山药多糖通过miR-98-5p/TGFβR1分子轴调控肝癌细胞凋亡的机制研究

目的探究山药多糖(CYPS)通过miR-98-5p/TGFβR1分子轴调控肝癌(HCC)细胞凋亡的分子机制。方法qPCR检测miR-98-5p在不同HCC细胞系中的表达情况,使用不同浓度的CYPS处理Huh-7细胞,CCK-8检测细胞增殖活力,Annexin V-FITC/PI流式细胞术检测细胞凋亡,Western blotting检测TGFβR1和细胞凋亡相关蛋白Caspase-3、Caspase-8、Bcl-2、Bax的表达,双荧光素酶报告基因系统验证miR-98-5p与TGFβR1的靶向关系。结果与人正常肝细胞HL-7702相比,miR-98-5p在HCC细胞系中表达升高(均P<0.05),且在Huh-7细胞中的表达高于其他HCC细胞(P<0.05);CYPS处理可明显抑制Huh-7细胞的增殖活力并诱导细胞凋亡(均P<0.05),且10-3 kg/L CYPS对细胞增殖活力的抑制作用比其他浓度明显。双荧光素酶报告基因实验证实,miR-98-5p靶向调控TGFβ1。与10-3 kg/L CYPS组相比,miR-98-5p mimics可下调10-3 kg/L CYPS对细胞增殖活力(P<0.05)的抑制作用和对细胞凋亡(P<0.01)的促进作用,CYPS+miR-98-5p mimics+pcDNA-TGFβR1组中细胞增殖活力与CYPS+miR-98-5p mimics组相比明显降低(P<0.05),细胞凋亡水平明显升高(P<0.01)。结论CYPS可下调miR-98-5p,促进TGFβR1的表达,抑制Huh-7细胞增殖并诱导细胞凋亡。     

MiR-490-3p靶向HMGA2在骨肉瘤细胞凋亡中的作用

目的以人骨肉瘤MG-63细胞为研究对象,探讨miR-490-3p诱导MG-63细胞凋亡的作用及其分子机制。方法分别设立miR-490-3p mimics组(对照组)、慢病毒lenti-sh miR-490-3p组(药物组),每组设6个复孔。采用MTT法检测miR-490-3p对细胞生长抑制作用;采用AnnexinV-FITC/PI双染法检测不同实验条件下miR-490-3p对MG-63细胞凋亡率的影响;Western blot检测miR-490-3p对HMGA2、Bcl-2、BAX和Caspase-3蛋白表达的影响。结果 MiR-490-3p能抑制MG-63细胞增殖,药物组与对照组比较差异有统计学意义(P0.05)。经3次实验结果,药物组的早期凋亡的细胞比例为58.90%±0.89%,明显高于对照组的5.34%±0.34%,差异有统计学意义(P0.05)。通过Western blot检测MG-63细胞中HMGA2、Bcl-2、Bax、Caspase-3、Survivin蛋白表达,结果显示miR-490-3p转染后,促进凋亡蛋白Bax表达增加,抗凋亡蛋白Bcl-2表达降低,Bax或Bcl-2比率增高,而线粒体凋亡途径下游的Caspase-3蛋白被激活,表达量降低。结论 MiR-490-3p能显著抑制肿瘤细胞增殖,诱导细胞凋亡,其作用机制可能与靶向HMGA2的表达、上调Bax/Bcl-2蛋白表达比例、激活Caspase-3蛋白级联反应和调控Survivin蛋白表达有关。     

miR-148-5p靶向H型血管调控因子Slit3的实验研究

目的 观察绝经后骨质疏松症中H型血管调控因子Slit3表达差异,进一步探讨其上游靶向调控miRNAs。方法 雌性SD大鼠随机分假手术组和模型组,Real-time PCR和免疫组化检测骨组织Slit3 mRNA和蛋白表达水平;生物信息学预测可能与Slit3-3’UTR互作的miRNAs,构建野生型和突变型Slit3-3’UTR重组荧光素酶报告载体,和miRNAs表达载体共转染293T细胞,双荧光素酶报告基因测定荧光素酶活性验证miRNA对Slit3-3’UTR的靶向作用。结果 与假手术组相比,模型组Slit3 mRNA表达水平略上调和平均光密度显著上调,生物信息学预测Slit3-3’UTR与miR-148a-5p、miR-148b-5p、miR-410-3p、miR-129-5p和miR-374-5p存在碱基结合位点,野生型Slit3-3’UTR共转染miR-148b-5p荧光活性下调至空白对照组的68.91%,突变型Slit3-3’UTR共转染miR-148b-5p荧光活性未见下调。结论 Slit3可能是绝经后骨质疏松症H型血管的调控基因,miR-148b-5p与Slit3-3’UTR的靶向结合直接调控Slit3的表达水平。     

circDUSP16调节miR-126-5p/SHH轴对结直肠癌细胞增殖、凋亡和上皮间质转化的影响

目的:探究circDUSP16对结直肠癌(CRC)细胞增殖、凋亡和上皮间质转化(EMT)的影响及其作用机制。方法:qRT-PCR检测circDUSP16、miR-126-5p、SHH表达水平,CCK-8法测定细胞增殖,流式细胞术检测细胞凋亡,Western blot检测SHH、PCNA、Bcl-2、Bax、E-cadherin、N-cadherin蛋白表达,荧光素酶实验检测circDUSP16和miR-126-5p/SHH的靶向关系。结果:沉默circDUSP16组SW480细胞circDUSP16、SHH mRNA表达、OD450值(24 h、48 h、72 h)、Bcl-2和N-cadherin蛋白表达降低,miR-126-5p、凋亡率、Bax、E-cadherin蛋白水平升高(P<0.05);下调miR-126-5p减弱了沉默circDUSP16对SW480细胞的增殖、EMT发生的抑制,降低了细胞的凋亡能力;circDUSP16靶向负调控miR-126-5p表达,miR-126-5p靶向负调控SHH表达。结论:沉默circDUSP16可能通过上调miR-126-5p并下调SHH抑制SW480细胞增殖和EMT发生,促进凋亡。     

miR-6827-3p靶向CXCL16调控结直肠癌细胞HT-29凋亡的机制研究

目的:研究miR-6827-3p靶向CXCL16调控结直肠癌细胞HT-29的凋亡的机制。方法 :使用MiRBase在线分析MiRBase与CXCL16信使RNA的结合位点;将分析出的结合位点克隆进PmirGLO质粒,使用luciferase assay分析miR-6827-3p是否靶向这些结合位点;在结直肠癌细胞HT-29中过表达或者敲低miR-6827-3p后,使用Western blot分析CXCL16以及细胞凋亡关键蛋白的表达情况。结果:MiRBase分析后,发现miR-6827-3p与CXCL16具有3处匹配区域;克隆PmirGLO-CXCL16-3’UTR后,luciferase assay发现miR-6827-3p靶向CXCL16;当过表达miR-6827-3p时,CXCL16的表达量下降,细胞凋亡标志蛋白cleaved-caspase3、cleaved-caspase9、BAX表达量下降,Bcl-2表达量上升(P0.05);当敲低miR-6827-3p时,CXCL16的表达量上升,细胞凋亡标志蛋白cleaved-caspase3、cleaved-caspase9、BAX表达量上升,Bcl-2表达量下降(P0.05)。结论:miR-6827-3p可以通过靶向CXCL16抑制结直肠癌细胞HT-29的凋亡。     

重楼皂苷Ⅰ通过调控LncRNA CEBPA-AS1/miR-195-5p通路抑制瘢痕疙瘩成纤维细胞增殖及促进细胞凋亡的初步研究

目的:探讨重楼皂苷Ⅰ (PPI)是否通过调控长链非编码RNA CEBPA-AS1 (LncRNA CEBPA-AS1)/微小RNA-19 5-5p(miR-195-5p)通路影响瘢痕疙瘩成纤维细胞增殖及凋亡。方法:原代培养人瘢痕疙瘩成纤维细胞,使用不同浓度的PPI处理细胞,s i-CEBPA-AS1、pcDNA-CEBPA-AS1分别转染至细胞;采用MTT检测细胞增殖;应用流式细胞仪检测细胞周期;采用流式细胞术检测细胞凋亡率;采用qRT-PCR法检测LncRNA CEBPA-AS1、miR-195-5p的表达量;双荧光素酶报告实验检测LncRNA CEBPA-AS1与miR-195-5p的靶向关系。结果:与Control组比较,PPⅠ可明显降低细胞存活率、S期细胞比例、Bc1-2蛋白水平、LncRNA CEBPA-AS1的表达水平,提高G_0/G_1期细胞比例、凋亡率、miR-195-5p的表达水平,差异均有统计学意义(p0.05)。与si-NC组比较,si-CEBPA-AS1组细胞存活率、S期细胞比例降低,G_0/G_1期细胞比例、凋亡率升高,差异均有统计学意义(p0.05)。双荧光素酶报告实验证实LncRNA CEBPA-AS1可靶向调控miR-195-5p;转染pcDNA-CEBPA-AS1可明显逆转PPⅠ对细胞增殖、细胞周期及凋亡的作用。结论:重楼皂苷Ⅰ可能通过下调LncRNA CEBPA-AS1的表达及上调miR-195-5p的表达从而抑制瘢痕疙瘩成纤维细胞增殖及促进细胞凋亡。     

lncRNA XIST靶向miR-302a-3p对IL-1β诱导软骨细胞损伤的调控机制

目的 探讨lncRNA X染色体失活特异转录因子（XIST）靶向miR-302a-3p对白细胞介素-1β（IL-1β）诱导软骨细胞损伤的调控机制。方法 SW1353细胞分为Control组、IL-1β组、IL-1β+si-NC组、IL-1β+si-XIST组、IL-1β+si-PDK1组、IL-1β+si-XIST+miR-NC inhibitor组、IL-1β+si-XIST+miR-302a-3p inhibitor组。检测XIST、miR-302a-3p以及3-磷酸肌醇依赖性蛋白激酶1（PDK1）mRNA表达;噻唑蓝（MTT）实验测定细胞活力;流式细胞术检测细胞凋亡;qRT-PCR检测炎症因子（TNF-α、IL-1β、IL-4、IL-6、IL-10）水平;双荧光素酶报告基因检测实验证实miR-302a-3p与XIST或PDK1的靶向关系;免疫印迹法检测凋亡蛋白（Bcl-2、Bax）及PDK1蛋白表达。结果 与IL-1β组相比,IL-1β+si-XIST组XIST表达下调,细胞活力升高,凋亡率降低,Bax表达及TNF-α、IL-1β、IL-6水平下降,Bcl-2表达及IL-4、IL-10水平升高（P<0.05）。XIST直接靶向下调miR-302a-3p表达,miR-302a-3p下调可恢复si-XIST介导的促增殖、抗凋亡和炎症反应作用（P<0.05）。PDK1是miR-302a-3p的靶基因,沉默PDK1可抑制细胞的炎症反应（P<0.05）。结论 敲低XIST可提高IL-1β诱导软骨细胞的细胞活力,减轻OA样软骨细胞损伤,其作用机制与靶向上调miR-302a-3p进而抑制PDK1表达有关。     

miR-124抑制结肠癌细胞增殖和侵袭与TET蛋白家族的关系

目的:探讨miR-124是否通过靶向调节TET蛋白家族的表达而抑制结肠癌细胞增殖与侵袭。方法:用双荧光素酶报告基因检测系统分别检测miR-124对TET家族(TET1、TET2、TET3)的3'UTR-荧光素酶活性的影响;用q RT-PCR与Western blot检测miR-124模拟物转染结肠癌HT29细胞后TET家族的m RNA与蛋白表达水平的变化;用MTS和Transwell实验观察HT29细胞转染miR-124模拟物及TET si RNA后增殖和侵袭能力的变化。结果:双荧光素酶报告基因检测结果显示,各TET m RNA的3'UTR均被miR-124特异性结合,其荧光素酶活性被明显抑制(均P0.05);转染miR-124模拟物后的HT29细胞TET的m RNA与蛋白表达水平明显减低(均P0.05);HT29细胞转染miR-124模拟物或TET si RNA后,增殖和侵袭能力均明显降低(均P0.05)。结论:miR-124可能通过直接靶向调控TET基因的表达,而抑制结肠癌细胞增殖和侵袭。     

microRNA-126靶向PIK3R2调控肝癌细胞SMMC-7721的增殖和凋亡

【摘要】 目的 研究microRNA-126(miR-126)对肝癌细胞SMMC-7721迁移能力的影响以及对靶基因PIK3R2表达的影响。方法 通过慢病毒转染肝癌细胞株SMMC-7721使其过表达miR-126,利用CCK-8法检测细胞增殖能力。流式细胞术检测细胞凋亡;采用实时定量PCR和蛋白印迹检测细胞PIK3R2表达水平的变化,并通过荧光素酶实验验证miR-126与PIK3R2基因的直接调控关系。结果 与对照组相比,转染miR-126组的SMMC-7721细胞的增殖能力减弱,凋亡增加。过表达miR-126后,SMMC-7721细胞PIK3R2的蛋白表达下调(P=0.0135)。荧光素酶报告基因实验显示,miR-126能明显抑制PIK3R2-3’UTR的荧光素酶活性(P=0.0016)。结论〓过表达miR-126可能通过靶向降低PIK3R2基因的蛋白表达,抑制肝癌细胞的迁移。     

miR-34b-5p promotes renal cell inflammation and apoptosis by inhibiting aquaporin-2 in sepsis-induced acute kidney injury

ObjectiveThis study was designed to uncover the mechanism of miR-34b-5p-mediated aquaporin-2 (AQP2) in sepsis-induced injury using human renal tubular epithelial cells (HK-2).MethodsSerum levels of miR-34b-5p, TNF-α, IL-1β, IL-6, serum creatinine (SCr), and blood urea nitrogen (BUN) in septic patients with acute kidney injury (AKI) and healthy controls were detected. Lipopolysaccharide (LPS) was used to induce sepsis in HK-2 cells. LPS-induced HK-2 cells were transfected with miR-34b-5p inhibitor, miR-34b-5p mimic, pcDNA3.1-AQP2, si-AQP2, miR-34b-5p inhibitor + si-NC, or miR-34b-5p inhibitor + si-AQP2. The expressions of miR-34b-5p, AQP2, Bax, Bcl-2, cleaved caspase-3, TNF-α, IL-1β, and IL-6 in HK-2 cells were detected. TUNEL staining revealed the apoptosis of HK-2 cells. Dual-luciferase reporter assay verified the binding between miR-34b-5p and AQP2.ResultsThe expression of miR-34b-5p and the inflammatory responses were augmented in septic AKI patients. miR-34b-5p was up-regulated and AQP2 was down-regulated in LPS-induced HK-2 cells. miR-34b-5p inhibition or AQP2 overexpression ameliorated apoptosis and inflammation in LPS-induced HK-2 cells. In contrast, overexpressing miR-34b-5p deteriorated LPS-induced injury in HK-2 cells. AQP2 was a downstream target of miR-34b-5p. AQP2 silencing abolished the suppressive effects of miR-34b-5p inhibition on LPS-induced apoptosis and inflammatory response in HK-2 cells.ConclusionmiR-34b-5p inhibits AQP2 to promote LPS-induced injury in HK-2 cells.     

miR-129-5p inhibits gemcitabine resistance and promotes cell apoptosis of bladder cancer cells by targeting Wnt5a

Objectives

Gemcitabine resistance is a major obstacle for effective treatment of bladder cancer. This study was aimed to investigate the potential role of miR-129-5p in the development of gemcitabine resistance in bladder cancer cells and its underlying mechanism.

Methods

The IC50 for gemcitabine in 20 bladder cancer cells was first profiled from Genomics of Drug Sensitivity in Cancer. miR-129-5p level and gene mRNA expression were detected using quantitative real-time PCR (qRT-PCR). Cell viability, apoptosis, and gene protein level were assessed by MTT, flow cytometry, and Western blot, respectively. Regulatory relationship between Wnt5a and miR-129-5p was determined using luciferase reporter assay.

Results

We found that down-regulated miR-129-5p level contributed to gemcitabine resistance in bladder cancer cells and tissues. We also observed restoration of miR-129-5p could significantly increase cell sensitivity to gemcitabine and promote cell apoptosis. Mechanism analysis revealed that Wnt5a is a direct target gene of miR-129-5p and knock-down of Wnt5a reversed gemcitabine resistance.

Conclusions

Taken together, our findings indicate that miR-129-5p and Wnt5a may be novel therapeutic targets for overcoming gemcitabine resistance in bladder cancer treatment.

     

miR-181a-5p通过HOXB4抑制骨肉瘤细胞HOS增殖和迁移

目的：研究miR-181a-5p对HOS骨肉瘤细胞增殖、周期和迁移的影响及其机制。方法 ：采用实时定量PCR检测hFOB1.19成骨细胞和HOS、U2OS、MG63骨肉瘤细胞系中miR-181a-5p及HOXB4的表达情况。利用Lipofectamine 2000将miR-181a-5p mimics和miR-181a-5p inhibitor分别转染至人骨肉瘤HOS细胞中（分别为过表达组和抑制剂组）,并设置miR阴性对照组;CCK-8法检测各组细胞的增殖能力变化,流式细胞术检测各组细胞的细胞周期变化,划痕愈合实验以及Transwell迁移实验检测各组细胞的迁移能力变化。Targetscan网站预测miR-181a-5p的靶向基因,并通过双荧光素酶报告基因系统及Western blot验证靶向关系。结果：与成骨细胞hFOB1.19相比,miR-181a-5p在骨肉瘤细胞HOS、U2OS和MG63中低表达（P<0.05）,而HOXB4在骨肉瘤中高表达（P<0.05）。与阴性对照组相比,过表达miR-181a-5p抑制骨肉瘤HOS细胞的增殖和迁移能力,并且处于细胞周期S期的细胞...     

MiR-129-5p/DLX1 signalling axis mediates functions of prostate cancer during malignant progression

We mainly corroborated the potential mechanism of DLX1 and miR-129-5p in prostate cancer cells. DLX1 was upregulated in cancer cells according to qRT-PCR assay. We evaluated the functional changes of the transfected cells via Transwell assay, CCK-8 assay and wound healing assay. DLX1 was confirmed as a cancer promoter. In addition, qRT-PCR showed down-regulated miR-129-5p expression in prostate cancer. We further used dual-luciferase reporter detection to elucidate the targeting between these two genes. The inhibition of miR-129-5p on tumour was verified. Besides, co-transfection of oe-DLX1 and miR-129-5p mimics attenuated this inhibition. These data demonstrated functions of DLX1/miR-129-5p axis in prostate cancer: miR-129-5p hindered the biological functions of cancer cells via inhibiting DLX1 expression. We provide a novel biomarker for prostate cancer.     

长链非编码RNA LINC00313与miR-342-3p/膜联蛋白A2轴在肝细胞癌细胞中的作用与机制

背景与目的 肝细胞癌（HCC）是造成癌症相关性死亡的常见原因之一，研究表明长链非编码RNA（lncRNA）调控微小RNA（miRNA）的表达，进而通过抑制靶mRNA翻译或促进mRNA降解来参与肿瘤发生及进展过程。LINC00313作为一种具有致癌活性的lncRNA参与肿瘤发生及进展过程；膜联蛋白A2（ANXA2）在包括HCC的多种恶性肿瘤中表达上调，促进恶性表型的发生，并可能受上游miR-342-3p的调控。因此，本研究探讨LINC00313、miR-342-3p、ANXA2在HCC细胞中的表达及其相互关系。方法 用qRT-PCR与Western blot检测人肝实质细胞及HCC细胞系（Li-7、HuH-7、Hep3B2.1-7）中LINC00313、miR-342-3p及ANXA2表达。将体外培养的Li-7细胞分为空白对照组（无处理）、LINC00313 siRNA组（转染LINC00313 siRNA）、miR-342-3p模拟物组（转染miR-342-3p模拟物）、共转染阴性对照组（转染阴性siRNA序列与阴性miRNA序列）、共转染组（转染LINC00313 siRNA及miR-342-3p抑制物），用qRT-PCR与Western blot检测各组细胞LINC00313、miR-342-3p及ANXA2表达；MTT实验及平板集落形成实验检测各组细胞增殖；进行TUNEL染色检测各组细胞凋亡；Transwell侵袭及Western blot分别检测各组细胞侵袭数目及上皮-间充质转化（EMT）相关蛋白波形蛋白（vimentin）、E-钙黏蛋白（E-cadherin）表达；免疫荧光染色检测各组细胞Bcl-2关联X蛋白（Bax）/B淋巴细胞瘤-2（Bcl-2）；双荧光素酶报告实验分析Li-7细胞中LINC00313对miR-342-3p、miR-342-3p对ANXA2的靶向调控。建立皮下裸鼠异种移植瘤模型，验证LINC00313沉默对Li-7细胞体内生长的影响。结果 与人肝实质细胞比较，Li-7、HuH-7、Hep3B2.1-7细胞的LINC00313、ANXA2 mRNA及蛋白表达均明显升高，而miR-342-3p表达均明显降低（均P<0.05）。与对照组比较，LINC00313 siRNA组、miR-342-3p模拟物组细胞ANXA2 mRNA及蛋白表达、增殖率、集落生成率、侵袭细胞数目、vimentin蛋白表达均明显降低（P<0.05），miR-342-3p表达、凋亡率、E-cadherin蛋白表达、Bax/Bcl-2比值均明显升高（均P<0.05）；与LINC00313 siRNA组比较，共转染组细胞ANXA2 mRNA及蛋白表达、增殖率、集落生成率、侵袭细胞数目、vimentin蛋白表达均明显升高，而miR-342-3p表达、凋亡率、E-cadherin蛋白表达、Bax/Bcl-2比值均明显降低（均P<0.05）。Li-7细胞中，LINC00313可靶向下调miR-342-3p表达，miR-342-3p可靶向下调其ANXA2表达（均P<0.05）。体内实验结果显示，与无处理的Li-7细胞移植瘤比较，LINC00313敲低的Li-7细胞移植瘤的体积与质量均明显降低，肿瘤组织中LINC00313、ANXA2 mRNA及蛋白表达均明显降低，而miR-342-3p表达明显升高（均P<0.05）。结论 LINC00313在HCC细胞中的表达上调，LINC00313可能通过抑制miR-342-3p而增加后者靶基因ANXA2的表达，进而促进HCC细胞的恶性表型。     

肝细胞癌中miR-574-5p的表达与作用及其与预后的关系

背景与目的:研究表明,miR-574-5p与多种肿瘤预后密切相关,但尚未见miR-574-5p与肝细胞癌(HCC)的关系报道.因此,本研究初步探讨miR-574-5p在HCC中表达及其与患者预后的关系.方法:用qRT-PCR检测130例HCC与癌旁组织及HCC细胞系(HepG2、MHCC-97H)与正常肝细胞系(L-0...     

Long non-coding RNA HOXA11-AS contributes to the formation of keloid by relieving the inhibition of miR-182-5p on ZNF217

BackgroundLong non-coding RNA (lncRNA) dysregulation is demonstrated to be associated with disease progression. Mounting studies show that lncRNA promotes or inhibits the development of keloid. We aimed to disclose the role of homebox A11 antisense RNA (HOXA11-AS) in the formation of keloid.MethodsQuantitative real-time PCR (qPCR) was adopted for expression analysis of HOXA11-AS, miR-182-5p and zinc finger protein 217 (ZNF217) mRNA, and the expression of ZNF protein and marker proteins was detected by western blot. Cell proliferation, cell migration and cell apoptosis were investigated using CCK-8 assay, wound healing assay and flow cytometry assay, respectively. The potential interplay between miR-182-5p and HOXA11-AS or ZNF217 was verified by dual-luciferase reporter assay, RIP assay and pull-down assay. The role of HOXA11 in vivo was studied by establishing animal models.ResultsHOXA11-AS was highly expressed in tissues and fibroblasts of keloid. Deficiency of HOXA11-AS blocked the proliferation and migration of keloid fibroblasts and induced fibroblast apoptosis. HOXA11-AS directly combined to miR-182-5p whose downregulation reversed the effects of HOXA11-AS knockdown. ZNF217 was a target of miR-182-5p, and HOXA11-AS indirectly promoted ZNF217 expression by binding to miR-182-5p. MiR-182-5p enrichment also blocked keloid fibroblast proliferation, survival and migration, while further ZNF217 overexpression abolished these effects. HOXA11-AS knockdown also hindered the growth of keloid in mouse models.ConclusionHigh expression of HOXA11-AS promoted the formation and growth of keloid through the upregulation of ZNF217 by targeting miR-182-5p, and the inhibition of HOXA11-AS might be a novel strategy to prevent keloid development.