LPS对骨肉瘤细胞迁移、侵袭及TLR4/HOTAIR通路的影响

目的探讨脂多糖(LPS)对骨肉瘤细胞迁移和侵袭的影响及其潜在的作用机制。方法将人MG-63骨肉瘤细胞随机分为2组:对照组和LPS组。LPS组细胞用10 g/ml的LPS干预24 h,对照组用生理盐水干预。ELISA检测干预后培养基中促炎因子的水平,Transwell实验检测细胞迁移和侵袭能力,Western Blot检测相关蛋白的表达。结果与对照组相比,LPS组培养基中促炎因子TNF-α、IL-1和IL-6的释放水平均显著增高(P<0.05),LPS组迁移细胞数和侵袭细胞数均显著增高(P<0.05),LPS组中E-cadherin的表达显著降低(P<0.05),而N-cadherin、α-SMA、波形蛋白、TLR4和HOTAIR的表达均显著增高(P<0.05)。结论LPS诱导的肿瘤微环境可促进骨肉瘤细胞的迁移和侵袭,其机制与TLR4/HOTAIR途径介导的EMT过程的发生有密切关系。     

Effect of Diacerein on HOTAIR/IL-6/STAT3, Wnt/β-Catenin and TLR-4/NF-κB/TNF-α axes in colon carcinogenesis

Colorectal cancer (CRC) is a common malignancy with high mortality and poor prognosis. Diacerein (DIA) is an anti-inflammatory used for treatment of osteoarthritis. We delineated some underlying molecular mechanisms of DIA’s anti-carcinogenic effect in CRC using in vivo and in vitro models. Human Caco-2 cells were treated with DIA followed by MTT and Annexin V assays and CRC was experimentally induced using 1,2-dimethylhydrazine. DIA (50 mg/kg/day, orally) was administrated for 8 weeks. The MTT assay confirmed cytotoxic effect of DIA in vitro and Annexin V confirmed its apoptotic effect. DIA resulted in regression of tumour lesions with reduced colonic TLR4, NF-κB and TNF-α protein levels and down-regulated VEGF expression, confirming anti-angiogenic impact. DIA triggered caspase-3 expression and regulated Wnt/β-Catenin pathway, by apparently interrupting the IL-6/STAT3/ lncRNA HOTAIR axis. In conclusion, DIA disrupted IL-6/STAT3/ lncRNA HOTAIR axis which could offer an effective therapeutic strategy for the management of CRC.     

Combined identification of long non-coding RNA CCAT1 and HOTAIR in serum as an effective screening for colorectal carcinoma

Long non-coding RNAs (lncRNAs) CCAT1 and HOTAIR have been shown to play an important regulatory role in cancer biology, and CCAT1 and HOTAIR are upregulated in several cancers, however, its value in the diagnosis of colorectal cancer (CRC) is unclear. Therefore, the aim of this study is to evaluate the clinical significance of plasma CCAT1 and HOTAIR as a biomarker in the screening of CRC. In our study, we found that the levels of HOTAIR (P < 0.05) and CCAT1 (P < 0.05) were significantly higher in plasma of CRC patients than that of the healthy control. Moreover, the levels of lincRNA-p21 (P < 0.05) were obviously decreased in plasma of CRC patients as compared to those of healthy control. There was highly correlated for CCAT1 (R = 0.752, mean differences = -0.06 ± 1.20), HOTAIR (R = 0.739, mean differences = -0.26 ± 0.76) and lincRNA-p21 (R = 0.848, mean differences = -0.41 ± 0.89) in plasma and serum. By receiver operating characteristic curve (ROC) analysis, plasma CCAT1 provided the higher diagnostic performance for detection of CRC (the area under the ROC curve (AUC), 0.836; P < 0.001; sensitivity, 75.7%; specificity, 85.3%). Moreover, CCAT1 combining with HOTAIR could provide a more effective diagnosis performance (AUC, 0.954, P < 0.001, sensitivity, 84.3%; specificity, 80.2%). Most importantly, this combination was effective to detect CRC at an early stage (85%). In conclusion, our results demonstrated that increased plasma HOTAIR and CCAT1 could be used as a predictive biomarker for CRC screening, and that combination of HOTAIR and CCAT1 had a higher positive diagnostic rate of CRC than HOTAIR or CCAT1 alone.     

Knockdown of long non-coding RNA HOTAIR inhibits malignant biological behaviors of human glioma cells via modulation of miR-326

Glioma is the most common and aggressive primary adult brain tumor. Long non-coding RNAs (lncRNAs) have important roles in a variety of biological properties of cancers. Here, we elucidated the function and the possible molecular mechanisms of lncRNA HOTAIR in human glioma U87 and U251 cell lines. Quantitative RT-PCR demonstrated that HOTAIR expression was up-regulated in glioma tissues and cell lines. Knockdown of HOTAIR exerted tumor-suppressive function in glioma cells. Further, HOTAIR was confirmed to be the target of miR-326 and miR-326 mediated the tumor-suppressive effects of HOTAIR knockdown on glioma cell lines. Moreover, over-expressed miR-326 reduced the FGF1 expression which played an oncogenic role in glioma by activating PI3K/AKT and MEK 1/2 pathways. In addition, the in vivo studies also supported the above findings. Taken together, knockdown of HOTAIR up-regulated miR-326 expression, and further inducing the decreased expression of FGF1, these results provided a comprehensive analysis of HOTAIR-miR-326-FGF1 axis in human glioma and provided a new potential therapeutic strategy for glioma treatment.     

类风湿关节炎患者血清长链非编码RNA HOTAIR、XIST和H19表达研究

目的探讨3种长链非编码RNA(lncRNA) HOTAIR、XIST和H19在类风湿关节炎(RA)、系统性红斑狼疮(SLE)和健康人对照组血清中的差异表达。方法收取50例RA疾病组、50例SLE疾病对照组和60例健康人对照组血清,共160例血清标本,提取血清总RNA,利用RT-qPCR方法分析HOTAIR、XIST和H19 3种lncRNA表达情况。用Spearman相关性分析探讨RA患者疾病活动指数(DAS28)与HOTAIR表达量的相关性。结果 RA组与健康人对照组和SLE疾病对照组相比,血清HOTAIR表达量显著增加(P0.01),XIST在RA和对照组之间表达量差异无统计学意义; RA患者血清中HOTAIR表达值与DAS28评分呈正相关(R~2=0.27,P=0.002),且DAS28评分≥5.1的疾病高度活动组的HOTAIR表达值显著高于3.2DAS28评分5.1的疾病活动组(P0.01);血清HOTAIR表达量与血清C反应蛋白(CRP)存在正相关关系(R~2=0.192,P=0.002)。结论LncRNA HOTAIR在RA患者血清中的表达量显著性升高且与DAS28评分呈显著正相关关系,血清中HOTAIR有可能成为诊断RA的潜在生物标志物。     

lncRNA HOTAIR靶向miR-126激活RhoA/ROCK通路促进喉癌Hep-2细胞增殖、侵袭与EMT

目的:探讨长链非编码RNA-Hox转录反义RNA（long non-coding RNA Hox antisense intergenic RNA,lncRNA HOTAIR）对喉癌Hep-2细胞增殖、侵袭及其上皮间质转化（epithelial-mesenchymal transition,EMT）的影响及机制。方法:利用实时荧光定量PCR（quantitative real-time PCR,RT-qPCR）检测喉癌组织、癌旁组织和Hep-2细胞中lncRNA HOTAIR、微小RNA-126（microRNA-126,miR-126）的表达量;下调lncRNA HOTAIR表达,分别通过细胞计数试剂盒（cell counting kit-8,CCK-8）、细胞侵袭实验和蛋白质印迹法（Western blot）检测Hep-2细胞增殖、侵袭和EMT能力。利用miRcode分析HOTAIR的靶基因,通过双荧光素酶报告基因实验分析lncRNA HOTAIR和miR-126之间的关系。同时上调lncRNA HOTAIR和miR-126的表达,检测lncRNA HOTAIR通过miR-126对Hep-2细胞增殖、侵袭和EMT的影响。下调miR-126表达后,Western blot检测RhoA、ROCK蛋白的表达量。Y-27632作用细胞后,检测下调miR-126对Hep-2细胞增殖、侵袭和EMT的影响。结果:在Hep-2细胞和喉癌组织中lncRNA HOTAIR的表达明显上调（P＜0.01）,miR-126表达明显下调（P＜0.01）。下调lncRNA HOTAIR表达后,抑制了Hep-2细胞增殖、侵袭和EMT;lncRNA HOTAIR与miR-126具有靶向和负调控关系;下调miR-126表达后,激活了Hep-2细胞内RhoA/ROCK通路,并且促进了Hep-2细胞增殖、侵袭和EMT。结论:上调lncRNA HOTAIR通过靶向miR-126激活RhoA/ROCK通路促进喉癌Hep-2细胞增殖、侵袭与EMT。     

肾癌细胞系中HOTAIR表达的测定及稳定高表达HOTAIR肾癌细胞株的建立

目的:检测常用肾癌细胞系中HOTAIR的表达情况及建立稳定高表达HOTAIR肾癌细胞株?方法:①q-PCR检测正常肾细胞系(HK-2)和4种肾癌细胞系(os-rc-2,kevt-3,achn和769-p)中HOTAIR的表达;②利用携带HOTAIR基因的慢病毒(pLenti GFP Puro-HOTAIR)感染肾癌细胞系(769-p)后,并采用嘌呤霉素(Puro)抗性筛选方法建立高表达HOTAIR肾癌细胞株?结果:①5种细胞系HOTAIR表达的高低依次是kevt-3?os-rc-2?achn?769-p?HK-2;②慢病毒(pLenti GFP Puro-HOTAIR)转染肾癌细胞系(769-p)后,HOTAIR表达明显升高?结论:①高侵袭性肾癌细胞株(kevt-3?os-rc-2)中HOTAIR的表达明显高于低侵袭性肾癌细胞株(achn?769-p)及正常肾细胞株(HK-2);②成功建立稳定表达的HOTAIR的769-p-HOTAIR细胞系?     

雌激素通过介导HOTAIR表达对子宫内膜癌细胞增生及裸鼠致瘤能力的影响

目的 探讨雌激素是否通过介导HOTAIR表达对子宫内膜癌细胞增生及裸鼠致瘤能力产生影响。方法 构建慢病毒介导干扰HOTAIR表达的shHOTAIR及阴性对照shNC稳定转染的子宫内膜癌Ishikawa细胞株,qRT-PCR检测HOTAIR的敲减水平。实验细胞分为4组：对照组（Ishikawa细胞）、雌二醇（17β-estradiol,E2）组（E2+Ishikawa细胞）、E2+shNC组（E2+shNC细胞）、E2+shHOTAIR组（E2+shHOTAIR细胞）,qRT-PCR检测E2对4组细胞中HOTAIR表达的影响。构建E2作用于HOTAIR表达不同的4组Ishikawa细胞的裸鼠移植瘤模型,记录成瘤时间、成瘤率及移植瘤生长曲线,剥离移植瘤,测量体积及质量,组织切片病理学检测。结果 成功构建慢病毒介导干扰HOTAIR表达的shHOTAIR及阴性对照shNC稳定转染的Ishikawa细胞株,HOTAIR表达被有效敲减（P<0.001）;E2组细胞中HOTAIR的相对表达量明显高于对照组（P<0.001）,E2+shHOTAIR组明显低于E2+shNC组（P<0.001）;成功构建E2作用于HOTAIR表达不同的4组Ishikawa细胞的裸鼠移植瘤模型;裸鼠致瘤能力对比：E2组>对照组,E2+shNC组>E2+shHOTAIR组;E2组移植瘤的终体积（P<0.001）及终质量（P<0.001）明显高于对照组,E2+shHOTAIR组移植瘤的终体积（P<0.01）及终质量（P<0.001）明显低于E2+shNC组;移植瘤HE染色符合子宫内膜癌细胞病理学特征。结论 雌激素通过上调HOTAIR表达促进子宫内膜癌细胞的增生及裸鼠致瘤能力,有望为子宫内膜癌的治疗提供新靶点。