环状RNA circMRPS35通过miR-130a-3p/ZNRF3轴调节胃癌细胞的增殖、凋亡、迁移和侵袭

摘要 目的：探讨环状RNA MRPS35（circMRPS35）对胃癌（GC）细胞增殖、凋亡、迁移和侵袭的调控机制。方法：体外培养人GC细胞系（HGC-27、MGC-803、MKN45和AGS）和正常胃上皮GES-1细胞,实时荧光定量PCR（RT-qPCR）检测circMRPS35、miR-130a-3p和锌环指蛋白3（ZNRF3）mRNA表达。另取MGC-803细胞,分为对照组、pc-NC组、pc-circMRPS35组、pc-circMRPS35+miR-NC组、pc-circMRPS35+miR-130a-3p组,采用Lipofectamine 3000进行质粒转染。RT-qPCR检测circMRPS35、miR-130a-3p和ZNRF3 mRNA表达,Western blot检测ZNRF3蛋白表达,CCK-8法、流式细胞术检测细胞增殖与凋亡,划痕实验和Transwell小室实验检测细胞迁移与侵袭能力,裸鼠移植瘤实验探究circMRPS35对GC细胞体内生长的影响。双荧光素酶报告基因检测miR-130a-3p与circMRPS35或ZNRF3的靶标关系。结果：GC细胞系中circMRPS35和ZNRF3 mRNA呈低表达,miR-130a-3p呈高表达（均P＜0.05）。过表达circMRPS35可降低miR-130a-3p,上调ZNRF3 mRNA和蛋白水平,抑制细胞增殖、迁移和侵袭,并促进细胞凋亡（均P＜0.05）;circMRPS35过表达对GC细胞恶性行为和裸鼠移植瘤生长的抑制作用可被miR-130a-3p mimic逆转（P＜0.05）。双荧光素酶实验结果显示,过表达miR-130a-3p可降低circMRPS35-WT和ZNRF3-WT的荧光素酶活性（P＜0.05）。结论：circMRPS35可能通过miR-130a-3p/ZNRF3轴抑制GC细胞的增殖、迁移和侵袭,并促进细胞凋亡。     

miR-375靶向MMP13抑制骨肉瘤细胞的迁移和侵袭

目的 为了探究miR-375是否通过影响基质金属蛋白酶13（MMP13）的表达来调控骨肉瘤（osteosarcoma,OS）恶性特征。方法 用Lipofectamine 3000试剂盒将质粒、miRNA转染至骨肉瘤细胞和HEK293细胞中。实时定量聚合酶链反应（real-time quantitative PCR,RT-qPCR）检测OS患者和OS细胞中miR-375和MMP13的表达。蛋白质印迹法（Western blot）分析OS患者和OS细胞中MMP13蛋白的表达。双荧光素酶法分析miR-375与MMP13的靶向关系。伤口愈合和transwell实验分别分析OS细胞的迁移和侵袭。结果 OS组织中miR-375的表达低于正常组织。MMP13在OS组织中表达上调。在OS患者中,MMP13的表达与miR-375呈负相关。与转染miRNA对照的OS细胞相比,转染miR-375模拟物OS细胞的迁移和侵袭明显被抑制。MMP13能部分逆转miR-375对OS细胞迁移和侵袭的抑制作用。结论 在OS细胞中,过表达miR-375通过调控MMP13的表达抑制细胞的迁移和侵袭。     

上调miR-216a-5p通过靶向双特异性磷酸酶10抑制胃癌细胞自噬并增强放射敏感性

摘要 目的：探究miR-216a-5p对胃癌细胞自噬和放射敏感性的调控机制及其对双特异性磷酸酶10（DUSP10）的调控作用。方法：采用直线加速器6-MV X射线照射SGC-7901细胞,剂量率为0.8Gy/min,总剂量为8Gy。用Lipofectamine 2000试剂将miR-216a-5p mimic、NC mimic、pcDNA DUSP10或pcDNA NC转染到SGC-7901细胞中。转染后,将细胞分为miR-216a-5p mimic组和NC mimic组,每组又分为0Gy和8Gy两个亚组。在拯救实验中,将细胞分为miR-216a-5p mimic+pcDNA DUSP10组和miR-216a-5p mimic+pcDNA NC组。通过qRT-PCR检测miR-216a-5p和DUSP10 mRNA水平。通过5-乙炔基-2''-脱氧尿苷（EdU）掺入实验和集落形成测定检测细胞增殖。通过流式细胞仪评估细胞凋亡。通过Western blot检测DUSP10、Bax、Bad、Bcl-2、LC3和p62的蛋白表达。通过免疫荧光法检测γH2AX的表达,用于评估细胞中的DNA双链断裂（DSB）。通过荧光素酶报告基因检测miR-216a-5p和DUSP10的靶向关系。通过GFP-mRFP-LC3检测自噬体。结果：与8Gy NC-mimic组相比,8Gy miR-216a-5p-mimic组的集落数量、EdU阳性率和Bcl-2蛋白表达水平降低,而γH2AX阳性率、细胞凋亡率和Bax和Bad蛋白表达水平升高（P<0.01）。与8Gy NC-mimic组相比,8Gy miR-216a-5p-mimic组的自噬体数量和LC3II蛋白表达水平降低,而p62蛋白表达水平升高（P<0.001）。与miR-216a-5p-mimic共培养后,与DUSP10-3''-UTR-MUT组相比,DUSP10-3''-UTR-WT的相对荧光素酶活性显著降低（P<0.001）。与NC-mimic组相比,miR-216a-5p-mimic组的DUSP10 mRNA和蛋白表达水平均降低（P<0.001）。与miR-216a-5p mimic+pcDNA NC组相比,miR-216a-5p mimic+pcDNA DUSP10组的集落数量和自噬体数量升高,而细胞凋亡率降低（P<0.001）。结论：miR-216a-5p通过抑制DUSP10来抑制细胞增殖、增加放射诱导的细胞凋亡并抑制放射诱导的自噬,从而增强胃癌细胞的放射敏感性。     

miR-1204表达对非小细胞肺癌细胞增殖凋亡、上皮间质转化和MAPKs信号通路的影响

摘要 目的：探究微小RNA-1204（miR-1204）表达对非小细胞肺癌细胞增殖凋亡、上皮间质转化（EMT）和丝裂原活化蛋白激酶（MAPKs）信号通路的影响。方法：将人非小细胞肺癌细胞A549随机分为miR-1204组（转染miR-1204mimic质粒）、NC组（转染空载质粒）和对照组（仅加转染试剂）。采用四甲基偶氮唑盐（MTT）法检测细胞增殖情况,采用流式细胞仪检测细胞凋亡情况,采用实时荧光定量聚合酶链式反应（RT-qPCR）检测细胞E-钙黏蛋白（E-cad）、N-钙黏蛋白（N-cad）和波形蛋白（Vim）mRNA的表达水平。采用RT-qPCR和蛋白免疫印迹（WB）法检测细胞p-P38、P38、p-ERK、ERK、p-JNK、JNK mRNA和蛋白的表达水平。结果：培养12、24、48 h,miR-1204组细胞增殖抑制率均高于对照组和NC组同期（P＜0.05）,对照组与NC组同期的细胞增殖抑制率比较差异无统计学意义（P＞0.05）。但三组细胞随着培养时间延长,细胞增殖抑制率均增加,两两时间点组内比较均有差异（P＜0.05）。miR-1204组细胞凋亡率高于对照组和NC组（P＜0.05）。miR-1204组E-cad mRNA的表达水平高于对照组和NC组（P＜0.05）,N-cad、Vim mRNA的表达水平低于对照组和NC组（P＜0.05）。miR-1204组p-P38、p-ERK、p-JNK mRNA和蛋白的表达水平均低于对照组和NC组（P＜0.05）。结论：上调miR-1204的表达可以抑制非小细胞肺癌细胞的增殖,促进其凋亡,还可以抑制其EMT,该作用可能是通过抑制MAPKs信号通路实现的。     

糖尿病病理性神经痛大鼠坐骨神经中miR-133a的上调对疼痛阈值的影响及机制

摘要 目的：探究糖尿病病理性神经痛大鼠坐骨神经中miR-133a的上调对疼痛阈值的影响及机制。方法：使用NGS测序和定量PCR分析糖尿病病理性神经痛大鼠坐骨神经中的相关 miRNA表达,预测潜在下游目标。使用miR-133a-3p转染的 RSC96细胞进行体外实验。使用微蛋白质印迹和蛋白质印迹以及免疫荧光分析验证miR-133a-3p的作用。使用miR-133a-3p拮抗剂分析miR-133a-3p与DNP之间的关联。结果：miR-133a-3p模拟物增加了RSC96细胞中VEGFR-2、p38αMAPK、TRAF-6和PIAS3的表达,并降低了NFκB p50和MKP3的表达（P＜0.05）。在正常大鼠中,AAV-miR-133a-3p通过神经内注射到坐骨神经中可诱导机械性异常性疼痛和p-p38 MAPK激活（P＜0.05）。在DM大鼠中,miR-133a-3p 拮抗剂给药可减轻DNP并下调p-p38磷酸化（P＜0.05）。结论：坐骨神经中miR-133a-3p的过度表达会引起这种疼痛,miR-133a-3p可能是患有神经性疼痛的患者的有用治疗靶点。     

miR-20a靶向CCND1作用PI3K/AKT信号通路抑制皮肤鳞状细胞癌的发展

摘要 目的：探究miR-20a与CCND1蛋白在皮肤鳞状细胞癌（CSCC）中的作用关系,以及其可能涉及的信号通路分子机制。方法：分别收集皮肤鳞状细胞癌患者的皮肤癌组织及其邻近正常皮肤组织,采用qRT-PCR分析组织中miR-20a和CCND1基因表达水平。为探究miR-20a对CSCC细胞的影响,将SCL-1细胞分为对照组（不转染）、miR-NC组（转染miR-NC）和miR-20a mimics组（转染miR-20a mimics）;为探究CCND1与PI3K/AKT信号通路的关系,将SCL-1细胞分为对照组（不转染）、si-NC组（转染si-NC）和si-CCND1组（转染si-CCND1）;为探究miR-20a与CCND1间的作用关系及对CSCC细胞的影响,将SCL-1细胞分为miR-NC组（转染miR-NC）、miR-20a mimics组（转染miR-20a mimics）、mimics+pcDNA组（共转染miR-20a mimics和pcDNA）和mimics+CCND1组（共转染miR-20a mimics和pcDNA-CCND1）。采用Western blot分析p-AKT、AKT、p-PI3K、PI3K和GSK-3β蛋白表达水平;采用MTT检测细胞增殖情况;采用流式细胞术检测细胞凋亡情况;采用Transwell分析细胞迁移和侵袭情况;采用双荧光素酶报告基因检测分析miR-20a与CCND1的靶向关系。结果：CSCC癌组织和SCL-1中miR-20a均低表达,CCND1高表达。与对照组和miR-NC组比较,miR-20a mimics组SCL-1细胞增殖水平以及侵袭和迁移数量均降低（P<0.05）,SCL-1细胞凋亡水平升高（P<0.05）,PI3K和AKT蛋白磷酸化水平降低（P<0.05）。TargetScanHuman数据库分析和双荧光素酶报告基因检测结果显示miR-20a与CCND1存在靶向作用关系。与对照组和si-NC组比较,si-CCND1组SCL-1细胞中CCND1和GSK-3β蛋白表达水平以及PI3K和AKT蛋白磷酸化水平均降低（P<0.01）。与miR-20a mimics组或mimics+pcDNA组比较,mimics+CCND1组SCL-1细胞增殖水平以及侵袭和迁移数量均升高（P<0.05）,SCL-1细胞凋亡水平降低（P<0.05）,PI3K和AKT蛋白磷酸化水平均升高（P<0.05）。结论：过表达miR-20a可能通过靶向抑制CCND1的表达而抑制PI3K/AKT信号通路的激活,从而抑制CSCC细胞的增殖、侵袭和迁移,并促进癌细胞凋亡。     

MiR-21靶向调控Mfn2对缺血再灌注损伤肾小管上皮NRK-52E细胞自噬及凋亡的影响

摘要 目的：探讨miR-21对缺血再灌注损伤肾小管上皮细胞自噬及凋亡的影响及其与线粒体融合素2（mitochondria fusion protein mitofusin2,Mfn2）的靶向关系。方法：将大鼠近端肾小管上皮细胞株NRK-52E细胞按处理方式不同分组：I/R+control mimics组（转染control mimics后缺氧3 h/复氧3 h）,I/R+miR-21mimics组（转染miR-21mimics后缺氧3 h/复氧3 h）,I/R组（缺氧3 h/复氧3 h）及对照组（正常培养）。选取30只Sprague-Dawley(SD)大鼠,随机分为假手术组、缺血再灌注模型组(I/R组)。取大鼠肾组织进行HE染色,自动生化分析仪检测大鼠血清尿素氮（BUN）、肌酐（Cr）,四甲基偶氮唑盐比色法（MTT）检测细胞增殖能力,TUNEL法检测细胞凋亡,实时荧光定量PCR检测细胞自噬和凋亡相关基因LC3-Ⅱ、LC3-Ⅰ、Beclin1、Bcl-2、Bax及Mfn2 mRNA表达,Western blot法检测细胞自噬和凋亡相关蛋白的表达,荧光素酶实验验证miR-21与Mfn2的靶向关系。结果：Sham组大鼠血清BUN、Cr水平,大鼠肾组织细胞凋亡率高于I/R组（P<0.05）。I/R组大鼠肾组织肾小管结构紊乱,大量炎症细胞浸润。Sham组大鼠肾组织miR-21水平高于I/R组（P<0.05）。48 和 72 h 时,I/R+miR-21 mimics组细胞活力明显低于I/R+control mimics组,I/R组及对照组（P<0.05）,I/R组细胞活力低于对照组（P<0.05）。I/R+miR-21mimics组凋亡率显著高于I/R+control mimics组,I/R组及对照组（P<0.05）,I/R组凋亡率显著高于对照组（P<0.05）。与对照组比较,I/R组细胞Beclin1、LC3-Ⅱ/LC3-Ⅰ、Bax蛋白及基因mRNA表达量升高,Bcl-2蛋白及基因mRNA表达量降低（P<0.05）;与I/R组比较,I/R+miR-21mimics组细胞Beclin1、LC3-Ⅱ/LC3-Ⅰ、Bax蛋白及基因mRNA表达量升高,Bcl-2蛋白及基因mRNA表达量降低（P<0.05）。miR-21与Mfn2具有靶向关系。结论：miR-21可靶向Mfn2促进肾缺血再灌注损伤引起的凋亡及自噬。     

叶酸抑制宫颈癌进展过程中miR-642a-5p的表达及其对肿瘤细胞的抑制作用研究

摘要 目的：探讨叶酸抑制宫颈癌进展中过程中miR-642a-5p的表达及其对肿瘤细胞的抑制作用。方法：选择宫颈癌细胞株SiHa进行传代培养,采用随机法分为4组：低剂量组、中剂量组、高剂量组分别加入1 μg/mL、10 μg/mL、100 μg/mL的叶酸,而空白对照组未加入叶酸处理。利用实时定量聚合酶链式反应（RT-qPCR）检测miR-642a-5p的表达;采用细胞增殖毒性试验（CCK-8法）、细胞迁移实验（划痕实验）和细胞侵袭实验（Transwell法）分别测量宫颈癌SiHa细胞的增殖活性、迁移和侵袭能力。结果：低、中、高剂量组中叶酸显著抑制了宫颈癌SiHa细胞中miR-642a-5p的表达（P<0.05）,而且随着叶酸浓度升高,其对miR-642a-5p的抑制作用逐渐增强。此外,低、中、高剂量组中叶酸对宫颈癌SiHa细胞的增殖、迁移以及侵袭具有显著抑制作用（P<0.05）,而且叶酸浓度越高,其抑制作用越强。结论：叶酸可以抑制宫颈癌进展过程中miR-642a-5p的表达,而且对宫颈癌SiHa细胞的增殖、迁移和侵袭具有一定的抑制作用。     

circ_0000267靶向miR-198对急性淋巴细胞白血病细胞KOCL44增殖和凋亡的影响

目的探讨circ_0000267对急性淋巴细胞白血病（ALL）KOCL44细胞增殖和凋亡的影响及其作用机制。 方法选择ALL细胞株KOCL44为研究对象,分别将小干扰RNA （siRNA）阴性对照（si-NC）、circ_0000267 siRNA （si-circ_0000267）、微小RNA （miRNA）阴性对照（miR-NC）、miR-198模拟物（miR-198）、circ_0000267 siRNA+miRNA抑制剂阴性对照（si-circ_0000267+anti-miR-NC）和circ_0000267 siRNA+miR-198抑制剂（si-circ_0000267+anti-miR-198）转染细胞,48 h后通过RT-qPCR检测细胞circ_0000267和miR-198相对表达水平,采用CCK-8法检测KOCL44细胞的增殖水平,流式细胞术实验检测KOCL44细胞的凋亡水平,Western blot检测KOCL44细胞Ki-67、Bcl-2和Bax蛋白表达水平,通过双荧光素酶报告实验验证circ_0000267和miR-198靶向关系。两组间比较采用独立样本t检验。 结果与健康志愿者比较,ALL患者circ_0000267表达水平（1.00±0.06比3.19±0.21）上调,miR-198表达水平（1.00±0.07比0.41±0.03）下调,差异有统计学意义（P < 0.05）。敲低circ_0000267或者过表达miR-198可抑制KOCL44细胞增殖（0.68±0.05比0.32±0.02、0.69±0.06比0.39±0.03）、Ki-67 （0.84±0.06比0.37±0.03、0.85±0.06比0.45±0.04）和Bcl-2蛋白表达（0.63±0.05比0.22±0.02、0.65±0.04比0.29±0.02）,促进细胞凋亡[（6.53±0.51）﹪比（24.29±2.06）﹪、（7.38±0.57）﹪比（20.03±1.66）﹪]和Bax蛋白表达（0.31±0.03比0.77±0.04、0.30±0.02比0.71±0.04）,差异有统计学意义（P < 0.05）。双荧光素酶报告实验验证circ_0000267可以靶向miR-198表达,干扰miR-198表达可以逆转抑制circ_0000267表达对KOCL44细胞的增殖和凋亡的作用,差异有统计学意义（P < 0.05）。 结论circ_0000267通过调控miR-198抑制ALL细胞增殖,并促进凋亡,为临床治疗ALL提供新的依据。     

下调lncRNA MCF2L-AS1靶向miR-33b-5p抑制胃癌细胞增殖、迁移、侵袭并促进凋亡

摘要 目的：探讨lncRNA MCF2L-AS1对胃癌细胞恶性生物学行为的影响及分子机制。方法：选取45例胃癌患者的癌组织及癌旁正常组织,或培养胃黏膜上皮细胞GES-1、胃癌细胞HGC-27,采用RT-qPCR检测MCF2L-AS1和miR-33b-5p的表达水平。采用双荧光素酶报告实验检测MCF2L-AS1和miR-33b-5p的靶向关系。将HGC-27细胞分为si-NC组、si-MCF2L-AS1组、mimic NC组、miR-33b-5p mimic组、si-MCF2L-AS1+inhibitor NC组、si-MCF2L-AS1+miR-33b-5p inhibitor组,分别转染si-NC、si-MCF2L-AS1、mimic NC、miR-33b-5p mimic或共转染si-MCF2L-AS1+inhibitor NC、si-MCF2L-AS1+miR-33b-5p inhibitor。采用MTT实验检测细胞增殖情况,流式细胞术检测细胞凋亡率,克隆形成实验检测细胞克隆形成数,Transwell实验检测迁移和侵袭细胞数。结果：与癌旁正常组织或GES-1细胞相比,胃癌组织或HGC-27细胞中MCF2L-AS1表达水平升高、miR-33b-5p表达水平降低,差异均有统计学意义（P<0.05）。MCF2L-AS1可靶向调控miR-33b-5p。下调MCF2L-AS1或过表达miR-33b-5p,miR-33b-5p表达水平升高,HGC-27细胞凋亡率升高,但细胞增殖、克隆形成数、迁移和侵袭数均减少,差异均有统计学意义（P<0.05）。抑制miR-33b-5p可减弱下调MCF2L-AS1对HGC-27细胞的生物学作用。结论：下调MCF2L-AS1通过上调miR-33b-5p抑制胃癌细胞增殖、迁移、侵袭并促进凋亡;MCF2L-AS1通过靶向调控miR-33b-5p表达进而参与胃癌细胞的恶性生物学行为。     

Circ_0011058 facilitates proliferation,angiogenesis and radioresistance in papillary thyroid cancer cells by positively regulating YAP1 via acting as miR-335-5p sponge

BackgroundCircular RNAs (circRNAs) are reported to be associated with multiple biological processes in human cancers. However, there are still numerous circRNAs whose functions remain unclear. The aim of this study was to investigate the role of circ_0011058 in papillary thyroid cancer (PTC).MethodsQuantitative real-time PCR (qPCR) was utilized to detect the expression of circ_0011058, microRNA-335-5p (miR-335-5p) and Yes-associated Protein 1 (YAP1). Cell proliferation was detected using cell counting kit-8 (CCK-8) assay and EdU assay. Cell apoptosis was detected by flow cytometry assay. Angiogenesis ability was assessed using tube formation assay. The expression of angiogenesis-related proteins and YAP1 protein was detected by western blot. Radioresistance was examined using colony formation assay. The binding relationship between miR-335-5p and circ_0011058 or YAP1 was verified by dual-luciferase reporter assay, pull-down assay and RIP assay. Xenograft models were constructed to ensure the role of circ_0011058.ResultsCirc_0011058 expression was aberrantly elevated in PTC tissues and cells. The downregulation of circ_0011058 suppressed proliferation, angiogenesis and radioresistance in PTC cells. MiR-335-5p was defined as a target of circ_0011058, and miR-335-5p inhibition reversed the effects of circ_0011058 downregulation. In addition, YAP1 was a target of miR-335-5p, and circ_0011058 positively regulated YAP1 expression by targeting miR-335-5p. MiR-335-5p restoration inhibited proliferation, angiogenesis and radioresistance in PTC cells, while YAP1 overexpression abolished these effects. Animal study showed that circ_0011058 knockdown inhibited tumor growth in vivo.ConclusionCirc_0011058 promoted PTC cell proliferation, angiogenesis and radioresistance by upregulating YAP1 via acting as miR-335-5p sponge.     

miR-15a-5p和miR-16-5p在骨肉瘤组织中的表达分析

目的:使用microRNAs基因芯片及实时定量PCR法测定骨肉瘤组织中miR-15a-5p和miR-16-5p的相对表达含量,并与瘤旁组织对比,分析骨肉瘤细胞内miR-15a-5p和miR-16-5p的表达变化。方法:选取34例骨肉瘤组织蜡块样本,使用microRNAs基因芯片观察miR-15a-5p和miR-16-5p在骨肉瘤和瘤旁组织内的表达差异;实时定量PCR法测定骨肉瘤组织和瘤旁组织中miR-15a-5p和miR-16-5p的相对表达含量,并将两种结果对比分析。结果:microRNAs基因芯片结果显示,在骨肉瘤组织中,miR-15a-5p在肿瘤中的表达较瘤旁组织低1.79倍,miR-16-5p较瘤旁组织低1.62倍。实时定量PCR实验结果表明,miR-15a-5p和miR-16-5p表达较瘤旁组织降低,差异有统计学意义(P0.05)。经过统计学计算,miR-15a-5p在肿瘤中的表达较瘤旁组织低3.14倍,miR-16-5p较瘤旁组织低5.65倍。结论:在骨肉瘤中,miR-15a-5p和miR-16-5p表达含量降低,提示这两种microRNAs在骨肉瘤中可能做为抑癌因子存在。     

Hsa_circ_0009910 promotes carcinogenesis by promoting the expression of miR-449a target IL6R in osteosarcoma

Circular RNAs (circRNAs) have recently shown capabilities as gene regulators in mammals. Some of them interact with microRNAs (miRNAs) and function as sponges to affect related miRNAs' activities. In this study, the molecular function of circRNA_0009910 and its potential downstream miRNA targets were explored. The expression levels of hsa_circ_0009910 were found to be overexpressed in osteosarcoma (OS) cells. Knockdown of circ_0009910 induced cell proliferation inhibition, cell cycle arrest, and apoptosis in OS cells. The target miRNA was predicted to be miR-449a, whose expression was downregulated in OS cells. Inhibition of miR-449a abolished the effect of circ_0009910 knockdown on cell growth and apoptosis. The expression of miR-449a were found to be negatively correlated with that of circ_0009910 in OS tissues. Direct interaction of circ_0009910 and miR-449a was confirmed through dual-luciferase assays. Moreover, IL6R was predicted as a potential target of miR-449a. Overexpression of miR-449a decreased the mRNA and protein levels of IL6R. Restoration of IL6R impaired the miR-449a induced inhibition of cell proliferation, cell cycle arrest, and apoptosis. The mRNA expression of IL6R was inversely correlated with miR-449a in OS tissues. In addition, JAK1/STAT3 signaling pathway was regulated by circ_0009910/miR-449a/IL6R axis. Taken together, we suggested that circ_0009910 acted as a sponge of miR-449a and upregulated miR-449a functional target IL6R, thereby contributed to carcinogenesis of OS.     

LncRNA TMPO-AS1 serves as a ceRNA to promote osteosarcoma tumorigenesis by regulating miR-199a-5p/WNT7B axis

Osteosarcoma (OS) is a common kind of aggressive tumor in bone which was mostly identified in children and adolescents with extremely high risk of death. Accumulating research works have displayed that long noncoding RNAs (lncRNAs) exert an essential role in the development of multiple cancers. It has been reported that TMPO-AS1 is an oncogene in cancers; nonetheless, its molecular mechanism in OS is totally unclear. Our present study elucidated that a remarkable overexpression of TMPO-AS1 was found in OS tissues and cells. Moreover, TMPO-AS1 depletion restrained Wnt/β-catenin pathway and cell proliferation as well as facilitated cell apoptosis. Further molecular mechanism investigations showed that TMPO-AS1 can sponge to miR-199a-5p. Moreover, miR-199a-5p was at a low level at OS cells. Importantly, miR-199a-5p's overexpression was associated with the OS cells' decreased proliferation and increased apoptosis. In addition, WNT7B was confirmed as a downstream gene of miR-199a-5p. Also the WNT7B expression was reversely modulated by miR-199a-5p and positively modulated by TMPO-AS1. Rescue experiments suggested that downregulated WNT7B rescued miR-199a-5p inhibitor-mediated repression on OS progression, but the treatment of LiCl counteracted the effect of WNT7B downregulation. In a word, TMPO-AS1 serves as a competing endogenous RNA to boost osteosarcoma tumorigenesis by regulating miR-199a-5p/WNT7B axis, which provided an underlying therapeutic target for patients with OS.     

miR-421 promotes apoptosis and suppresses metastasis of osteosarcoma cells via targeting LTBP2

Increasing evidence has confirmed that microRNAs (miRs) are involved in tumor development and progression. A previous study reported that miR-421 could serve as a diagnostic marker in patients with osteosarcoma (OS). The present study explored the potential roles of miR-421 in the regulation of cell proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition of OS cells. Our results showed that miR-421 was upregulated in OS tissues and cell lines (MG63, U2OS, HOS, and Saos-2) compared with the corresponding adjacent tissues or human osteoblast cells hFOB1.19, while the latent transforming growth factor β-binding protein 2 (LTBP2) expression was reduced. In MG63 and U2OS cells, CCK8 assay displayed that cell proliferation was repressed by the miR-421 inhibitor, conversely increased by miR-421 mimics. Inhibition of miR-421 promoted cell apoptosis rate, caspase 3 activity, cleaved-caspase 3 (c-caspase 3) expression, and Bax/Bcl-2 ratio, restoration of miR-421 showed the opposite functions. Suppression of miR-421 blocked migration and invasion, whereas miR-421 overexpression promoted the migration and invasion of MG63 and U2OS cells. In addition, real-time polymerase chain reaction and Western blot analysis revealed that miR-421 negatively regulated E-cadherin expression, and positively regulated the expression of N-cadherin and vimentin. The luciferase reporter assay determined that miR-421 could target LTBP2-3′-UTR, and LTBP2 expression was regulated negatively by miR-421 both in mRNA and protein levels. Depletion of LTBP2 partly abolished the biological functions of miR-421 inhibitor in OS. In conclusion, miR-421 plays an oncogenic role in OS via targeting LTBP2, suggesting that miR-421 may be a potential therapeutic target against OS.     

miR-17-5p promotes proliferation and epithelial-mesenchymal transition in human osteosarcoma cells by targeting SRC kinase signaling inhibitor 1

MicroRNA-17-5p (miR-17-5p) and epithelial-mesenchymal transition (EMT) have been reported to participate in the development and progression of multiple cancers. However, the relationship between the miR-17-5p and EMT in osteosarcoma (OS) is still poorly understood. This study was to investigate the effects of the miR-17-5p and its potential mechanism in regulating proliferation, apoptosis, and EMT of human OS. Quantitative real-time PCR was used to detect the miR-17-5p and SRC kinase signaling inhibitor 1 (SRCIN1) messenger RNA expression in OS specimens and cell lines. After transfection with miR-17-5p inhibitors, proliferation, apoptosis, migration, and invasion of OS cells were assessed by using the Cell Counting Kit-8, the annexin V-FITC apoptosis, wound-healing, and transwell assays. The SRCIN1 was validated as a target of the miR-17-5p through bioinformatics algorithms and luciferase reporter assay. Moreover, the expression of EMT markers, E-cadherin, N-cadherin, and Snail was identified by the Western blot analysis. MiR-17-5p was significantly upregulated in OS tumor samples and cell lines. It inhibited proliferation and EMT, and promoted apoptosis in OS. The SRCIN1 was identified as a direct target of the miR-17-5p. Silenced miR-17-5p could change the expression of EMT markers, such as upregulating the expression of E-cadherin, and downregulating the expression of N-cadherin and Snail through targeting the antioncogenic SRCIN1. These findings suggest that the miR-17-5p promotes cell proliferation, and EMT in human OS by directly targeting the SRCIN1, and reveal a branch of the miR-17-5p/SRCIN1/EMT signaling pathway involved in the progression of OS.     

Hsa_circ_0088212-mediated miR-520 h/APOA1 axis inhibits osteosarcoma progression

BackgroundIt has been known for decades that circRNAs are deregulated in cancer. Here, we characterized the role and underlying mechanism of circ_0088212 in osteosarcoma.MethodsThe expression levels of circ_0088212, miR-520 h, and APOA1 were determined by RT-qPCR. RNase R digestion was performed to verify the circular structure of circ_0088212. CCK8 and transwell invasion assays were conducted to examine the in vitro malignancy of osteosarcoma. Caspase-3 activity was also measured.An in vivo model of osteosarcoma was constructed to examine the in vivo effect of circ_0088212 on osteosarcoma. Luciferase reporter, RNA RIP, and RNA pull-down assays were performed to verify the interaction between miR-520 h and APOA1 or circ_0088212.ResultsCirc_0088212 and APOA1 were expressed at low levels in osteosarcoma tissues and cells, while miR-520 h was highly expressed. Overexpression of circ_0088212 was found to inhibit the in vitro and in vivo growth of osteosarcoma. Mechanistically, miR-520 h was the target of circ_0088212 and APOA1 was the target of miR-520 h. Circ_0088212 downregulated miR-520 h expression, while miR-520 h overexpression abolished the inhibitory effect of circ_0088212 on osteosarcoma cell proliferation and migration. Furthermore, miR-520 h overexpression led to reduced APOA1 expression, while APOA1 overexpression counteracted the oncogenic effect of miR-520 h in osteosarcoma cells.ConclusionOur findings demonstrated that circ_0088212 might exert a tumor-suppressive activity in osteosarcoma by sponging and sequestering miR-520 h away from APOA1. This suggests that the circ_0088212/miR-520 h/APOA1 axis may be a promising therapeutic target for osteosarcoma intervention.