Tumor necrosis factor alpha delivers exogenous inflammation-related microRNAs to recipient cells with functional targeting capabilities.

Tumor necrosis factor alpha (TNF-α) is a critical pro-inflammatory cytokine in a wide range of tumors and infectious diseases. This study showed for the first time that TNF-α could specifically bind to certain intracellular or circulating inflammation-related microRNAs both in vitro and in vivo. The binding sites of TNF-α to microRNAs are located at the N-terminal of TNF-α and the 3'-GGUU motif of microRNAs. TNF-α could deliver exogenous unmodified single-stranded microRNAs into recipient cells through the TNF-α receptors (TNFRs) and stabilize them from being degraded by RNase in cells. Exogenous miR-146a or let-7c delivered into HCT116 cells by TNF-α could escape from lysosomes and specifically downregulate their target genes and then affect cell proliferation and migration in vitro, as well as tumorigenesis in vivo. Based on the above findings, the concept of "non-conjugated ligand-mediated RNA delivery (ncLMRD)" was proposed, which may serve as a promising strategy for therapeutic microRNA delivery in the future.

Systematic study of cis-antisense miRNAs in animal species reveals miR-3661 to target PPP2CA in human cells.

MicroRNAs (miRNAs) suppress targeting gene expression through blocking translation or triggering mRNA degradation and, in general, act in trans, through a partially complementary interaction with the 3' untranslated region (3' UTR) or coding regions of a target gene. Although it has been reported previously that some miRNAs suppress their target genes on the opposite strand with a fully complementary sequence (i.e., natural antisense miRNAs that act in cis), there is no report to systematically study such cis-antisense miRNAs in different animal species. Here we report that cis-antisense miRNAs do exist in different animal species: 48 in Caenorhabditis elegans, 17 in Drosophila, 36 in Mus musculus, and 52 in Homo sapiens using a systematical bioinformatics approach. We show that most of these cis-antisense miRNAs can efficiently reduce the expression levels of their target genes in human cells. We further investigate hsa-miR-3661, one of the predicted cis-antisense miRNAs, in detail and demonstrate that this miRNA directly targets the coding sequence of PPP2CA located on the opposite DNA strand and inhibits the PPP2CA expression. Taken together, these results indicate that cis-antisense miRNAs are conservative and functional in animal species including humans.

d-/l-Isothymidine incorporation in the core sequence of aptamer BC15 enhanced its binding affinity to the hnRNP A1 protein.

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.

MiR-138 downregulates miRNA processing in HeLa cells by targeting RMND5A and decreasing Exportin-5 stability.

MicroRNAs (miRNAs) are a class of non-coding small RNAs that consist of ≈ 22 nt and are involved in several biological processes by regulating target gene expression. MiR-138 has many biological functions and is often downregulated in cancers. Our results showed that overexpression of miR-138 downregulated target RMND5A (required for meiotic nuclear division 5 homolog A) and reduced Exportin-5 stability, which results in decreased levels of pre-miRNA nuclear export in HeLa cells. We also found that miR-138 could significantly inhibit HeLa cell migration by targeting RMND5A. Our study therefore identifies miR-138-RMND5A-Exportin-5 as a previously unknown miRNA processing regulatory pathway in HeLa cells.

Comparison of the methods for generating single-stranded DNA in SELEX.

The generation of single-stranded DNA (ssDNA) from double-stranded PCR products is an essential step in the selection of aptamers by systematic evolution of ligands by exponential enrichment (SELEX). Magnetic separation with streptavidin-coated beads is always the most commonly used method. Recently, two size separation methods derived from unequal primers with chemical or structural modification were designed in SELEX. In this report, we made a comparison between magnetic separation and the two size separation methods for generation of ssDNA from double-stranded PCR products. Our results showed that all the methods produced ssDNA of good purity. Compared to the magnetic separation, size separation derived from unequal primers with chemical modification achieved an almost equivalent recovery rate of ssDNA, whereas size separation derived from unequal primers with structural modification showed a lower recovery rate of ssDNA. Considering the low cost, size separation derived from unequal primers with chemical modification could be a satisfactory alternative to the classic magnetic separation for the generation of ssDNA in SELEX.

Clinical value of hematologic test in predicting tumor response to neoadjuvant chemotherapy with esophageal squamous cell carcinoma.

BACKGROUND: To investigate the relationship between hematologic test results and the predictive effect of regression of esophageal cancer after neoadjuvant chemotherapy (NACT), we analyzed pre-NACT hematologic data and their relationship to tumor regression. METHODS: Thirty-eight consecutive patients with locally advanced squamous cell esophageal carcinoma who had undergone two cycles of paclitaxel/carboplatin NACT were enrolled. On the day prior to the first cycle of chemotherapy, hematologic tests, including routine blood test and biochemical examinations, were recorded. All patients were confirmed to have no history of hepatitis. Surgical resection was performed when clinical restaging showed effective regression. Histopathological examination was routinely performed to evaluate the postoperative effects of chemotherapy. RESULTS: After two cycles of NACT, tumor imaging evaluation showed that 27 of the 38 patients had CR and PR, including 25 patients who underwent radical esophagectomies. Six patients had stable disease and five patients had progressive disease. According to the hematologic test results before NACT, patients with higher white blood cell counts, lymphocyte percentages, mononuclear cell counts, neutrophilic granulocyte counts, and eosinophilic granulocyte counts and lower alanine aminotransferase (ALT) level had a significantly greater opportunity for an effective response. CONCLUSION: Basal host immunologic function and hepatic function are associated with tumor response to NACT in patients with esophageal cancer. These parameters may have a certain predictive efficacy on NACT for esophageal squamous cell carcinoma.

Rapid and Sensitive Detection of Inactivated SARS-CoV-2 Virus via Fiber-Optic and Electrochemical Impedance Spectroscopy Based Aptasensors.

The development of rapid detection tools for viruses is vital for the prevention of pandemics and biothreats. Aptamers that target inactivated viruses are attractive for sensors due to their improved biosafety. Here, we evaluated a DNA aptamer (named as 6.9) that specifically binds to the inactivated SARS-CoV-2 virus with a low dissociation constant (KD = 9.6 nM) for the first time. Based on aptamer 6.9, we developed a fiber-optic evanescent wave (FOEW) biosensor. Inactivated SARS-CoV-2 and the Cy5.5-tagged short complementary strand competitively bound with the aptamer immobilized on the surface of the sensor. The detection of the inactivated SARS-CoV-2 virus was realized within six minutes with a limit of detection (LOD, S/N = 3) of 740 fg/mL. We also developed an electrochemical impedance aptasensor which exhibited an LOD of 5.1 fg/mL and high specificity. We further demonstrated that the LODs of the FOEW and electrochemical impedance aptasensors were, respectively, more than 1000 and 100,000 times lower than those of commercial colloidal gold test strips. We foresee that the facile aptamer isolation process and sensor design can be easily extended for the detection of other inactivated viruses.

Angiogenesis and bone regeneration by allogeneic mesenchymal stem cell intravenous transplantation in rabbit model of avascular necrotic femoral head.

AIM: To explore the feasibility of allogeneic mesenchymal stem cells (MSCs) transplanted intravenously for angiogenesis and bone repair in a rabbit model of avascular necrosis of femoral head (ANFH). MATERIALS AND METHODS: Forty-five rabbits were randomized into three groups: a blank control group (without treatment), a necrotic control group (ANFH induced but without therapy), and an MSC transplantation group (ANFH induced and treated with MSC transplantation). The biopsies, blood sampling, and imaging examinations were performed on each animal at different time points (2, 4, and 6 wk). To monitor angiogenesis and bone repair progress, examinations included real-time polymerase chain reaction, Western blot analysis, x-ray, computed tomography, Masson trichrome staining, picrosirius red staining, and immunohistochemical staining. RESULTS: Necrosis and bone collapse were observed in bilateral femoral heads of necrotic rabbits of the necrotic control group, whereas the femoral head morphology was generally restored in the MSC transplantation group. The mRNA levels of Cbfa1, BMP, VEGF, and OPN in bone tissue were significantly higher in the MSC transplantation group than in the necrotic control group. In addition, the total protein amount of Cbfa1 in the MSC transplantation group was also significantly higher than that in the necrotic control group (P < 0.05). CONCLUSION: Intravenous transplantation of allogeneic MSCs can promote vascular and bone regeneration in the necrotic region of the femoral head in a rabbit model of ANFH. The results of our study suggest that the intravenous transplantation of MSCs could be a potential and minimally invasive treatment option for ANFH patients.

Mutual regulation between TRIM21 and TRIM8 via K48-linked ubiquitination.

Tripartite motif (TRIM)-containing proteins, one of the largest subfamilies of the RING type E3 ubiquitin ligases, control important biological processes such as cell apoptosis, autophagy, signal transduction, innate immunity and tumorigenesis. So far, the mutual regulation between TRIM family members has rarely been reported. Here, we found for the first time that there was a direct mutual regulation between TRIM21 and TRIM8 in lung and renal cancer cells, mechanistically by activating their proteasome pathway via Lys48 (K48)- linked ubiquitination. Subsequent studies verified that negatively correlated expressions existed in clinical non-small cell lung cancer (NSCLC) and renal cell carcinoma (RCC) tissues, which were closely related to tumor progression. Our findings highlighted a possible homeostasis between TRIM21 and TRIM8 that might possibly affect cell stemness and was expected to provide a new idea for cancer therapy.

Selection of regioselective DNA aptamer for detection of homocysteine in nondeproteinized human plasma.

Small molecule-binding aptamers often suffer from high cross reactivity to structure analogues in biological samples, limiting their value for clinical diagnosis. Herein, we present a method to overcome this issue, by performing binding-inhibited organic reaction-based regioselective selection of aptamers against homocysteine (Hcy), which is a marker for diagnosing many disorders including stroke and Alzheimer's. This approach has led to isolation of a DNA aptamer that binds to the alkane thiol chain of Hcy with exceptional specificity against cysteine. It also binds with oxidized Hcy at weaker affinity. Using this new aptamer, we produced a reusable fluorescent optical fiber aptasensor for direct and validated detection of both free and total Hcy in nondeproteinized patient plasma in the diagnostic concentration range. The binding site-specific aptamer selection and optical-fiber-sensing strategy can expand the practical utility of aptamers in clinical diagnosis.

AGO4 suppresses tumor growth by modulating autophagy and apoptosis via enhancing TRIM21-mediated ubiquitination of GRP78 in a p53-independent manner.

Argonaute proteins, which consist of AGO1, AGO2, AGO3 and AGO4, are key players in microRNA-mediated gene silencing. So far, few non-microRNA related biological roles of AGO4 have been reported. Here, we first found that AGO4 had low expression in non-small cell lung cancer (NSCLC) patient tumor tissues and could suppress NSCLC cell proliferation and metastasis. Subsequent studies on the mechanism showed that AGO4 could interact with the tripartite motif-containing protein 21 (TRIM21) and the glucose-regulated protein 78 (GRP78). AGO4 promoted ubiquitination of GRP78 by stabilizing TRIM21, a new specific ubiquitin E3 ligase for promoting K48-linked polyubiquitination of GRP78 confirmed in this paper, which resulted in induced cell apoptosis and inhibited autophagy by activating mTOR signal pathway. Further studies showed that p53 had dominant effects on TRIM21-GRP78 axis by directly increasing the expression of TRIM21 in p53 wild-type cells and AGO4 may alternatively regulate TRIM21-GRP78 axis in p53-deficient cells. We also found that overexpression of AGO4 results in suppression of multiple p53-deficient cell growth both in vivo and vitro. Together, we showed for the first time that the AGO4-TRIM21-GRP78 axis, as a new regulatory pathway, may be a novel potential therapeutic target for p53-deficient tumor treatment.

Small ncRNA expression and regulation under hypoxia in neural progenitor cells.

Small non-coding RNA (ncRNA) plays critical roles in a large number of cellular processes, including neural development, cell survival and cell determination. Our previous work showed that low oxygen promoted the survival and proliferation of neural progenitor cells (NPCs) in vitro. In this study, we examine the expression and regulation of small ncRNAs in the hypoxia-driven proliferation of NPCs. The expression profiles of ncRNAs in NPCs under hypoxia were detected using microarray analysis. Results of significance analysis of microarrays (SAM) revealed that 15 small RNAs were up-regulated at least threefold and 11 were down-regulated under hypoxic conditions. The differentially expressed small ncRNAs were confirmed by quantitative RT-PCR, and miR-210 was observed to be highly expressed in NPCs under hypoxic conditions. Further study showed that hypoxia-inducible factor (HIF)-1α had a direct impact on the putative promoter regions of miR-210. From these results, we conclude that some small ncRNAs participate in the regulation of the proliferation of NPCs under hypoxia and that miR-210 is directly regulated by HIF-1α.

Intravenous transplantation of allogeneic bone marrow mesenchymal stem cells and its directional migration to the necrotic femoral head.

In this study, we investigated the feasibility and safety of intravenous transplantation of allogeneic bone marrow mesenchymal stem cells (MSCs) for femoral head repair, and observed the migration and distribution of MSCs in hosts. MSCs were labeled with green fluorescent protein (GFP) in vitro and injected into nude mice via vena caudalis, and the distribution of MSCs was dynamically monitored at 0, 6, 24, 48, 72 and 96 h after transplantation. Two weeks after the establishment of a rabbit model of femoral head necrosis, GFP labeled MSCs were injected into these rabbits via ear vein, immunological rejection and graft versus host disease were observed and necrotic and normal femoral heads, bone marrows, lungs, and livers were harvested at 2, 4 and 6 w after transplantation. The sections of these tissues were observed under fluorescent microscope. More than 70 % MSCs were successfully labeled with GFP at 72 h after labeling. MSCs were uniformly distributed in multiple organs and tissues including brain, lungs, heart, kidneys, intestine and bilateral hip joints of nude mice. In rabbits, at 6 w after intravenous transplantation, GFP labeled MSCs were noted in the lungs, liver, bone marrow and normal and necrotic femoral heads of rabbits, and the number of MSCs in bone marrow was higher than that in the, femoral head, liver and lungs. Furthermore, the number of MSCs peaked at 6 w after transplantation. Moreover, no immunological rejection and graft versus host disease were found after transplantation in rabbits. Our results revealed intravenously implanted MSCs could migrate into the femoral head of hosts, and especially migrate directionally and survive in the necrotic femoral heads. Thus, it is feasible and safe to treat femoral head necrosis by intravenous transplantation of allogeneic MSCs.

Inhibiting platelets aggregation could aggravate the acute infection caused by Staphylococcus aureus.

Several fibrinogen binding proteins (Fibs) play important roles in the pathogenesis of Staphylococcus aureus (S. aureus). Most Fibs can promote the aggregation of platelets during infection, but the extracellular fibrinogen-binding protein (Efb) is an exception. It is reported that Efb can specifically bind fibrinogen and inhibit the aggregation of platelet with its N terminal. However, the biological significance of platelet aggregation inhibition in the infection caused by S. aureus is unclear until now. Here, we demonstrated that the persistence and aggregation of platelets were important for killing S. aureus in whole blood. It was found that the N terminal of Efb (EfbN) and platelets inhibitors could increase the survival of S. aureus in whole blood. The study in vivo also showed that EfbN and platelets inhibitors could reduce the killing of S. aureus and increase the lethality rate of S. aureus in the acute infection mouse model.

Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus.

In this article, a panel of ssDNA aptamers specific to Staphylococcus aureus was obtained by a whole bacterium-based SELEX procedure and applied to probing S. aureus. After several rounds of selection with S. aureus as the target and Streptococcus and S. epidermidis as counter targets, the highly enriched oligonucleic acid pool was sequenced and then grouped under different families on the basis of the homology of the primary sequence and the similarity of the secondary structure. Eleven sequences from different families were selected for further characterization by confocal imaging and flow cytometry analysis. Results showed that five aptamers demonstrated high specificity and affinity to S. aureus individually. The five aptamers recognize different molecular targets by competitive experiment. Combining these five aptamers had a much better effect than the individual aptamer in the recognition of different S. aureus strains. In addition, the combined aptamers can probe single S. aureus in pyogenic fluids. Our work demonstrates that a set of aptamers specific to one bacterium can be used in combination for the identification of the bacterium instead of a single aptamer.

Intronic miR-301 feedback regulates its host gene, ska2, in A549 cells by targeting MEOX2 to affect ERK/CREB pathways.

miR-301 is localized in the first intron of ska2, whose function has not been clarified. Here, a new circuit model in which intronic miR-301 regulates the transcription and function of its host gene through a feedback mechanism has been described. Our results showed that blocking of miR-301 in A549 cells leads to a decrease in the expression of the host gene, ska2. Further analysis showed that miR-301 targets MEOX2 to affect the ERK/CREB pathway. CREB directly regulates the expression of the host gene, ska2. In addition, the inhibition of miR-301 or ska2 resulted in an increase of the mitotic index and a decrease in colony formation in soft agar, which may be related to lung tumorigenesis.

Aptamer-Based Sensitive Detection of Target Molecules via RT-PCR Signal Amplification.

In the efforts to explore an aptamer-based approach for target sensing and detection with higher sensitivity and specificity, instead of directly labeling aptamer with fluorophores, we proposed a new strategy by attaching a polymerase chain reaction (PCR) template to an oligonucleotide aptamer selected by systematic evolution of ligands by exponential enrichment (SELEX), so that after aptamer target binding, the template moiety serves as the PCR template in real-time quantitative PCR (RT-PCR), and therefore, the binding event can be reported by the following RT-PCR signals. Using the subtractive SELEX method, the oligonucleotide aptamers specific for the Fc fragment of mouse IgG were selected and subjected to coupling with the PCR dsDNA template by using overlap and the asymmetric extension PCR method. The target binding affinity of the PCR template tethered aptamer has been proven by electrophoretic mobility shift assay (EMSA), and further template tethered aptamer mediated real-time quantitative PCR (A-PCR) was conducted to validate the application for such a template tethered aptamer to be a sensitive probe for IgG detection. The results show that the protocols of A-PCR can detect 10-fold serial dilutions of the target, demonstrating a new mechanism to convert aptamer target binding events to amplified RT-PCR signal, and the feasibility of the PCR template tethered aptamer as a facile, specific, and sensitive target probing and detection is established. This new approach also has potential applications in multiple parallel target detection and analysis in a wide range of research fields.

MicroRNA-200b regulates cyclin D1 expression and promotes S-phase entry by targeting RND3 in HeLa cells.

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that inhibit gene expression post-transcriptionally. By regulating their target genes, miRNAs play important roles in tumor generation and development. Recently, the mir-200 family was revealed to inhibit the epithelial-mesenchymal transition, which is viewed as an essential step in early tumor metastasis. Here, we used luciferase assays to demonstrate that mir-200b interacts with predicted target sites in the 3' untranslated region of RND3. In HeLa cells, mir-200b directly reduced the expression of RND3 at the mRNA and protein levels, which thereby promoted expression of the downstream protein cyclin D1 and increased S-phase entry. In conclusion, our study demonstrates a novel role for mir-200b in cell cycle progression and identifies RND3 as a novel mir-200b target.

Identification of an aptamer targeting hnRNP A1 by tissue slide-based SELEX.

We report a new in situ tissue slide-based SELEX strategy targeting neoplastic tissues from breast cancer patients. The methodology, using the molecular differences between clinical specimens, can evolve aptamers to all fractions of tissue. The aptamers may be used as new molecular probes for pathological diagnosis and tumour imaging, and also to reveal the molecular differences that are responsible for the diseases. The specific aptamers were enriched by unequal length strand PCR employing a structured (-) strand primer. After 12 rounds of selection, using the paraffin tissue sections from infiltrating ductal carcinomas as targets, and using the adjacent normal tissue from the same case as controls, one of the enriched ssDNA aptamers, BC15, was selected from a nucleic acid library and characterized as recognizing breast cancer cells either within the tissue sections or from the culture medium, but only weakly binding to adjacent normal cells or immortalized breast cell line MCF10A. The calculated equilibrium dissociation constants (K(d)) of BC15 bound to MCF7 cells was 111.0 +/- 36.9 nM. Through streptavidin magnetic beads mediated affinity purification assay followed by mass spectrometry identification and western blot confirmation, the target of BC15 was characterized to be hnRNP A1, which was further verified to be specifically and highly expressed in cancerous tissues of breast by hnRNP A1 antibody immunostaining as well as western blot. BC15 aptamer was also used to probe cancer cells in tissues from other pathological types of breast cancers including lobular carcinoma, ductal carcinoma complicated with lobular carcinoma, comedo carcinoma, and lymph node metastasis of breast ductal carcinoma origin or breast lobular carcinoma origin. Therefore, tissue slide-based SELEX holds promise in identifying tumour markers and developing specific molecular probes for cancer diagnosis.

Transforming growth factor-beta-regulated miR-24 promotes skeletal muscle differentiation.

MicroRNAs (miRNAs) have recently been proposed as a versatile class of molecules involved in regulation of a variety of biological processes. However, the role of miRNAs in TGF-beta-regulated biological processes is poorly addressed. In this study, we found that miR-24 was upregulated during myoblast differentiation and could be inhibited by TGF-beta1. Using both a reporter assay and Northern blot analysis, we showed that TGF-beta1 repressed miR-24 transcription which was dependent on the presence of Smad3 and a Smads binding site in the promoter region of miR-24. TGF-beta1 was unable to inhibit miR-24 expression in Smad3-deficient myoblasts, which exhibited accelerated myogenesis. Knockdown of miR-24 led to reduced expression of myogenic differentiation markers in C2C12 cells, while ectopic expression of miR-24 enhanced differentiation, and partially rescued inhibited myogenesis by TGF-beta1. This is the first study demonstrating a critical role for miRNAs in modulating TGF-beta-dependent inhibition of myogenesis, and provides a novel mechanism of the genetic regulation of TGF-beta signaling during skeletal muscle differentiation.