脑胶质瘤组织长链非编码RNA FTX、RHPN1-AS1表达与预后的关系

摘要 目的：探讨脑胶质瘤组织长链非编码核糖核酸(LncRNA) FTX、RHPN1-AS1表达与预后的关系。方法：选取我院105例脑胶质瘤患者手术切除的癌组织和癌旁组织（距离肿瘤边缘3~5 cm）。采用实时荧光定量PCR（qRT-PCR）检测组织中LncRNA FTX、RHPN1-AS1表达。分析LncRNA FTX、RHPN1-AS1表达与脑胶质瘤患者临床病理特征的关系。K-M法绘制不同LncRNA FTX、RHPN1-AS1表达脑胶质瘤患者术后5年无进展生存期和总生存期曲线。Cox回归分析脑胶质瘤患者预后不良的影响因素。结果：脑胶质瘤组织中LncRNA FTX、RHPN1-AS1表达水平高于癌旁组织(P＜0.05)。LncRNA FTX、RHPN1-AS1表达与脑胶质瘤患者卡氏体力状态(KPS)评分和世界卫生组织(WHO)分级相关(P＜0.05)。LncRNA FTX、RHPN1-AS1高表达组无进展生存期和总生存期均短于低表达组(P＜0.05)。KPS评分(HR=2.621,95%CI：1.284～5.348)、WHO分级(HR=2.264,95%CI：1.152～4.449)、LncRNA FTX(HR=1.997,95%CI：1.017～3.922)、LncRNA RHPN1-AS1(HR=2.431,95%CI：1.257～4.701)均是脑胶质瘤患者预后不良的影响因素(P＜0.05)。结论：脑胶质瘤组织中LncRNA FTX、RHPN1-AS1表达水平升高,且二者与KPS评分、WHO分级均是患者预后不良的影响因素,可用于脑胶质瘤患者预后评估。     

circPPP1R12A激活p53信号通路调控骨关节炎中软骨细胞增殖和凋亡

摘要 目的：探讨circPPP1R12A（circ_0000423）调控p53信号通路对骨关节炎（osteoarthritis,OA）中软骨细胞增殖和凋亡的影响。方法：采用qRT-PCR检测circPPP1R12A在OA软骨细胞中的表达水平。在OA软骨细胞中分别转染oe-circPPP1R12A和sh-circPPP1R12A后,采用CCK-8检测细胞增殖情况;免疫荧光检测Ki-67阳性细胞表达率;流式细胞术检测细胞凋亡情况;qRT-PCR检测Ki-67和p53表达水平;Western Blot检测Cleaved-caspase3、P53、BCL-2和BAX的表达水平。结果：OA软骨细胞中circPPP1R12A的表达水平明显高于正常软骨细胞。过表达circPPP1R12A能够抑制OA软骨细胞增殖和促进细胞凋亡,通过上调p53表达激活p53信号通路,低表达circPPP1R12A能够促进OA软骨细胞增殖和抑制细胞凋亡,通过下调p53表达阻滞p53信号通路。在OA软骨细胞中同时低表达circPPP1R12A和过表达p53能够反转单独低表达circPPP1R12A对OA软骨细胞增殖和凋亡的影响。结论：circPPP1R12A在OA软骨细胞中明显高表达,circPPP1R12A能够通过激活p53信号通路抑制骨OA软骨细胞增殖和促进软骨细胞凋亡。circPPP1R12A可能成为OA治疗的干预靶点。     

LncRNA MALAT1、miR-145表达与早产儿视网膜病变严重程度的关系研究

摘要 目的：探讨长链非编码核糖核酸（RNA）肺腺癌转移相关转录本1（LncRNA MALAT1）、微小RNA-145（miR-145）表达与早产儿视网膜病变（ROP）严重程度的关系。方法：选择2021年1月至2023年12月我院收治的ROP早产儿82例,根据病情严重程度分为重度组、轻度组,例数分别为为38例、44例。另选择同期我院健康早产儿40例纳入对照组。比较三组血清LncRNA MALAT1、miR-145表达,应用Pearson相关性分析血清LncRNA MALAT1、miR-145表达与ROP严重程度的关系。早产儿ROP的影响因素应用多因素Logistic回归模型分析。血清LncRNA MALAT1、miR-145对早产儿ROP的预测价值应用受试者工作特征（ROC）曲线分析。结果：重度组血清LncRNA MALAT1表达显著高于轻度组和对照组,miR-145表达显著低于轻度组和对照组;轻度组LncRNA MALAT1表达显著高于对照组,miR-145表达显著低于对照组（P<0.05）。血清miR-145表达与ROP严重程度呈负相关（r=-0.412,P<0.05）,LncRNA MALAT1表达与ROP严重程度呈正相关（r=0.434,P<0.05）。三组胎龄、出生体重<1500 g构成比、出生1min Apgar评分<7分、出生5 min Apgar评分<7分、母亲妊娠期糖尿病发生率有统计学意义（P<0.05）。LncRNA MALAT1升高、miR-145降低、胎龄≤31周、出生体重<1500 g、母亲妊娠期糖尿病是早产儿ROP发生的危险因素（P<0.05）。血清LncRNA MALAT1、miR-145对ROP严重程度的预测价值具有较高的敏感度、特异度,联合检测对ROP严重程度的曲线下面积（AUC）大于上述指标单独检测。结论：ROP早产儿血清LncRNA MALAT1表达升高、miR-145表达降低与ROP严重程度加重有关,血清LncRNA MALAT1联合miR-145检测对ROP严重程度预测具有一定价值。     

宫颈鳞状细胞癌组织LncRNA DCST1-AS1、LncRNA MCM3AP-AS1表达与增殖侵袭基因和预后的关系

摘要 目的：探讨宫颈鳞状细胞癌（CSCC）组织长链非编码核糖核酸（LncRNA）DC-STAMP结构域1-反义1（DCST1-AS1）、LncRNA MCM3AP反义RNA 1（MCM3AP-AS1）表达与增殖侵袭基因和预后的关系。方法：选择2018年1月至2020年1月苏州大学第二附属医院收治的124例CSCC患者,取CSCC组织及其癌旁正常组织,实时荧光定量聚合酶链反应（qRT-PCR）检测LncRNA DCST1-AS1、LncRNA MCM3AP-AS1以及增殖基因（YAP1、Piwil2、EZH2、PKCε）和侵袭基因（Rab11、TUG1、Gli1、FoxM1）mRNA表达。患者出院后随访3年。Pearson相关性分析Lnc RNA DCST1-AS1、Lnc RNA MCM3AP-AS1表达与增殖基因、侵袭基因的相关性。分析Lnc RNA DCST1-AS1、Lnc RNA MCM3AP-AS1表达与临床病理特征的关系。绘制Kaplan-Meier曲线分析预后情况,多因素COX回归分析影响CSCC患者预后的因素。结果：CSCC癌组织中Lnc RNA DCST1-AS1表达高于癌旁组织（P＜0.05）,Lnc RNA MCM3AP-AS1表达低于癌旁组织（P＜0.05）。YAP1、Piwil2、EZH2、PKCε、Rab11、TUG1、Gli1、FoxM1 mRNA水平与CSCC癌组织中Lnc RNA DCST1-AS1表达呈正相关（P＜0.05）,与Lnc RNA MCM3AP-AS1呈负相关（P＜0.05）。低中度分化、FIGO分期Ⅲa期、盆腔淋巴结转移CSCC患者癌组织中Lnc RNA DCST1-AS1表达高于高度分化、FIGO分期Ⅰ～Ⅱ期、无盆腔淋巴结转移宫颈癌患者（P＜0.05）,Lnc RNA MCM3AP-AS1表达低于高度分化、FIGO分期Ⅰ～Ⅱ期、无盆腔淋巴结转移宫颈癌患者（P＜0.05）。Lnc RNA DCST1-AS1高表达、Lnc RNA MCM3AP-AS1低表达CSCC患者3年总生存率低于Lnc RNA DCST1-AS1低表达、Lnc RNA MCM3AP-AS1高表达患者（P＜0.05）。多因素COX回归分析显示盆腔淋巴结转移、Lnc RNA DCST1-AS1高表达是CSCC患者预后不良的危险因素（P＜0.05）,Lnc RNA MCM3AP-AS1高表达是保护因素（P＜0.05）。结论：CSCC组织中LncRNA DCST1-AS1表达上调,LncRNA MCM3AP-AS1表达下调,且与CSCC增殖侵袭和预后不良有关。     

长链非编码RNA SNHG14/miR-211对宫颈癌细胞的增殖、侵袭能力的影响

摘要 目的：探讨长链非编码(Long chain non-coding,Lnc)RNA SNHG14/微小RNA(microRNA,miR)-211对宫颈癌细胞的增殖、侵袭能力的影响。方法：宫颈癌细胞株SiHa设三组：空白组(不进行转染)、对照组(转染miR-NC)与miR-211组(转染miR-211 mimic), 噻唑蓝[3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide,MTT] 检测细胞增殖活性,Transwell检测细胞侵袭及转移,实时荧光定量核酸扩增检测系统(Real-time Quantitative PCR, qPCR)检测LncRNA SNHG14与miR-211mRNA水平,Westem印迹法检测黏蛋白4(mucin 4, MUC4)蛋白水平。结果：转染后24 h与48 h,miR-211组的细胞增殖、侵袭、转移指数与LncRNA SNHG14与MUC4蛋白相对表达水平低于空白组和对照组(P<0.05),miR-211 mRNA表达水平高于空白组(P<0.05),空白组与对照组对比差异无统计学意义(P>0.05)。结论：过表达miR-211可抑制LncRNA SNHG14的表达,也能抑制MUC4表达,从而能抑制宫颈癌细胞的增殖、侵袭及转移。     

MiR-1-3p通过抑制CAPRIN1调控胰腺癌发生发展

摘要 目的：探讨miR-1-3p在胰腺癌发生发展中的分子机制。方法：以MIA-PaCa-2,SW 1990为研究目标,通过qRT-PCR技术检测miR-1-3p的表达量,利用TargetScan和miRDB数据库预测miR-1-3p的下游靶基因及结合位点,并通过构建双荧光素酶报告基因,进一步确认miR-1-3p与靶基因的结合。利用CCK8细胞增殖实验及平板克隆形成实验检测过表达miR-1-3p及敲低CAPRIN1对细胞增殖的作用;利用流式检测细胞周期;利用蛋白质免疫印迹方法检测miR-1-3p对CAPRIN1及其下游基因的影响;通过流式来确认,过表达miR-1-3p及敲减CAPRIN1基因对细胞周期的影响。结果：miR-1-3p在胰腺癌细胞MIA-PaCa-2,SW 1990中低表达;miR-1-3p直接与CAPRIN1的3''-untranslated region (3''- UTR)结合;过表达miR-1-3p或抑制CAPRIN1基因的表达可明显抑制胰腺癌细胞的增殖能力,同时也产生细胞周期阻滞。结论：miR-1-3p通过抑制CAPRIN1基因表达,而产生细胞周期阻滞进而抑制胰腺癌细胞的增殖能力。     

SLC2A1-AS1在卵巢癌中的表达及临床意义

摘要 目的：探究lncRNA SLC2A1反义RNA 1（SLC2A1 antisense RNA 1,SLC2A1-AS1）在卵巢癌中的表达情况及与卵巢癌患者预后之间的关系,为卵巢癌的诊断和预后提供一种新的生物标志物。方法：通过多个数据库中的卵巢癌样本信息及其实时荧光定量PCR（Real Time Quantitative PCR,RT-qPCR）分别探究SLC2A1-AS1在卵巢癌中的表达情况及其与卵巢癌患者预后之间的关系,通过免疫荧光实验和划痕实验探究SLC2A1-AS1的表达对卵巢癌细胞的增殖和迁移的影响。通过基因本体(Gene ontology,GO)和京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes,KEGG）分析寻找SLC2A1-AS1影响卵巢癌恶性进程的可能机制。结果：基于多个数据库中的生物信息学分析和RT-qPCR验证发现SLC2A1-AS1在卵巢癌中异常低表达,且SLC2A1-AS1低表达与卵巢癌患者的不良预后密切相关。SLC2A1-AS1过表达可明显抑制卵巢癌细胞的增殖和迁移能力。基于GO和KEGG分析,发现SLC2A1-AS1可能通过调控细胞外基质（extracellular matrix,ECM）组分以及ECM受体的相互作用通路抑制卵巢癌的恶性进程。结论：SLC2A1-AS1可能作为一种关键的潜在的生物标志物抑制着卵巢癌的恶性进展。     

LncRNA Terc在心肌纤维化过程中的功能研究

摘要 目的：探讨lncRNA端粒酶RNA组分（Terc）在心肌纤维化（MF） 过程中的作用。方法：使用不同浓度的TGF-β1诱导心肌成纤维细胞（CFs）转分化,通过免疫荧光染色、western blot检测α-SMA、Vimentin、Collagen I、Collagen III蛋白的表达水平,qRT-PCR检测lncRNA Terc表达水平。过表达和敲减Terc后,通过western blot、CCK-8和流式细胞术观察模型细胞的胞外基质产生、细胞增殖、凋亡和Smads信号传导情况。皮下注射异丙肾上腺素（ISO）构建小鼠心肌纤维化模型,并使用Terc敲低慢病毒干预,其后多普勒超声仪检测小鼠的心脏射血分数（EF）和左室短轴缩短率（FS）,称量小鼠心脏湿重,HE、Masson染色检测小鼠心脏的病理改变,IHC检测α-SMA、Vimentin蛋白的表达水平,qRT-PCR检测lncRNA Terc表达水平。结果：TGF-β1处理增加CFs的α-SMA、Vimentin、Collagen I、Collagen III的蛋白表达以及Terc水平;过表达Terc促进α-SMA、Vimentin、Collagen I、Collagen III的蛋白表达以及Smad2/3的磷酸化水平,同时还可促进CFs的增殖、抑制CFs的凋亡;敲减Terc则起相反的作用;动物模型中,ISO可抑制EF和FS,增加心脏湿重,加重心肌的病理损伤,而敲减Terc可有效缓解上述过程。结论：LncRNA Terc可通过促进Smads信号传导,加速心肌成纤维细胞转分化和心肌纤维化进展。     

LNCRNA FAR2P1对乳腺癌细胞增殖迁移和凋亡的影响研究

摘要 目的：探讨新型 LncRNA FAR2P1在乳腺癌发生发展中的作用和影响。方法：癌症基因组图谱（The cancer genome atlas, TCGA, https://portal.gdc.cancer.gov/）数据库分析LncRNA FAR2P1差异表达量。根据169例LncRNA FAR2P1高表达的乳腺癌患者和1180例LncRNA FAR2P1 低表达患者随访资料,分析LncRNA FAR2P1与乳腺癌患者预后的相关性。首先利用实时定量荧光聚合酶链反应(qRT-PCR)检测LncRNA FAR2P1在乳腺癌细胞系（MDA-MB-231、SKBR3）和乳腺正常上皮细胞（MCF-10A）中的表达水平;转染小干扰（si-RNA）获得低表达 FAR2P1的细胞株, Cell Counting Kit-8（CCK8）实验、EDU实验、克隆形成评估细胞增殖活力,Transwell 实验探究 FAR2P1 对细胞迁移的影响;通过流式分析FAR2P1 是否调节细胞凋亡;体内异种成瘤模型评估 FAR2P1 在体内对乳腺癌增值的调节作用。结果：LncRNA FAR2P1在MDA-MB-231、SKBR3中表达显著升高,并且与乳腺癌患者预后负相关。体外实验表明,敲低MDA-MB-231和SKBR3细胞中的 FAR2P1 后,细胞的增殖能力和迁移能力减弱,但是凋亡率明显升高。体内异种成瘤模型显示沉默 FAR2P1相比对照,乳腺癌细胞增殖能力显著降低。结论：LncRNA FAR2P1 在乳腺癌中高表达,并且参与调控乳腺癌增殖、凋亡和迁移。     

LncRNA KCNQ1OT1靶向调控miR-124-3p/HMGB1轴对高糖诱导肾小球系膜细胞增殖、凋亡及纤维化的影响

摘要 目的：探讨长链非编码核糖核酸（LncRNA）KCNQ1OT1靶向调控miR-124-3p/高迁移率族蛋白B1（HMGB1）轴对高糖诱导肾小球系膜细胞（HMC）增殖、凋亡及纤维化的影响。方法：将人HMC分为对照组（NC组）、高糖组（30 mmol/L葡萄糖）、阴性序列（si-NC）组、KCNQ1OT1小干扰核糖核酸（RNA）（si-KCNQ1OT1）组、si-KCNQ1OT1+模拟对照序列（miR-NC）组、si-KCNQ1OT1+miR-124-3p抑制剂（miR-124-3p inhibitor）组,各组在转染后进行高糖处理。实时荧光定量聚合酶链式反应（RT-qPCR）检测LncRNA KCNQ1OT1信使核糖核酸（mRNA）、miR-124-3p mRNA、HMGB1 mRNA表达;四甲基偶氮唑盐比色法（MTT）检测细胞增殖活性;流式细胞术检测细胞凋亡率;蛋白印迹法（Western blot）检测HMGB1蛋白、增殖相关蛋白[细胞周期蛋白1（CyclinD1）]、细胞凋亡蛋白[半胱氨酸蛋白酶-3（caspase-3）、半胱氨酸蛋白酶蛋白9（caspase-9）]、细胞纤维化蛋白[纤维连接蛋白（FN）、细胞黏附分子-1（ICAM-1）]表达;双荧光素酶报告基因实验验证LncRNA KCNQ1OT1与miR-124-3p与HMGB1之间的靶向关系。结果：与NC组比较,高糖组KCNQ1OT1 mRNA、HMGB1 mRNA及HMGB1蛋白、CyclinD1蛋白、FN蛋白、ICAM-1蛋白表达、HMC活性（24 h、48 h和72 h）明显上升,miR-124-3p mRNA、caspase-3蛋白及caspase-9蛋白表达、HMC凋亡率明显下降（P<0.05）;与高糖组、si-NC组比较,si-KCNQ1OT1组KCNQ1OT1 mRNA、HMGB1 mRNA及HMGB1蛋白、CyclinD1蛋白、FN蛋白、ICAM-1蛋白表达、HMC活性（24 h、48 h和72 h）明显下降,miR-124-3p mRNA、caspase-3蛋白及caspase-9蛋白表达、HMC凋亡率明显上升（P<0.05）;与si-KCNQ1OT1组、si-KCNQ1OT1+miR-NC组比较,si-KCNQ1OT1+miR-124-3p inhibitor组HMGB1 mRNA及HMGB1蛋白、CyclinD1蛋白、FN蛋白、ICAM-1蛋白表达、HMC活性（24 h、48 h和72 h）明显上升,miR-124-3p mRNA、caspase-3蛋白及caspase-9蛋白表达、HMC凋亡率明显下降（P<0.05）。较miR-NC组与KCNQ1OT1-WT共转染组而言,miR-124-3p mimic组与KCNQ1OT1-WT共转染组细胞荧光素酶活性明显降低（P<0.05）;较miR-NC组与HMGB1-WT共转染组而言,miR-124-3p mimic组与HMGB1-WT共转染组细胞荧光素酶活性明显降低（P<0.05）。结论：LncRNA KCNQ1OT1可以靶向下调miR-124-3p mRNA表达,上调HMGB1 mRNA及HMGB1蛋白表达,促进高糖诱导HMC增殖,抑制凋亡,促进细胞纤维化发展。     

miR-29b inhibits TGF-β1-induced cell proliferation in articular chondrocytes

Transforming growth factor β1 (TGF-β1) is a known regulator of chondrocyte proliferation and promotes cartilage repair in osteoarthritis (OA). microRNA-29b-3p (miR-29b-3p) is downregulated by TGF-β1 and overexpressed in OA cartilage. However, the ability of miR-29b-3p to mediate the chondrocyte pro-proliferative effects of TGF-β1 is not yet understood. This current study aimed to investigate the effect of miR-29b-3p on TGF-β1-induced cell proliferation in murine articular chondrocytes. The stimulation of chondrocytes by TGF-β1 for 24 h resulted in the downregulation of miR-29b-3p expression. The ratio of G0/G1 phase cells decreased in response to TGF-β1 whereas the ratio of S phase cells was increased. Consistent with this observation, miR-29b-3p overexpression inhibited TGF-β1’s ability to promote the ratio of S phase cells and downregulate the ratio of G0/G1 phase cells. These findings suggest that the downregulation of miR-29b-3p is a likely requirement for TGF-β1-mediated proliferation of murine articular chondrocytes. Furthermore, implying that miR-29b-3p expression may be involved in reduced chondrocyte proliferation in OA.     

The miR-302c/transforming growth factor-β receptor type-2 axis modulates interleukin-1β-induced degenerative changes in osteoarthritic chondrocytes

Chondrocyte production of catabolic and inflammatory mediators participating in extracellular matrix degradation has been regarded as a central event in osteoarthritis (OA) development. During OA pathogenesis, interleukin-1β (IL-1β) decreases the mRNA expression and protein levels of transforming growth factor-β receptor type-2 (TGFBR2), thus disrupting transforming growth factor-β signaling and promoting OA development. In the present study, we attempted to identify the differentially expressed genes in OA chondrocytes upon IL-1β treatment, investigate their specific roles in OA development, and reveal the underlying mechanism. As shown by online data analysis and experimental results, TGFBR2 expression was significantly downregulated in IL-1β-treated human primary OA chondrocytes. IL-1β treatment induced degenerative changes in OA chondrocytes, as manifested by increased matrix metalloproteinase 13 and a disintegrin and metalloproteinase with thrombospondin motifs 5 proteins, decreased Aggrecan and Collagen II proteins, and suppressed OA chondrocyte proliferation. These degenerative changes were significantly reversed by TGFBR2 overexpression. miR-302c expression was markedly induced by IL-1β treatment in OA chondrocytes. miR-302c suppressed the expression of TGFBR2 via direct binding to its 3′- untranslated region. Similar to TGFBR2 overexpression, miR-302c inhibition significantly improved IL-1β-induced degenerative changes in OA chondrocytes. Conversely, TGFBR2 silencing enhanced IL-1β-induced degenerative changes and significantly reversed the effects of miR-302c inhibition in response to IL-1β treatment. In conclusion, the miR-302c/TGFBR2 axis could modulate IL-1β-induced degenerative changes in OA chondrocytes and might become a novel target for OA treatment.Electronic supplementary materialThe online version of this article (10.1007/s12079-020-00591-2) contains supplementary material, which is available to authorized users.     

microRNA-186 inhibition of PI3K–AKT pathway via SPP1 inhibits chondrocyte apoptosis in mice with osteoarthritis

Chondrocyte apoptosis has been implicated as a major pathological osteoarthritis (OA) change in humans and experimental animals. We evaluate the ability of miR-186 on chondrocyte apoptosis and proliferation in OA and elucidate the underlying mechanism concerning the regulation of miR-186 in OA. Gene expression microarray analysis was performed to screen differentially expressed messenger RNAs (mRNAs) in OA. To validate the effect of miR-186 on chondrocyte apoptosis, we upregulated or downregulated endogenous miR-186 using mimics or inhibitors. Next, to better understand the regulatory mechanism for miR-186 governing SPP1, we suppressed the endogenous expression of SPP1 by small interfering RNA (siRNA) against SPP1 in chondrocytes. We identified SPP1 is highly expressed in OA according to an mRNA microarray data set GSE82107. After intra-articular injection of papain into mice, the miR-186 is downregulated while the SPP1 is reciprocal, with dysregulated PI3K–AKT pathway in OA cartilages. Intriguingly, miR-186 was shown to increase chondrocyte survival, facilitate cell cycle entry in OA chondrocytes, and inhibit chondrocyte apoptosis in vitro by modulation of pro- and antiapoptotic factors. The determination of luciferase activity suggested that miR-186 negatively targets SPP1. Furthermore, we found that the effect of miR-186 suppression on OA chondrocytes was lost when SPP1 was suppressed by siRNA, suggesting that miR-186 affected chondrocytes by targeting and depleting SPP1, a regulator of PI3K–AKT pathway. Our findings reveal a novel mechanism by which miR-186 inhibits chondrocyte apoptosis in OA by interacting with SPP1 and regulating PI3K–AKT pathway. Restoring miR-186 might be a future therapeutic strategy for OA.     

miR-382-3p靶向RASA1基因调控骨关节炎软骨细胞增殖和凋亡

目的:探讨mi R-382-3p对骨关节炎软骨细胞增殖和凋亡的影响及其机制。方法:用100 ng/mL的脂多糖(LPS)处理软骨细胞,记为LPS组,以正常培养的软骨细胞作为正常对照(NC)组。mi R-NC、mi R-382-3p、anti-miR-NC、anti-miR-382-3p转染至软骨细胞中,记为mi R-NC组、mi R-382-3p组、anti-miR-NC组、anti-miR-382-3p组;将mi R-NC、mi R-382-3p、si-NC、si-RASA1转染至软骨细胞后再用100 ng/mL的LPS处理,记为mi R-NC+LPS组、mi R-382-3p+LPS组、si-NC+LPS组、si-RASA1+LPS组;将mi R-382-3p分别与pcDNA-NC、pcDNA-RASA1共转染至软骨细胞后再用100 ng/mL的LPS处理,记为mi R-382-3p+pcDNA-NC+LPS组、mi R-382-3p+pcDNA-RASA1+LPS组。实时荧光定量PCR(RT-qPCR)检测mi R-382-3p和Ras p21蛋白活化因子1(RASA1)m RNA表达水平;蛋白质印迹(Western blot)法检测RASA1、细胞周期蛋白D1(CyclinD1)、裂解的半胱氨酸天冬氨酸蛋白酶-3(Cleaved-caspase-3)蛋白表达;四甲基偶氮唑盐比色法(MTT)检测细胞存活率;流式细胞术检测细胞凋亡;荧光素酶报告实验检测mi R-382-3p和RASA1的靶向关系。结果:LPS诱导的软骨细胞中mi R-382-3p表达水平显著降低,RASA1表达水平显著升高,CyclinD1表达水平显著降低,Cleaved-caspase-3表达水平显著升高,细胞存活率显著降低,细胞凋亡率显著升高(P0.05)。过表达mi R-382-3p和敲减RASA1,LPS诱导的软骨细胞中CyclinD1表达水平显著升高,Cleaved-caspase-3表达水平显著降低,细胞存活率显著升高,细胞凋亡率显著降低(P0.05)。mi R-382-3p靶向调控RASA1,高表达RASA1部分逆转了mi R-382-3p高表达对LPS处理的软骨细胞增殖和凋亡的影响。结论:过表达mi R-382-3p促进软骨细胞增殖,抑制LPS诱导的软骨细胞凋亡,其机制可能与RASA1有关。     

Selective loading of exosomal HULC and miR-372 is responsible for chondrocyte death during OA pathogenesis

Exosomes are membranous vesicles containing various biomolecules, including non-coding RNAs (ncRNAs). ncRNAs are secreted from several cell types and are involved in various biological functions, including cellular communication. The aim of this study was to identify and illustrate the significance of the osteoarthritis (OA)-specific packaging of exosomal ncRNAs. In this study, we hypothesized that selective packaging of ncRNAs into exosomes would reflect the cellular response to chondrocyte death during OA pathogenesis. Exosomal HULC level significantly decreased in OA exosomes, whereas exosomal miR-372-3p level significantly increased in OA exosomes. In addition, chondrocytes with high HULC levels in the cytosol showed lower overall proliferation and higher apoptotic cell death than normal chondrocytes, whereas chondrocytes with high miR-372-3p in the cytosol showed higher overall proliferation and lower cell death than OA chondrocytes. Among the signaling molecules known to be involved in OA pathogenesis, GSK is one of the regulators of the selective exosomal packaging observed in OA chondrocytes. Inhibition of GSK observed in OA chondrocytes was responsible for enriched uploading of miR-372-3p and suppressed uploading of HULC during OA pathogenesis. In conclusion, we show that selective ncRNAs observed in OA play a critical role in chondrocyte proliferation/apoptosis.     

Silencing of circ_0000205 mitigates interleukin-1β-induced apoptosis and extracellular matrix degradation in chondrocytes via targeting miR-766-3p/ADAMTS5 axis

The aim of this study was to explore the role of hsa_circRNA_0000205 (circ_0000205) in chondrocyte injury in osteoarthritis (OA) and the underlying mechanism. Expression of circ_0000205, microRNA (miR)-766-3p and a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-5 was detected by quantitative real time (qRT)-polymerase chain reaction (PCR) and Western blot assays. Cell proliferation, apoptosis, and extracellular matrix (ECM) synthesis were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 5-ethynyl-2-deoxyuridine assays, flow cytometry, and qRT-PCR and Western blot assays. The target relationship between miR-766-3p and circ_0000205 or ADAMTS5 was confirmed by luciferase reporter assay and RNA immunoprecipitation. IL-1β treatment could attenuate cell viability of primary chondrocytes and proliferating cell nuclear antigen (PCNA) and collagen II type alpha-1 (COL2A1) levels, and elevate apoptosis rate and cleaved caspase-3, ADAMTS5 and matrix metalloproteinase-13 (MMP13) levels, suggesting that IL-1β induced chondrocyte apoptosis and ECM degradation. Expression of circ_0000205 was up-regulated in OA tissues and IL-1β-induced primary chondrocytes, accompanied with miR-766-3p down-regulation and ADAMTS5 up-regulation. Knockdown of circ_0000205 could mitigate IL-1β-induced above effects and improve cell proliferation. Moreover, both depleting miR-766-3p and promoting ADAMTS5 could partially counteract circ_0000205 knockdown roles in IL-1β-cultured primary chondrocytes. Notably, circ_0000205 was verified as a sponge for miR-766-3p via targeting, and ADAMTS5 was a direct target for miR-766-3p. Silencing circ_0000205 could protect chondrocytes from IL-1β-induced proliferation reduction, apoptosis, and ECM degradation by targeting miR-766-3p/ADAMTS5 axis.     

MiR-127-5p通过靶向调节adipoR1促进骨关节炎软骨细胞的增殖

脂联素（adiponection）与骨关节炎（osteoarthritis, OA）的发病密切相关,且主要通过其受体adipoR1发挥作用。而骨关节炎中脂联素的表达是否受miRNA表达的影响却未见报道。本文旨在研究miR-127-5p对骨关节炎软骨细胞中脂联素及细胞增殖的影响。分离培养人原代OA软骨细胞及对应正常细胞,甲苯胺蓝染色和II型胶原免疫细胞化学染色进行鉴定。 Real-time PCR结果表明,OA软骨细胞中miR-127-5p的表达与正常软骨细胞中的相比较显著下降。MiR-127-5p转染可显著降低荧光素酶报告基因的荧光强度（P<0.05）,表明adipoR1为miR-127-5p的靶向基因。MiR-127-5p mimic转染软骨细胞后,MTT法研究结果表明,miR-127-5p mimic 可显著促进软骨细胞增殖,Western 印迹结果表明,脂联素及其受体（adipoR1）表达显著上升,p65的表达以及p38、ERK1/2以及IkBα的磷酸化水平显著下降。ELISA结果表明,MMP-1、MMP-3、MMP-13的含量显著下降。实验结果提示,miR-127-5p通过靶向下调adipoR1及脂联素的表达,促进软骨细胞增殖,并且抑制NF-κB信号通路,进而抑制炎性反应。     

MiR-526b targets lncRNA SLC16A1-AS1 to suppress cell proliferation in triple-negative breast cancer

The present study investigated the potential interaction between miR-526b and lncRNA SLC16A1-AS1 in triple-negative breast cancer (TNBC). Expression of miR-526b and SLC16A1-AS1 in TNBC tumor tissues and paired nontumor tissues from 60 TNBC patients was detected by real-time polymerase chain reaction (RT-qPCR). The interaction between miR-526b and SLC16A1-AS1 was evaluated with overexpression experiments, followed by RT-qPCR. The proliferation and migration of cells were detected with cell counting kit-8 assay and Transwell assay, respectively. Apoptosis of cells was assessed by cell apoptosis assay. The expression of apoptosis-related proteins was quantified by Western blot analysis. MiR-526b was predicted to bind with SLC16A1-AS1. Overexpression of miR-526b in TNBC cells decreased the expression levels of SLC16A1-AS1, while overexpression of SLC16A1-AS1 did not affect the expression of miR-526b. In TNBC tissues, miR-526b was downregulated in TNBC tissues, while SLC16A1-AS1 was upregulated in TNBC tissues compared to that in nontumor tissues. The expression of SLC16A1-AS1 and miR-526b were inversely correlated. In vitro experiments showed that overexpression of SLC16A1-AS1 promoted cell proliferation and invasion but suppressed cell apoptosis. MiR-526b played an opposite role and suppressed the function of SLC16A1-AS1. MiR-526b is downregulated in TNBC and it targets SLC16A1-AS1 to regulate proliferation, apoptosis, and invasion of TNBC cells.