Objective:To establish the HepG2 cell line with human vasorin(VASN) gene stably knockout by CRISPR/Cas9 technology,and address biological functions of VASN protein.Methods:The small guidance RNA of VASN was cloned into the eukaryotic expression vector and the recombinant pCas-gRNA was acquired.The left,right homologous arms of VASN gene were amplificated and combined by PCR,then cloned into pBackZero-T vector to obtain the recombinant pBackZero-T-VASN.The two recombinant plasmids were transfected into HepG2 cells and screened by hygromycin B,then the expression of VASN was assayed by genomic PCR,RT-qPCR and Western blotting.The proliferation and migration ability of the VASN stably knockout cell lines were detected by CCK-8 and transwell assay.Results:The plasmids pCas-gRNA and pBackZero-T-VASN were constructed successfully.The result of genomic PCR showed that the hygromycin B gene was correctly reorganized into the VASN gene.The result of RT-qPCR showed that the level of VASN mRNA was significantly reduced,and the result of Western blotting showed a significant decrease in the protein expression level.The proliferation and migration of the VASN stably knockout cell lines were significantly decreased than those of wild-type cells.Conclusion:The plasmids pCas-gRNA and pBackZero-T-VASN were constructed successfully.The VASN knockout cell line was established successfully.And it was suggested that the VASN protein was involved in cell proliferation and migration process.These results might lay a foundation for in-depth researches on VASN biological functions.

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) was reported to participate in the development of a variety of tumors. BC15 is a DNA aptamer targeting hnRNP A1. Firstly, through sequence truncation, we identified 31-mer sequence BC15-31 as the core sequence of BC15 with a strong binding affinity and high selectivity to the hnRNP A1 protein. Isothymidine (isoT) modification was then applied for the structural optimization of BC15-31, systematic modification and biological evaluation were carried out. Incorporation of isoT in the 1,3 sites at the 5-end of BC15-31 can significantly enhance the protein affinity. Chemical modifications close to the 3-end can greatly improve the stability of the aptamer. Furthermore, BC15-31 modified with isoT at both the 5-end and 3-end displayed an additive effect with enhanced bioactivity and stability at the same time. Our study strategy on BC15 provides a useful guideline for chemical modification and optimization of the aptamer for further clinical application.

目的:筛选能特异识别vasorin (VASN)蛋白的单链DNA(ssDNA)适配体并对其进行鉴定.方法:利用SPRSELEX技术筛选VASN蛋白特异ssDNA适配体,通过基因克隆测序、MEME在线软件和RNA structure软件分析富集文库序列并挑选出候选适配体序列,利用EMSA、ELISA、流式细胞术对候选适配体进行特异性与亲和力鉴定.结果:经过5轮SELEX筛选,获得了与VASN蛋白特异结合的ssDNA适配体富集文库,合成候选适配体,经EMSA和ELISA检测分析,证实适配体V4-2能与VASN蛋白特异结合,而不与无关对照牛血清白蛋白结合,其平衡解离常数为281.3±103.7 nmol/L;流式细胞术证实V4-2能够特异识别高表达VASN蛋白的HepG2细胞.结论:筛选获得特异识别VASN蛋白的ssDNA适配体V4-2.

Human vasorin (VASN) as a type I transmembrane protein, is a potential biomarker of hepatocellular carcinoma, which could expedite HepG2 cell proliferation and migration significantly in vitro. The ectodomain of VASN was proteolytically released to generate soluble VASN (sVASN), which was validated to be the active form. Among several monoclonal antibodies produced against sVASN, the clone V21 was found to bind with the recombinant human sVASN (rhsVASN) with the highest affinity and specificity, and also have inhibitory effects on proliferation and migration of HepG2 cells. Hence the phage-displayed peptide library was screened against the antibody V21. The positive phage clones were isolated and sequenced, and one unique consensus motifs was obtained. The result of sequence alignment showed that the conserved motif had similarity to VASN(Cys432-Cys441), embedded in the epidermal growth factor (EGF)-like domain. The synthetic mimotope peptide V21P1 and V21P2 were confirmed to bind with V21 and could compete with rhsVASN in ELISA assay. And they could also almost completely reverse the inhibitory effect of V21 on HepG2 migration and proliferation. Furthermore, the antibodies produced against V21P1 were able to bind not only with the peptide V21P1, but also with rhsVASN and the natural VASN from HepG2 cell. Our results showed that V21 seemed to be a functional antibody. The mimotopes toward V21 might mimic the functional domain of VASN, which would be helpful to exploit VASN functions and act as a candidate target for developing therapeutic antibodies against VASN.

The type A influenza viruses are the most virulent and variable human pathogens with epidemic or even pandemic threat. The development of sensitive, specific and safe field testing methods is in particular need and quite challenging. We report here the selection and practical utilization of the inactivated influenza virus-specific aptamers. The DNA aptamers against inactivated intact H1N1 virus particles were identified through the systematic evolution of ligands by exponential enrichment (SELEX) procedure. The discriminative aptamers and their truncated sequences showed selectively high affinity to inactive H1N1 virus and H3N2 virus with the Kd in the low nanomolar range and collective binding properties. The truncated sequences were first applied in a sandwich enzyme-linked oligonucleotide assay (ELONA) with a H1N1 detection limit (LOD, S/N = 3) of 0.3 ng/μL and then in an electrochemical impedance (EIS) aptasensor with more than 300 times improved LOD (0.9 pg/μL) and the excellent selectivity over other viruses (> 100 times). Therefore the developed aptasensors represent the safer, simpler, and possibly better virus-variation adaptable means of virus diagnostics.

Objective:To knock out Ku70 gene of HeLa cells by CRISPR/Cas9 technology and explore the biological functions of Ku70 knockout cell lines.Methods:The guide RNA expressing vector pCas-gRNA and homologous recombination donor DNA vector pBackZero-T-Ku70 were designed and obtained.After co-transfection of the two plasmids,cells were screened with hygromycin B for obtaining Ku70 gene knockout cells.Genomic PCR and Western blot assays were used to identify Ku70 gene knockout cells.The proliferation and migration of Ku70 knockout cell lines were assayed.In order to detect the levels of five miRNAs(hsa-miR-649,hsa-miR-544a,hsamiR-562,hsa-miR-548a and hsa-miR-492),total RNA of Ku70 gene knockout cell lines was extracted and reversely transcribed to cDNA.qPCR assay was used to quantify the expression of these miRNAs in the cDNA preparation.Results:We constructed the plasmids pCas-gRNAs and pBackZero-T-Ku70 successfully,and after cotransfection and screening,we obtained Ku70 gene knockout HeLa cell lines.The results of cell proliferation and migration assays showed that after knockout of Ku70 gene,the proliferation and migration of HeLa cells were attenuated.qPCR results indicated that among the five kinds of miRNAs we chose,the levels of three miRNAs(hsamiR-649,hsa-miR-544a,hsa-miR-562) were increased in Ku70 knockout cells than those in the wide type cells,while the level of hsa-miR-548a and hsa-miR-492 had no obvious changes.Conclusion:Ku70 gene knockout HeLa was obtained by using CRISPR/Cas9 technology.Ku70 protein appears to have effects on proliferation and migration of Hela cells.Moreover,it appeared that Ku70 protein could regulate expression of some miRNAs.

LncRNA-GAS5 can act as a "sponge" for microRNA-21 (miR-21) by competitively sequestering miR-21 from binding target mRNAs. Moreover, miR-21 directly regulates PTEN and TPM1 via imperfect complementary target base pairing. We confirmed miR-21 regulates PTEN and TPM1 protein expression without significantly affecting PTEN and TPM1 mRNA expression via imperfect complementary target binding in HEK293T and HeLa cells. Furthermore, Overexpressing miR-21 could significantly shorten half-lives of PTEN and TPM1, miR-21 enhanced PTEN and TPM1 decay. Cells were transfected with 1ncRNA-GAS5 expression vector, we found 1ncRNA-GAS5 competitively bound miR-21 and increased the half-lives of PTEN and TPM1. Besides, miR-21 bound with 1ncRNA-GAS5 could lead rapid decay of 1ncRNA-GAS5. This study indicates 1ncRNA-GAS5 functions as a miR-21 "sponge" that inhibits mRNA degradation of the miR-21 imperfect complementary targets PTEN and TPM1, and miR-21 also could regulate lncRNA-GAS5 stability by base pairing. Further exploration of the mechanisms may improve our understanding of the IncRNA-miRNA-mRNA sophisticated regulatory feedback loop.

Objective To screen an ssDNA aptamer for rabbit mesenchymal stem cells (MSCs) and to identify the ability of the aptamer to recognize MSCs of a variety of species origin.Methods MSCs were isolated from the thigh bone of immature rabbits and identified by induced osteogenic and adipogenic differentiation,respectively.Aptamers were screened by cell SELEX (systematic evolution of ligands by exponential enrichment) technique targeting to isolated MSCs.Enrichment of the 5th pool was evaluated through binding assay of FAM modified pool to MSCs by confocal microscopy.The enriched 5th pool was then cloned into pGE-T vector and the cloned sequences were determined randomly.The candidates were chosen based on primary sequence conservation and predicted secondary structure by RNA structure and MEME online analysis.Flow cytometry analysis was used to identify the aptamers binding to MSCs of rabbit, rat, and human origin.Results The isolated MSCs had the potential of osteogenic differentiation and adipogenic differentiation under certain conditions.Aptamer 5-1-12 from 5th enriched pool was characterized as MSCs recognizing aptamer binding to MSCs of rabbit, rat and human origin.Conclusion Aptamer 5-1-12 that recognizes MSCs of different species origin is obtained through live cell-SELEX.

GADD45A (growth arrest and DNA damage inducible alpha), a stress response gene induced by genotoxic and nongenotoxic stresses, is implicated in various key processes, including the control of cell cycle checkpoints and DNA repair. The expression of GADD45A is directly regulated by numerous transcription factors, with p53 being the most representative. Moreover, post-transcriptional regulation also plays a role in GADD45A expression. However, little is known about the regulatory effects of microRNAs (miRNAs) on GADD45A expression. As a potential tumour suppressor, miR-138 has pleiotropic biological functions in various cancers. We have previously reported p53-mediated activation of miR-138 in human non-small-cell lung cancer (NSCLC) cells. In this study, we found that miR-138 specifically targeted AGO2, which affects the stability and maturation of miR-130b. Decreased expression of miR-130b promoted the expression of GADD45A and resulted in the G2/M phase arrest and proliferation inhibition in human NSCLC cells. Our results suggested that p53 could alternatively upregulate GADD45A in human NSCLC cells through a post-transcriptional pathway in which miR-138 is involved.

Objective:To explore the effects of miR-216b on lncRNA-UCAl after miR-216b binding with UCA1.Methods:Genome was extracted from HEK293T cell,and from which UCA1-coding sequence was amplified by PCR with specific primers,then the gene fragment was cloned into pcDNA6.0b vector.Cells were transfected with miR-216b mimic and treated with actinomycin D,then total RNA were extracted at three time points and qRT-PCR was performed to detect the half-life of UCA1.The recombinant pcDNA6-UCA1 was transfected into cells for 24 h,then we transfected miR-216b mimic or miR-216b inhibitor into cells and treated cells with actinomycin D,three time points total cell RNA was harvested,and UCA1 half-life was detected by qRT-PCR.Results:UCA1 RNA level was remarkably increased after transfection of pcDNA6-UCA1.Overexpression of miR216b significantly reduced the half-life of both endogenous and exogenous UCA1 compared to the control group.miR-216b inhibitor could prolong UCA1 half-life and enhance UCA1 RNA stability.Conclusion:miR-216b could accelerate decay of UCA1 which provided a new thought for interaction between miRNA with lncRNA.

目的:纯化人vasorin(VASN)蛋白胞外结构域的单克隆抗体并鉴定.方法:用表达人VASN蛋白胞外结构域单克隆抗体的杂交瘤细胞免疫小鼠,收集腹水,纯化抗体;ELISA检测单抗的特异性和亲和力,Western印迹、免疫共沉淀、细胞免疫荧光等实验检测单抗的特异性与可能的用途.结果:纯化获得2株抗VASN胞外结构域单抗V20和V21,ELISA结果显示二者均特异性强、亲和力高;Western印迹显示2株单抗均可结合HepG2细胞中的VASN蛋白,以V20为佳;免疫共沉淀实验结果显示V21能够钓取HepG2细胞中的VASN蛋白及细胞培养上清中的分泌型VASN;免疫荧光实验结果显示V21能与HepG2细胞的VASN蛋白结合.结论:纯化获得2株抗人VASN胞外结构域单抗,为进一步研究VASN蛋白的生物学功能提供了实验工具.

目的:探讨人肿瘤坏死因子α(TNF-α)作为microRNA(miRNA)结合蛋白的生物学特性.方法:Western印迹检测LPS激活的U937细胞中TNF-α蛋白表达水平,RT-PCR检测几种炎症相关的miRNA在激活前后表达水平的变化;用RNA结合蛋白免疫共沉淀(RIP)技术钓取U937细胞中TNF-α结合的miRNA,RT-PCR检测RIP产物中上述几种miRNA的含量;用凝胶阻滞实验检测TNF-α与miRNA是否直接结合.结果:LPS激活的U937细胞中能检测到膜结合型和可溶性2种形式的TNF-α,细胞激活前后miR-16和miR-21的表达水平在检测的几种miRNA中最高,miR-146b和miR155次之;而RIP产物中miR-146b的水平在实验组和对照组中有显著差异,其他miRNA差异不明显;凝胶阻滞实验结果显示miR-146b能与人TNF-α直接结合.结论:首次证实人TNF-α能够直接特异地结合miR-146b,提示TNF-α可能作为miRNA结合蛋白发挥生物学功能.

This study proposes a facile method for synthesis of Au-coated magnetic nanoparticles (AuMNPs) core/shell nanocomposites with nanoscale rough surfaces. MnFe2O4 nanoparticles (NPs) were first modified with a uniform polyethylenimine layer (2 nm) through self-assembly under sonication. The negatively charged Au seeds were then adsorbed on the surface of the MnFe2O4 NPs through electrostatic interaction for Au shell formation. Our newly developed sonochemically assisted hydroxylamine seeding growth method was used to grow the adsorbed gold seeds into large Au nanoparticles (AuNPs) to form a nanoscale rough Au shell. Au-coated magnetic nanoparticles (AuMNPs) were obtained from the intermediate product (Au seeds decorated magnetic core) under sonication within 5 min. The AuMNPs were highly uniform in size and shape and exhibited satisfactory surface-enhanced Raman scattering (SERS) activity and strong magnetic responsivity. PATP was used as a probe molecule to evaluate the SERS performance of the synthesized AuMNPs with a detection limit of 10(-9) M. The synthesized AuMNPs were conjugated with Staphylococcus aureus (S. aureus) antibody for bacteria capture and separation. The synthesized plasmonic AuNR-DTNB NPs, whose LSPR wavelength was adjusted to the given laser excitation wavelength (785 nm), were conjugated with S. aureus antibody to form a SERS tag for specific recognition and report of the target bacteria. S. aureus was indirectly detected through SERS based on sandwich-structured immunoassay, with a detection limit of 10 cells/mL. Moreover, the SERS intensity at Raman peak of 1331 cm(-1) exhibited a linear relationship to the logarithm of bacteria concentrations ranging from 10(1) cells/mL to 10(5) cells/mL.

MicroRNAs are small non-coding RNAs involved in pathogenesis and progression of human malignancies. MicroRNA-491-5p (miR-491-5p) is down-regulated in many human cancers where it would serve as a tumour suppressor. However, the role played by miR-491-5p in pathogenesis of human osteosarcoma has remained largely unknown. This study has been conducted to examine effects of miR-491-5p on migration and proliferation of cells of the SAOS-2 and MG63 osteosarcoma lines, and mechanisms of those effects. Levels of miR-491-5p expression in osteosarcoma tissues and in human osteosarcoma cell lines were studied using qualitative real-time polymerase chain reaction (qRT-PCR) methods. Cell viability was detected using the CCK-8 and EdU assays, while the transwell assay was used to evaluate migration and invasion. Apoptosis was analysed uing flow cytometry and the Hoechst 33342 nuclear staining method. A dual-luciferase reporter system was used to confirm the target gene of miR-491-5p. The electrophoretic mobility shift assay (EMSA) with DIG-labelled double-stranded FOXP4 oligonucleotides was used to confirm whether or not miR-491-5p suppressed FOXP4 activation. Cells of osteosarcoma tissues and cell lines had low levels of miR-491-5p expression, but high levels of forkhead-box P4 (FOXP4) expression. Transfection of SAOS-2 and MG63 cells with miR-491-5p mimics inhibited expression of FOXP4 protein, which suppressed cell growth and migration, but induced apoptosis. Dual-luciferase reporter assays confirmed as the target gene for miR-491-5p. Overexpression of miR-491-5p suppressed FOXP4 activity in SAOS-2 and MG63 cells. Knockdown of in SAOS-2 and MG63 cells using an RNAi strategy resulted in reduced levels of cell proliferation and migration, but increased levels of apoptosis. Our in vitro studies showed that up-regulation of miR-491-5p suppressed proliferation of the human osteosarcoma cells and induced apoptosis by targeting . These findings suggest that miR-491-5p could be further studied as a potential clinical diagnostic or predictive biomarker for human osteosarcoma.

Objective To explore the effect of ethanol on the expression and secretion of VASN protein in HepG2 cells and its influence on HUVEC cell migration.Methods The expression level of VASN mRNA in HepG2 cells stimulated by ethanol was detected by quantitative real-time PCR ( qRT-PCR) .Western blotting was used to detect VASN protein levels in HepG2 cells and the cell culture supernatants.The wound-healing assay was employed to examine the migration of HU-VECs when co-cultured with ethanol-treated HepG2 cells.Results The results of qPCR showed that ethanol could up-regulate the mRNA level of VASN in HepG2 cells.Western blotting results also showed that ethanol could increase the expression and secretion of VASN protein in HepG2 cells.Wound-healing assay results showed that the increased production of VASN protein in ethanol-treated HepG2 cells could promote the migration of HUVECs.Conclusion Ethanol can increase the expression and secretion of VASN in HepG2 cells, and thereby promote the migration of HUVECs co-cultured with HepG2 cells.

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.

AIMS:The aim of the study was to explore the effect of miR-200b on the development of human peripheral blood monocyte-deriveddendritic-cell (DC) and its mechanisms.MAIN METHODS:Expression levels of miR-200b and its predicted targets were measured by real time-PCR. Protein expression of WASF3 was determined by Western blot and immunohistochemistry. Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation from the buffy coat fraction of anticoagulated blood. Monocytes were purified from PBMCs using anti-CD14 microbeads. The immunophenotypes of DCs were tested by flow cytometry.KEY FINDINGS:A strong reduction in miR-200b expression was associated with human peripheral blood monocyte-derivedDC differentiation. The overexpression of miR-200b significantly reduced the numbers of protruding veils in mature DCs (mDCs) that are critical for promoting antigen-specificT-cell activation. Further experiments showed that miR-200b could regulate the function of DCs by targeting WASF3, a protein involved in cell movement and invasion.SIGNIFICANCE:Our results define an important function of miR-200b in the negative regulation of DC development and provide a potential form of miRNA-mediated cell therapy for diseases that range from auto-immunity to graft-versus-host disease.

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.