,and the cells were collected after 72 hours.The expression of NDRG1,STAT3,Snai1,E-cadherin and α-SMA were analyzed by q RT-PCR,Western-blot and immunofluorescence.The lentiviral plasmids p SLenti-SFH-NDRG1,p SLenti-U6-sh RNA-NDRG1 were constructed and transfected into HK2 cells,respectively.Cells were collected after 72 hours of 30mmol/L high glucose DMEM culture,the m RNA and protein expressions changes of NDRG1,STAT3,Snai1,E-cadherin and α-SMA were examined.Then,STAT3 inhibitor JSI-124 were used to inhibit STAT3 in HK2 cells,the expression levels of NDRG1,STAT3,Snai1,E-cadherin and α-SMA were detected.Db/db mice were adopted to establish diabetic kidney disease model.Body weight and blood glucose were measured continuely in each two-week period.At 20-week-age,24-hour urine protein was examined.The mice were sacrificed,and the renal tissues were obtained for observation under light microscopy(HE and Masson staining)and electron microscopy(EM).Immunofluorescence stain assay was adopted for detecting of NDRG1,STAT3,Snai1,E-cadherin and α-SMA.The m RNA expressions level of NDRG1,STAT3,Snai1,E-cadherin and α-SMA in renal tissues were analyzed by q RT-PCR method.Db/m and C57BL/6 mice were selected as controls.A standard diet and free water were given to all mice in the experiment process.Part III.circRNA 1860/hsa-miR-125b-5p/NDRG1 Ce RNA regulatory network for EMT Firstly,we tested candidate circRNAs and miRNAs by transcriptome gene sequencing by q RT-PCR in HK2 cells cultured in high glucose.The final target Ce RNA regulatory network was identified as circRNA1860/hsa-miR-125b-5p/NDRG1.Circular structure of circRNA1860 was identified by RT-PCR using divergent primer and convergent primer.Subcellular localization of circRNA1860 and hsa-miR-125b-5p was tested under high glucose.FISH suggested that circRNA1860/hsa-miR-125b-5p regulated the expression of NDRG1 by Ce RNA mechanism.The overexpression and knockout plasmids of circRNA1860 were constructed and transfected into HK2 cells.q RT-PCR was adopted to analyze the expression changes of hsa-miR-125b-5p,NDRG1,E-cadherin and α-SMA.Western-blot analysis assay was used to detected protein expressions of NDRG1,E-cadherin and α-SMA.The hsa-miR-125b-5p mimic and hsa-miR-125b-5p inhibitor were transfected into HK2 cells.72 hours later,cells were collected,and the m RNA expressions of hsa-miR-125b-5p,NDRG1,E-cadherin and α-SMA were detected by q RT-PCR.Western-blot method was used to examine the protein exression of NDRG1,E-cadherin and α-SMA.The binding of circRNA 1860 with hsa-miR-125b-5p were detected by dual luciferase method.Results: Part Ⅰ: Transcriptome sequencing analysis A total of 66072 m RNA expressions were obtained in the transcriptome sequencing.Among them,when compared with the HK2 cells cultured in control DMEM medium,194 genes were differentially expressed in high glucose DMEM medium,among which 131 genes were up-regulated and 63 genes were down-regulated.24 miRNAs were differentially expressed in the high glucose group,including 10 up-regulated and 14 down-regulated.39 circRNAs were differentially expressed cultured in the high glucose medium,including 22 up-regulated and 17 down-regulated circRNAs.The top five significant up-and down-regulated m RNAs were selected for preliminary verification.The five up-regulation m RNAs were EIF2S3 B,SMIM15,SLC28A3,BASP1 and C12ORF45;and the five down-regulated m RNAs were RIMBP3 b,HSPA6,HID1,LAMP3 and NDRG1.q RT-PCR results showed that EIF2S3 B,SMIM15,BASP1 were up-regulated genes and NDRG1 was down-regulated genes.Functions and pathways were searched for the above proteins in public databases,and NDRG1 was found to be associated with EMT.Therefore,the role of NDRG1 for EMT in renal tubular epithelial cells was further explored in the follow-up studies.According to Targetscan and Miranda databases,the predicted candidate circRNA-miRNA-m RNA Ce RNA network had 26 circRNA nodes,24 miRNA nodes,and 677 m RNAs,constituting 5870 possible Ce RNA interaction networks.After screening the NDRG1-related nodes,5 circRNA nodes and 5 miRNA nodes were obtained,forming 6 kinds of interaction networks.Therefore,the candidate Ce RNA network will be explored for further studies.Part II: NDRG1 participated in the process of epithelial-mesenchymal transition of renal epithelial cells under high glucose.Under the condition of high glucose DMEM medium,in which the concentration of glucose was 30mmol/L,the expression of E-cadherin m RNA decreased and α-SMA m RNA increased in HK2 cells.Meanwhile,the protein expression of E-cadherin decreased and α-SMA increased in HK2 cells.HK2 cells underwent wedge-shaped deformation,and the migration ability of HK2 cells was enhanced by wound-healing and transwell test.All the above results demonstrated that HK2 cells underwent EMT under high glucose.The results of q RT-PCR and Western-blot analysis showed that the m RNA and protein expression of NDRG1 decreased under high glucose.These results suggest that NDRG1 may be involved in the EMT process of HK2 cells under high glucose condition.NDRG1 overexpression was designed in HK2 cells transfected with p SLenti-SFH-NDRG1 lentivirus.After overexpression of NDRG1,the expression of NDRG1 and E-cadherin increased signigicantly,STAT3,Snai1 and α-SMA decreased by examined by q RT-PCR and Western-blot assays.These results suggested that NDRG1 overexpression can inhibit EMT and may be related to STAT3/Snai1 pathway.NDRG1 was knocked down by transfected with p SLenti-U6-sh RNA-NDRG1 vector.The expression of STAT3,Snai1,E-cadherin and α-SMA in HK2 cells was detecterd by q RT-PCR and Western-blot method.Results showed that the expression of NDRG1 and E-cadherin decreased signigicantly.However,the expression of STAT3,Snai1 and α-SMA increased after NDRG1 knocked out.Then,JSI-124,a STAT3 inhibitor was used to inhibit STAT3.Results showed the expression of Snai1 and α-SMA decreased and the expression of E-cadherin increased.Thus,NDRG1 inhibited EMT in HK2 cells under high glucose concentration via STAT3/Snai1 pathway.Spontaneous DKD was constructed by db/db mice model.The db/db mice were in obese,with polydipsia,polyphagia and polyuria.In H-E staining,enlarged glomerular,diminished renal capsule,and thickened glomerular basement membrane were observed.In tubular cells,extensive edema,granular and vacuolar degeneration,and thickened basement membrane were observed.Masson’s staining showed increased basement membrane stroma in glomerulus and increased fibrinogen in tubulobasement membrane with thickened basement membrane.Under the electron microscope,db/db kidney glomerular cells had homogeneous thickened glomerular basement membrane and increased mesangial matrix.The number and morphology of mitochondria in renal tubular epithelial cells were mainly decreased.In db/db mice,the expression of NDRG1 and E-cadherin decreased,while the expression of α-SMA,STAT3 and Snai1 increased in kidney.The immunofluorescence results showed that NDRG1 gene was expressed specifically in renal tubules,but not in glomeruli.And the NDRG1 expression level decreased significantly in db/db mice kidney.Part III.The circRNA 1860/hsa-miR-125b-5p/NDRG1 Ce RNA regulatory network for EMT In the study,five circRNAs and five miRNAs were screened by q RT-PCR,which might be related to NDRG1 gene expression.Ultimately,we selected circRNA1860 and hsa-miR-125b-5p for follow-up studies.The convergent primers and divergent primers were designed and amplified by RT-PCR.After detection,the c DNA could be amplified by divergent primer,while not g DNA.After RNase treatment,agarose gel electrophoresis showed that circRNA1860 bands still existed.Sequencing results showed that circRNA 1860 was formed by reverse splicing cyclization of exon 2-10 of FBXL5.Therefore,circRNA1860 is a ring structure.After overexpression of circRNA 1860,the expression of NDRG1 m RNA was increased.However,the expression of NDRG1 was decreased after knockdown of circRNA 1860.The results of Western-blot were in accordance with those of q RT-PCR.The m RNA expression of hsa-miR-125b-5p was significantly increased by hsa-miR-125b-5p mimic,and NDRG1 m RNA and NDRG1 protein was significantly decreased afterward when testing by q RT-PCR and Western-blot analysis.Conversely,both NDRG1 m RNA and protein expression levels increased after inhibition of hsa-miR-125b-5p by hsa-miR-125b-5p inhibitor.Results of in situ hybridization of circRNA 1860 and hsa-miR-125b-5p showed that circRNA 1860 and hsa-miR-125b-5p were mainly located within the cytoplasm.Under high glucose condition,the expression of circRNA 1860 decreased and hsa-miR-125b-5p increased significantly,suggesting that they may regulate expression of NDRG1 by competitive inhibition.Dual-luciferase assay showed that circRNA 1860 was able to bind specifically to hsa-miR-125b-5p,and hsa-miR-125b-5p could bind with NDRG1 m RNA.Therefore,circRNA 1860/hsa-miR-125b-5p regulated the expression of NDRG1 gene through Ce RNA mechanism.Conclusion: NDRG1 gene can inhibit EMT in tubular cells induced by high glucose.Under high glucose,the expression of NDRG1 decreased,EMT was actived via STAT3/SNAI1 pathway by decreased NDRG1.The reduction of NDRG1 was related to decreasing of circRNA 1860.circRNA 1860 regulated the transduction of NDRG1 by competing combining with hsa-miR-125b-5p,thus causing the occurrence of EMT.This is the first study to demonstrate that NDRG1 is involved in the occurance of EMT in renal tubular epithelial cells under high glucose,which provides a new potential direction for the study of DKD.更多还原