背景 麦冬皂苷D(ophiopogoninD,OPD)是中药麦冬提取物中重要的单体成分且具有抗癌作用,但是否具有抗肝细胞癌(hepatocellular carcinoma,HCC)作用还未知.本研究假设OPD能够通过上调miR-519d-3p表达进而下调真核细胞翻译起始因子4E(eukaryotic translation initiation factor 4E,EIF4E)表达发挥抗HCC作用.目的 探讨OPD对HCC细胞增殖、迁移和侵袭的影响及可能的作用机制.方法 培养HCC细胞HepG2和MHCC97,不同浓度(2.5 μmol/L、5 μmol/L、10tmol/L)的OPD作用48 h后,四甲基噻唑蓝染色法(methylthiazoletrazolium,MTT)检测细胞增殖,Transwell检测细胞迁移和侵袭,实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)检测细胞中miR-519d-3p和EIF4E mRNA表达,Western blot 检测细胞周期蛋白Dl(CyclinD1)、p21、基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)、MMP-9和EIF4E蛋白表达,双荧光素酶报告基因实验验证miR-519d-3p和EIF4E之间关系.转染miR-519d-3pmimics、si-EIF4E构建miR-519d-3p过表达或EIF4E表达抑制的HepG2和MHCC97细胞,RT-qPCR检测细胞中miR-519d-3p表达或Western blot检测EIF4E蛋白表达验证转染效率.MTT、Transwell、Western blot分别检测过表达miR-519d-3p或抑制EIF4E表达对HepG2和MHCC97细胞增殖、迁移和侵袭,及CyclinD1、p21、MMP-2和MMP-9蛋白表达的影响.结果 与对照组比,OPD组HepG2细胞抑制率显著升高(P＜0.05),迁移和侵袭数显著降低(P＜0.05),HepG2细胞中CyclinD1、MMP-2和MMP-9蛋白表达显著降低(P＜0.05),p21蛋白表达显著升高(P＜0.05),miR-519d-3p表达显著升高(P＜0.05),EIF4E mRNA和蛋白表达显著降低(P＜0.05),且呈浓度依赖性.miR-519d-3p在HepG2细胞中靶向负调控EIF4E表达.miR-519d-3p过表达或抑制EIF4E表达均可抑制HepG2细胞增殖、迁移和侵袭.抑制miR-519d-3p表达部分逆转了OPD对HepG2细胞增殖、迁移和侵袭的抑制作用.结论 OPD可能通过调控miR-519d-3p/EIF4E表达抑制HCC细胞的增殖、迁移和侵袭.

Objective: MicroRNA (miRNA), a short noncoding RNA, is claimed to be a potential blood-based biomarker. We aimed to identify and evaluate miRNAs as diagnostic biomarkers for non-small cell lung cancer (NSCLC). Methods: Profiles of 745 miRNAs were screened in the serum of 8 patients with NSCLC and 8 age- and sex-matched controls using TaqMan low-density arrays (TLDAs) and validated in 25 patients with NSCLC and 30 with other lung diseases (OLs) as well as in 19 healthy persons (HPs). The diagnostic performance of the candidate miRNAs was assessed in 117 cases of NSCLC and 113 OLs using quantitative real-time polymerase chain reaction (qRT-PCR). Differences in miRNA expression between patients with NSCLC and controls were assessed using the Mann–Whitney U test. The area under receiver operating characteristic (ROC) curve (AUC) was obtained based on the logistic regression model. Results: Ten miRNAs were found to be differentialy expressed between patients with NSCLC and controls, including miR-769, miR-339-3p, miR-339-5p, miR-519a, miR-1238, miR-99a#, miR-134, miR-604, miR-539, and miR-342. The expression of miR-339-3p was significantly higher in patients with NSCLC than in those with OLs (P < 0.001) and HPs (P = 0.020). ROC analysis revealed an miR-339-3p expression AUC of 0.616 [95％ confidence interval (CI): 0.561–0.702]. The diagnostic prediction was increased (AUC = 0.706, 95％ CI: 0.649–0.779) in the model combining miR-339-3p expression and other known risk factors (i.e., age, smoking status, and drinking status). Conclusions: MiR-339-3p was significantly upregulated in patients with NSCLC compared with participants without cancer, suggesting a diagnostic prediction value for high-risk individuals. Therefore, miR-339-3p expression could be a potential blood-based biomarker for NSCLC.

该研究探讨了 lncRNA SOX2-OT对乳腺癌细胞增殖和放射敏感性的影响及可能机制.采用qRT-PCR法检测了正常乳腺上皮细胞MCF-10A及乳腺癌细胞系(MCF-7、T47D和Bcap-37)中lncRNA SOX2-OT和miR-519e-5p表达.乳腺癌 T47D细胞中 lncRNA SOX2-OT和miR-519e-5p表达较MCF-10A细胞差异最显著(P＜0.05),选择乳腺癌T47D细胞为研究对象.采用核质分离及RNA荧光原位杂交分析lncRNA SOX2-OT在T47D细胞内的定位.分别向T47D细胞转染lncRNA SOX2-OT小干扰RNA、miR-519e-5p模拟物、过表达RNA SOX2-OT、共转染lncRNA SOX2-OT小干扰RNA与miR-519e-5p抑制剂、共转染过表达RNA SOX2-OT与miR-519e-5p模拟物,即得si-lncRNA SOX2-OT组、si-NC组、miR-519e-5p组、miR-NC组、si-lncRNA SOX2-OT+anti-miR-519e-5p组、si-lncRNA SOX2-OT+anti-miR-NC组、RNA SOX2-OT+miR-519e-5p mimics组、si-lncRNA SOX2-OT+miR-NC组.采用CCK-8法观察细胞增殖活性,克隆形成实验计算细胞存活分数,并用单击多靶数学模型计算细胞增敏比,蛋白质印迹法检测细胞中CyclinD1和y-H2AX蛋白表达.双荧光素酶报告基因实验验证lncRNA SOX2-OT和miR-519e-5p的调控关系.结果显示,lncRNA SOX2-OT主要定位于T47D的细胞质.敲减lncRNA SOX2-OT或上调miR-519e-5p后,T47D细胞增殖活性和存活分数均降低(P＜0.05),增敏比分别为1.195、1.343,细胞中CyclinD1蛋白表达降低(P＜0.05),γ-H2AX蛋白表达升高(P＜0.05).lncRNA SOX2-OT在T47D细胞中靶向负调控miR-519e-5p.下调或上调miR-519e-5p可分别逆转敲减或上调lncRNA SOX2-OT对T47D细胞增殖和放射敏感性的影响.总之,lncRNA SOX2-OT可能通过靶向负调控miR-519e-5p促进乳腺癌细胞增殖并降低乳腺细胞对放射的敏感性.

目的 探讨黄癸素(luteolin)是否通过长链非编码RNA(long noncoding RNA,LncRNA)ZF-PM2反义RNA 1(ZFPM2-AS1)/微小RNA(miRNA)-519e-5p轴影响神经母细胞瘤细胞的增殖和凋亡.方法 将神经母细胞瘤细胞分为对照组,黄癸素低、中、高剂量组(1、2、8 mg·L-1 黄癸素),si-NC 组(转染 si-NC),si-ZFPM2-AS1 组(转染 si-ZFPM2-AS1),黄癸素高剂量+pcDNA 组(8 mg·L-1 黄癸素处理前转染pcDNA),黄癸素高剂量+pcDNA-ZFPM2-AS1组(8 mg·L-1 黄癸素处理前转染 pcDNA-ZFPM2-AS1),miR-NC 组(转染 miR-NC),miR-519e-5p 组(转染 miR-519e-5p mimic).采用细胞计数试剂盒8(CCK-8)法、集落形成实验、流式细胞术、免疫印迹实验(Western blotting)和定量逆转录聚合酶链反应(qRT-PCR)检测神经母细胞瘤细胞的活力、集落形成、凋亡率、Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤/白血病-2(Bcl-2)和ZFPM2-AS1、miR-519e-5p表达.生物信息学StarBase软件与双荧光素酶报告实验分析ZFPM2-AS1与miR-519e-5p之间的靶向结合.结果 与对照组或黄癸素低剂量组相比,黄癸素中剂量组和黄癸素高剂量组神经母细胞瘤细胞OD值、集落形成数、Bcl-2蛋白、ZFPM2-AS1表达量降低,细胞凋亡率、Bax蛋白、miR-519e-5p表达量升高(P＜0.05),对照组与黄癸素低剂量组各指标比较差异不显著.si-ZFPM2-AS1组神经母细胞瘤细胞中miR-519e-5p表达量、凋亡率和Bax蛋白表达量比si-NC组高,OD值、集落形成数、Bcl-2蛋白表达量比si-NC组低(P＜0.05).与黄癸素高剂量+pcDNA组相比,黄癸素高剂量+pcDNA-ZFPM2-AS1组神经母细胞瘤细胞miR-519e-5p表达量、凋亡率、Bax蛋白表达量降低,OD值、集落形成数、Bcl-2蛋白表达量升高(P＜0.05).ZFPM2-AS1可以靶向miR-519e-5p.miR-519e-5p组神经母细胞瘤细胞OD值、集落形成数、Bcl-2蛋白表达量比miR-NC组低,凋亡率、Bax蛋白表达量比miR-NC组高(P＜0.05).结论 黄癸素可以通过调控ZFPM2-AS1/miR-519e-5p轴抑制神经母细胞瘤细胞的增殖并诱导其凋亡.

目的:探讨长链非编码RNA(long non-coding RNA,lncRNA)LINC00461对骨肉瘤细胞增殖、迁移和侵袭的影响及分子机制.方法:选取40例骨肉瘤患者的瘤组织及瘤旁组织,培养人成骨细胞hFOB 1.19,骨肉瘤细胞系Saos2、U2OS和HOS,用real-time RT-PCR检测LINC00461和微RNA-519e-5p(microRNA-519e-5p,miR-519e-5p)表达水平.将Saos2细胞分为NC组、si-LINC00461组、si-NC组、miR-519e-5p组(miR-519e-5p类似物)、miR-NC组、si-LINC00461+anti-miR-519e-5p组、si-LINC00461+anti-miR-NC组.蛋白质印迹法检测蛋白质表达;CCK-8检测细胞活性;克隆形成实验检测细胞克隆形成数;Transwell检测细胞迁移和侵袭数;双荧光素酶报告实验和RNA pull-down实验检测LINC00461和miR-519e-5p的靶向关系.结果:与瘤旁组织比较,骨肉瘤组织中LINC00461高表达,miR-519e-5p低表达(P<0.05).与hFOB 1.19细胞比较,骨肉瘤细胞系Saos2、U2OS和HOS中LINC00461表达水平升高,miR-519e-5p表达水平降低(P<0.05).低表达LINC00461或过表达miR-519e-5p,Ki-67、基质金属蛋白酶(matrix metalloproteinase,MMP)2、MMP9蛋白表达水平,Saos2细胞活性,细胞克隆形成数及迁移侵袭数降低(P<0.05).LINC00461靶向负调控miR-519e-5p的表达.低表达miR-519e-5p可以逆转LINC00461低表达对Saos2细胞增殖、迁移和侵袭的影响.结论:低表达LINC00461通过靶向上调miR-519e-5p抑制骨肉瘤细胞的增殖、迁移和侵袭.

目的:探讨lncRNAZFPM2-AS1靶向miR-519e-5p对脊索瘤U-CH1细胞生物学行为的影响.方法:体外培养U-CH1细胞,将ZFPM2-AS1的小干扰RNA以及miR-519e-5p的模拟物分别转入U-CH1细胞中,并随机分组:si-NC组、si-ZFPM2-AS1组、miR-NC组、miR-519e-5p 组、si-ZFPM2-AS 1+anti-miR-NC 组、si-ZFPM2-AS 1+anti-miR-519e-5p 组;实时荧光定量聚合酶链式反应(RT-qPCR)检测U-CH1细胞中lncRNAZFPM2-AS1与miR-519e-5p表达量;MTT、流式细胞术、划痕实验、Transwell实验分别检测U-CH1细胞增殖、凋亡、迁移、侵袭;双荧光素酶报告实验检测lncRNAZFPM2-AS1与miR-519e-5p的靶向调控关系;蛋白印迹(western blotting)法分析蛋白 E-cadherin、N-cadherin表达.结果:与 si-NC 组、miR-NC 组比较,si-ZFPM2-AS 1 组、miR-519e-5p组细胞活力、划痕愈合率、侵袭细胞数及蛋白N-cadherin水平下降,凋亡率、E-cadherin水平增高(P＜0.05);ln-cRNA ZFPM2-AS1 可负向调控 miR-519e-5p 表达;si-ZFPM2-AS1+anti-miR-519e-5p 组与 si-ZFPM2-AS1+anti-miR-NC 组比较,细胞活力、划痕愈合率、侵袭细胞数及蛋白N-cadherin水平增高,凋亡率、E-cadherin水平下降(P＜0.05).结论:下调lncRNA ZFPM2-AS1可促进U-CH1细胞凋亡并阻滞增殖、迁移、侵袭,其机制与上调miR-519e-5p表达有关.