目的 证明肺癌细胞miRNA芯片中不存在高表达的miR-4454,而是来源于tRNAHis的同源序列16 nt小RNA,并对该16 nt小RNA生物学功能进行初步预测分析.方法 利用生物素标记的anti-miR-4454成熟体探针Northern印迹(NB)检测肺癌H460细胞内miR-4454成熟体及其前体,利用该探针钓取与之特异结合的RNA,测序确定该RNA的性质.Dicer酶体外切割tRNAHis,检测其切割产物并对切割产生的小RNA进行测序鉴定.在H460细胞中过表达tRNAHis来源的16 nt小RNA,通过转录组测序预测其生物学功能.结果 肺癌H460细胞中预测的miR-4454成熟体不存在,其前体序列与tRNAHis高度一致.Dicer酶体外切割tRNAHis能产生一条16 nt小RNA,序列鉴定与tRNAHis 3'端完全匹配,是一条新的tRNAHis来源小RNA.H460细胞中过表达tRNAHis来源的16 nt小RNA,转录组测序结果分析显示,tRNAHis来源的16 nt小RNA可能参与有丝分裂、细胞周期调控及碱基错配修复.结论 首次发现数据库预测的miR-4454在非小细胞肺癌细胞中不存在,证明它是来源于tRNAHis 3'端由Dicer酶切割产生的一种16 nt小RNA.过表达16 nt小RNA的转录组测序结果提示其可能参与细胞有丝分裂、周期调控及碱基错配修复.

In recent years, p53 was identified to regulate the expression of many miRNAs and was also regulated by miRNAs. In this paper, we found that miR-138 showed a pronounced increase after p53 activation in human non-small cell lung cancer (NSCLC) cells, which is mediated by p53 binding sites in the promoter region of its host gene, but this did not happen with rat and mouse cells. More interestingly, we found that p53 could be also regulated by miR-138 in mouse and rat cells, but not in the human NSCLC cells. Our results suggest the existence of species-specific differences of the regulations of miRNA against its targets and the regulations of miRNA itself by other proteins.

Vasorin (VASN) is a type I transmembrane protein that plays important roles in tumor development and vasculogenesis. In this paper, we showed that VASN could be a key mediator of communication between tumor cells and endothelial cells. We confirmed for the first time that HepG2-derived VASN can be transferred to human umbilical vein endothelial cells (HUVECs) via receptor mediated endocytosis of exosomes, at least in part through HSPGs. The HepG2-derived VASN containing exosomes promote migration of recipient HUVECs cells. Our results identify a novel pathway by which a functional protein expressed in tumor cells affects the biological fate of endothelial cells via exosomes.

In this study, we further investigated a previously developed aptamer targeting ROS 17/2.8 (rat osteosarcoma) cells. We found that this C6-8 aptamer specifically binds to heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 and that it specifically labeled multiple tumor-cell lines as effectively as hnRNP A2/B1 monoclonal antibodies. When conjugated with fluorescent carbon nanodots (CDots) it could freely enter multiple living tumor cell lines (HepG2, MCF-7, H1299, and HeLa), whose growth it inhibited by targeting hnRNP A2/B1. Similar inhibitory effects were observed when the GFP-HepG2 hepatocarcinoma cells treated with C6-8-conjugated CDots were implanted in nude mice. Our work provides a new aptamer for targeting/labeling multiple tumor cell types, and its nanoparticle conjugates bring further advantages that increase its potential for use in cancer diagnosis and therapy.

We report a new biomarker of hepatocarcinoma, vasorin (VASN), screened by a subtractive EMSA-SELEX strategy from AFP negative serum of hepatocellular carcinoma (HCC) patients with extrahepatic metastases. VASN was verified to be highly expressed in sera of 100 cases of HCC patients compared with 97 cases of normal persons and 129 cases of hepatitis patients. Further validation by Q-PCR, IFA and Western blot showed higher expression of VASN at mRNA and protein levels in HCC cell lines and HCC tissues than in normal controls. RNA interference and forced overexpression assays verified that VASN promotes cell proliferation and migration and inhibits apoptosis. Down-regulation of microRNA miR145 and miR146a is an important mechanism leading to high expression of VASN. Conclusion: As a membrane protein and/or as free protein, VASN may be an effective target for biological treatment of liver cancer and is a potential biomarker for HCC diagnosis. Small molecular nucleotides targeting VASN are promising biological therapies to HCC.

Objective: Using non-isotope electrophoretic mobility shift assay(EMSA) to replace isotope-labeled EMSA method for detecting aptamer specifically binding to its target protein. Methods: Biotin-labeled ssDNA ap?tamer incubated with the target protein(or unrelated protein) at room temperature, the ssDNA-protein complex were run onto a non-denaturing polyacrylamide gel electrophoresis and then transfered to nylon membrane. After UV cross-linking, proteinase K digestion, the shift bands were observed by chemiluminescence method. Results:After proteinase K digestion, the shift bands of ssDNA aptamer to its target protein could be clearly observed and the sensitivity is similar to the isotope-labeled EMSA method. Conclusion: The biotin-labeled EMSA method with proteinase K digestion could be used for the detection of binding of ssDNA aptamer to its target.

目的:构建含p53保守结合位点的microRNA(miRNA)表达载体,促进相关miRNA在具有野生型p53蛋白细胞中的高效表达.方法:改构miRNA表达载体pCMV-miR,在其多克隆位点前插入p53保守结合位点,分别将miR-138、miR-34a和miR-21前体序列pre-miR-138、pre-miR-34a和pre-miR-21插入上述改构的载体pCMV/p53-miR,将构建的pCMV/p53-miR-138、pCMV/p53-miR-34a和pCMV/p53-miR-21表达载体转染具有野生型p53的HeLa细胞和不表达p53的H1299细胞,分析p53对上述miRNA表达调控的影响.结果:转染改构的miRNA表达载体后,HeLa细胞中miR-138、miR-34a和miR-21的表达水平明显提高,它们对应的已知靶基因Cyclin D3、CDK2和PTEN的表达同时被显著下调.结论:在p53转录调控作用下,具有p53保守结合位点的miRNA表达载体能够更加有效地提高miRNA的表达水平;构建的载体不但可用于促进相关miRNA的表达,也能用于miRNA是否受p53调控的检测.

As a cleavage enzyme of precursor TNF‐α, the high expression level of ADAM17 in endothelial cells is an important factor in atherosclerosis. In this study, we demonstrate that ADAM17 is the target of miR‐152. We found that miR‐152 could reduce TNF precursor cleavage and inhibit cell proliferation and migration by targeting ADAM17 in human umbilical vein endothelial cells (HUVECs). Furthermore, the expression pattern of miR‐152 and corresponding target ADAM17 was opposite in HUVECs under hypoxic conditions. The levels of circulating miR‐152 in AS patient sera were lower than those detected in the sera of normal individuals. Our results indicate that miR‐152 may be involved in the development of human atherosclerosis and could be used as diagnostic biomarker or therapeutic target in atherosclerosis.