Complementary Binding Sites Research Articles

Hsa_circ_0048764 facilitates the progression of non-small cell lung cancer by targeting miR-1178-3p/HMGA1 axis

Non-small cell lung cancer (NSCLC) remains a highly lethal disease, with a lack of fully established biomarkers and therapies. Circular RNAs (circRNAs) have emerged as powerful regulators of gene expression in multiple cancers. The role of circRNAs in NSCLC progression is still not well understood. In this study, GEO database analysis and qRT-PCR results revealed that hsa_circ_0048764 (circ_0048764) was overexpressed in NSCLC tissues and associated with poorer overall survival in patients with NSCLC. Functional assays demonstrated that silencing circ_0048764 inhibited NSCLC cell proliferation and metastasis. Bioinformatics analysis identified miR-1178-3p as having complementary binding sites with circ_0048764, a finding further validated by the dual-luciferase reporter assay. Additionally, predictions from the Starbase3.0 database, along with cellular experiments, revealed that miR-1178-3p regulates HMGA1 expression in NSCLC. Taken together, our findings suggest that circ_0048764 promotes NSCLC progression by enhancing HMGA1 expression through sponging miR-1178-3p, offering potential therapeutic targets for NSCLC treatment.

Electro-responsive Molecularly Imprinted Polymers (E-MIPs) for Rapid Detection of Progesterone in Human Serum Samples.

Rapid and reagent-free detection of progesterone (P4) is crucial in point-of-care (POC) measurement due to its important role in the human endocrine and central nervous system. Currently available technologies for P4 detection are often not rapid or require reagents, limiting their use in POC settings. In this work, a self-signaling electrochemical sensing platform for rapid detection of P4 is developed by using electroactive molecularly imprinted polymers (E-MIPs). E-MIPs possess the ability to transduce the molecular recognition event into a measurable electrochemical signal. The E-MIPs are prepared by bulk copolymerization of acrylic acid (AA) and ferrocenylmethyl methacrylate (FcMA) in the presence of P4 followed by its removal from the polymer matrix. By incorporation of ferrocene moieties, the MIP gains intrinsic electroactivity, enabling label-free and direct electrochemical detection of P4 levels. Electrochemical techniques, including cyclic voltammetry and electrochemical impedance spectroscopy, were employed to elucidate the electron transport reaction responsible for the MIP's intrinsic electroactivity. Additionally, the predetermined complementary binding sites confined to the small working area of the screen-printed carbon electrode (SPCE) within the E-MIP matrix facilitate rapid P4 binding, achieving completion within 120 s. The developed E-MIP sensing platform was used for the rapid detection of P4 levels in human serum samples. The short incubation times reported for the E-MIP sensing platform would be beneficial in minimizing the nonspecific binding in the real-world samples. We believe that the proposed E-MIP sensing platform can be applied to clinical samples for detecting fluctuations in P4 levels.

Adult zymosan re-exposure exacerbates the molecular alterations in the brainstem rostral ventromedial medulla of rats with early life zymosan-induced cystitis

Recent evidence suggests that the descending modulatory pathways from the brainstem rostral ventromedial medulla (RVM) are important for bladder inflammatory pain. This study aimed to identify the long-term molecular changes in RVM neurons due to early life cystitis during neuronal development and the effect of reexposure later in adulthood. RVM tissues from two treatment protocols were used: (1) neonatal zymosan exposures with acute adult rechallenge (RC) and (2) only neonatal zymosan exposures (NRC). RNAseq analysis showed upregulation of several genes associated with synaptic plasticity (Grin1, Grip2, Notch1, Arc, and Scn2b) in the cystitis groups compared to controls in both protocols. The RC protocol exhibited a stronger treatment effect with significantly higher fold differences between the groups compared to the NRC protocol (p < 0.001, fold differences RC vs NRC). In microarrays, miR-34a-5p showed cystitis-induced downregulation in both protocols. Bioinformatics analysis identified multiple 3′UTRs complementary binding sites for miR-34a-5p on Grin2b, Notch1, Grip2, Scn2b, and Arc genes. The enhanced response in the RC protocol indicates a possible priming effect of early life cystitis on rechallenge in adulthood. These long-term molecular alterations may play a critical role in the development of chronic bladder pain conditions as seen in patients with Interstitial Cystitis/Bladder pain syndrome.

LINC-p21 Regulates Pancreatic β-Cell Function in Type 2 Diabetes Mellitus.

This study aimed to investigate the underlying mechanism and assess the biological role of long intergenic non-coding RNA (LINCRNA)-p21 in type 2 diabetes mellitus (T2DM). LINC-p21 and miR-335-3p expression levels were evaluated in blood from T2DM patients, healthy individuals, and mouse islet β-cell line MIN6 cells grown in a high glucose environment. Apoptosis-related proteins, iNOS, and IGF-1 were detected in vitro and in vivo. Bioinformatics was used to predict that miR-335-3p had complementary binding sites to IGF-1, and a dual-luciferase reporter confirmed the targeting link between LINC-p21 and miR-335-3p. LINC-p21 was highly expressed in the T2DM serum and cells, and LINC-p21 was significantly associated with T2DM prognosis. In vitro and in vivo dysfunction of β-cells was reduced by LINC-p21 knockdown. MiR-335-3p and IGF-1 may be potential targets of LINC-p21 and miR-335-3p, respectively, after the prediction of the target of LINC-p21 was verified by dual-luciferase assay. Anti-miR-335-3p made LINC-p21 knockdown function again; however, interference of IGF-1 mRNA restored the function of LINC-p21. The miR-335-3p/IGF-1 axis may have a role in the functional protection of pancreatic β-cells by LINC-p21 silencing, boosting insulin production, and slowing the course of diabetes.

Synergy of Post-Crosslinked imprinting strategy and silanol chemistry for the anisotropic construction of molecularly imprinted polymers onto Halloysite nanotubes

Searching for alternative porous substrates which can realize anisotropic preparation of “tailor-made” complementary binding sites is an important way to improve the identification efficiency and interfacial stability of the surface molecularly imprinted polymers (SMIPs). In this work, a functional polymer (FP) with multi-functional properties was firstly synthesized using 3-(isobutenyloxy) propyltrimethoxysilane, 1-(vinylbenzyl) thymidine and cinnamoyloxyethyl methacrylate (CEMA) as monomers, and then FP with post-crosslinking property was covalently deposited onto theoutersurfaceof halloysite nanotubes (HNTs) using silanol chemistry to prepare 2′-deoxyadenosine (dA) imprinted sorbents (HNTs-SMIPs). More than 65 % of dA molecules was captured within the first 2.0 h. The number of high affinity site from the total binding sites of HNTs-SMIPs is estimated to be as high as 73.25 %, and the maximum adsorption amount of HNTs-SMIPs is 48.78 μmol g -1 at 25 °C. The imprinting factor (IF) of HNTs-SMIPs towards dA is 1.89, and 79.49 % the adsorption capacity is remained after five adsorption–desorption cycles. In addition, 70.2 % of the dA from spiked human urine samples can be selectively extracted by HNTs-SMIPs. HNTs-SMIPs have excellent interfacial stability and strong affinity for the template molecule dA, and this work provides a new strategy for the specific separation and enrichment of dA.

CircANXA4 (hsa_circ_0055087) regulates the miR-1256/PRM1 axis to promote tumor progression in colorectal cancer

Colorectal cancer (CRC) incidence ranks third among malignant cancers with a high propensity for distant metastasis. Despite continuous efforts to improve treatment, the prognosis especially in patients with advanced distant metastasis is low. The mechanism of development and progression of CRC is not fully understood. Non-coding RNAs (ncRNAs) have emerged as essential regulators in cancer progression. Here, we aim to dissect the role of one critical ncRNA, circANXA4, in CRC progression. CircANXA4 expression was analyzed by the GEO database. Differentially expressed circRNAs were identified by the Limma package R software. Expression of circANXA4 and miR-1256 was detected by qRT-PCR. The regulation of circANXA4 on cell proliferation and progression was confirmed with the cell viability assay using cell counting kit-8 (CCK-8) and transwell migration assay. RNA pull-down assay, RNA immunoprecipitation (RIP), and western blot were used to determine the interaction between circANXA4, miR-1256, and protamine1 (PRM1). CircANXA4 was upregulated in both CRC tissues and cell lines. Knockdown of circANXA4 effectively reduced cell proliferation, progression, and migration. Additionally, silencing circANXA4 remarkably increased miR-1256 expression, while reducing PRM1 expression, thereby demonstrating that circANXA4 downregulates miR-1256 expression through a complementary binding site. Rescue experiments revealed the interactions between circANXA4, miR-1256, and PRM1. Pearson correlation analysis revealed that circANXA4 expression positively correlated with PRM1 expression and miR-1256 expression inversely correlated with PRM1 expression. In sum, we demonstrated that circANXA4 promotes cancer cell proliferation and progression by sponging miR-1256 and upregulating PRM1 in CRC.

Effects of miR-214 on adenosine A2A receptor and carboxymethyl chitosan nanoparticles on the function of keloid fibroblasts and their mechanisms

This work prepared and investigated the impact of carboxymethyl chitosan nanoparticles (MC-NPs) on the proliferative capability of keloid fibroblasts (KFBs) while analyzing the mechanistic roles of miR-214 and adenosine A2A receptor (A2AR) in fibroblasts within hypertrophic scars. MC-NPs were synthesized through ion cross-linking, were characterized using transmission electron microscopy (TEM) and laser particle size scattering. The influence of MC-NPs on the proliferation capacity of KFBs was assessed using the MTT method. Changes in the expression levels of miR-214 and A2AR in KFBs, normal skin fibroblasts (NFBs), hypertrophic scar tissue, and normal skin tissue were analyzed. KFBs were categorized into anti-miR-214, anti-miR-NC, miR-214 mimics, miR-NC, si-A2AR, si-con, anti-miR-214+ si-con, and anti-miR-214+ si-A2AR groups. Bioinformatics target prediction was conducted to explore the interaction between miR-214 and A2AR. Real-time quantitative PCR and immunoblotting (WB) were employed to detect the expression levels of miR-214, A2AR, apoptotic protein Bax, and TGF-β in different cells. Cell counting kit-8 (CCK8) and flow cytometry were employed to assess cell proliferation activity and apoptosis. The results indicated that MC-NPs exhibited spherical particles with an average diameter of 236.47 ± 4.98 nm. The cell OD value in the MC-NPs group was lower than that in KFBs (P < 0.05). The mRNA levels of miR-214 in KFBs and hypertrophic scar tissue were lower than those in NFBs and normal tissue (P < 0.001), while the mRNA and protein levels of A2AR were significantly elevated (P < 0.05). Compared to the control group and anti-miR-NC, the anti-miR-214 group showed significantly increased cell OD values and Bcl-2 protein expression (P < 0.001), decreased levels of apoptotic gene Bax protein, TGF-β gene mRNA, and protein expression (P < 0.001). Continuous complementary binding sites were identified between miR-214 and A2AR. Compared to the control group, the si-A2AR group exhibited a significant decrease in A2AR gene mRNA and protein expression levels (P < 0.001), reduced cell viability (P < 0.001), increased apoptosis rate (P < 0.001), and a significant elevation in TGF-β protein expression (P < 0.001). miR-214 targetedly regulated the expression of A2AR, inducing changes in TGF-β content, promoting the proliferation of keloid fibroblasts, and inhibiting cell apoptosis.

Identification of Neural (N)-Cadherin as a Novel Endothelial Cell Receptor for Fibrin and Localization of the Complementary Binding Sites.

Based on the high structural homology between vascular endothelial (VE)-cadherin and neural (N)-cadherin, we hypothesized that fibrin, which is known to interact with VE-cadherin and promote angiogenesis through this interaction, may also interact with N-cadherin. To test this hypothesis, we prepared fibrin and its plasmin-produced and recombinant fragments covering practically all parts of the fibrin molecule. We also prepared the soluble extracellular portion of N-cadherin (sN-cadherin), which includes all five extracellular N-cadherin domains, and studied its interaction with fibrinogen, fibrin, and the aforementioned fibrin fragments using two independent methods, ELISA and SPR. The experiments confirmed our hypothesis, revealing that fibrin interacts with sN-cadherin with high affinity. Furthermore, the experiments localized the N-cadherin binding site within the fibrin βN-domains. Notably, the recombinant dimeric (β15-66)2 fragment, corresponding to these domains and mimicking their dimeric arrangement in fibrin, preserved the N-cadherin-binding properties of fibrin. To localize the fibrin binding site within N-cadherin, we performed ELISA and SPR experiments with (β15-66)2 and recombinant N-cadherin fragments representing its individual extracellular domains and combinations thereof. The results obtained indicate that the interaction of fibrin with N-cadherin occurs through the third and fifth extracellular domains of the latter. This is in contrast to our previous study, which revealed that fibrin interacts only with the third extracellular domain of VE-cadherin. In conclusion, our study identified N-cadherin as a novel receptor for fibrin and localized complementary binding sites within both fibrin and N-cadherin. The pathophysiological role of this interaction remains to be established.

Single-monomer dual templated MIP based electrochemical sensor for tartrazine and brilliant blue FCF

In this study, a dual-templated molecularly imprinted polymer-based electrochemical sensor was developed for the simultaneous analysis of two food additive dyes, brilliant blue FCF and tartrazine. Using a 3-aminophenyl boronic acid (3-APBA) monomer and the dual templates of brilliant blue FCF (BB) and tartrazine (TZ), the molecularly imprinted polymer (MIP) layer was electropolymerized on the carbon fibre paper (CFP) electrode. By using BB and TZ as template molecules along the electro-polymerization of 3-APBA, then removing both template molecules, the MIP film was generated on the surface of the CFP electrode. Due to the high surface area provided by modification, several complementary binding sites for template molecules are formed on the surface of the MIP sensor during this process of sensor fabrication. On the MIP/CFP electrode, the electrochemical behavior of BB and TZ was assessed. The monomer/template ratio, pH values, and influencing parameters like the electro-polymerization scanning cycles were all optimized. This sensor was applied to detect brilliant blue FCF and tartrazine in beverage and food samples using MIPAPBA/CFP electrode.

Mechanism of LEF1-AS1 regulating HUVEC cells by targeting miR-489-3p/S100A11 axis.

The venous malformation is the most common congenital vascular malformation and exhibits the characteristics of local invasion and lifelong progressive development. Long noncoding RNA (lncRNA) regulates endothelial cells, vascular smooth muscle cells, macrophages, vascular inflammation, and metabolism and also affects the development of venous malformations. This study aimed to elucidate the role of the lncRNA LEF1-AS1 in the development of venous malformations and examine the interaction among LEF1-AS1, miR-489-3p, and S100A11 in HUVEC cells. Venous malformation tissues, corresponding normal venous tissues, and HUVEC cells were used. Agilent human lncRNA microarray gene chip was used to screen differential genes, RNA expression was detected using quantitative reverse transcription PCR, and protein expression was detected using Western blotting. The proliferation, migration, and angiogenesis of HUVEC cells were assessed using CCK8, transwell, and in vitro angiogenesis tests. A total of 1,651 lncRNAs were screened using gene chip analysis, of which 1015 were upregulated and 636 were downregulated. The lncRNA LEF1-AS1 was upregulated with an obvious difference multiple, and the fold-change value was 11.03273. The results of the analysis performed using the StarBase bioinformatics prediction website showed that LEF1-AS1 and miR-489-3p possessed complementary binding sites and that miR-489-3p and S100A11 also had complementary binding sites. The findings of tissue experiments revealed that the expressions of LEF1-AS1 and S100A11 were higher in tissues with venous malformations than in normal tissues, whereas the expression of miR-489-3p was lower in venous malformations than in normal tissues. Cell culture experiments indicated that LEF1-AS1 promoted the proliferation, migration, and angiogenesis of HUVEC cells. In these cells, LEF1-AS1 targeted miR-489-3p, which in turn targeted S100A11. LEF1-AS1 acted as a competitive endogenous RNA and promoted the expression of S100A11 by competitively binding to miR-489-3p and enhancing the proliferation, migration, and angiogenesis of HUVEC cells. Thus, LEF1-AS1 participated in the occurrence and development of venous malformation. The expression of LEF1-AS1 was upregulated in venous malformations, and the expression of S100A11 was increased by the adsorption of miR-489-3p to venous endothelial cells, thus enhancing the proliferation, migration, and angiogenesis of HUVEC cells. In conclusion, LEF1-AS1 is involved in the occurrence and development of venous malformations by regulating the miR-489-3p/S100A11 axis, which provides valuable insights into the pathogenesis of this disease and opens new avenues for its treatment.

Matrine inhibits inflammatory response induced by TNF-α in human umbilical vein endothelial cells through miR-25-3p-mediated Klf4 pathway

This study aimed to analyze the effect of matrine on tumor necrosis factor-α(TNF-α)-induced inflammatory response in human umbilical vein endothelial cells(HUVECs) and explore whether the underlying mechanism was related to the miR-25-3p-mediated Krüppel-like factor 4(Klf4) pathway. The HUVEC cell inflammation model was induced by TNF-α stimulation. After 24 or 48 hours of incubation with different concentrations of matrine(0.625, 1.25, and 2.5 mmol·L~(-1)), CCK-8 assay was used to detect cell proliferation. After treatment with 2.5 mmol·L~(-1) matrine for 48 h, the expression of TNF-α, interleukin-6(IL-6), interleukin-1β(IL-1β), and Klf4 mRNA and miR-25-3p was detected by real-time fluorescence-based quantitative PCR, and the protein expression of TNF-α, IL-6, IL-1β, and Klf4 was detected by Western blot. The anti-miR-25-3p was transfected into HUVECs, and the effect of anti-miR-25-3p on TNF-α-induced cell proliferation and inflammatory factors was detected by the above method. The cells were further transfected with miR-25-3p and incubated with matrine to detect the changes in proliferation and expression of related inflammatory factors, miR-25-3p, and Klf4. The targeting relationship between miR-25-3p and Klf4 was verified by bioinformatics analysis and dual luciferase reporter gene assay. The results displayed that matrine could inhibit TNF-α-induced HUVEC proliferation, decrease the mRNA and protein expression of TNF-α, IL-6, and IL-1β, increase the mRNA and protein expression of Klf4, and reduce the expression of miR-25-3p. Bioinformatics analysis showed that there were specific complementary binding sites between miR-25-3p and Klf4 sequences. Dual luciferase reporter gene assay confirmed that miR-25-3p negatively regulated Klf4 expression in HUVECs by targeting. The inhibition of miR-25-3p expression can reduce TNF-α-induced cell proliferation and mRNA and protein expression of TNF-α, IL-6, and IL-1β. MiR-25-3p overexpression could reverse the effect of matrine on TNF-α-induced cell proliferation and the mRNA and protein expression of TNF-α, IL-6, IL-1β, and Klf4. This study shows that matrine inhibits the inflammatory response induced by TNF-α in HUVECs through miR-25-3p-mediated Klf4 pathway.

Label-free MIP-SERS biosensor for sensitive detection of colorectal cancer biomarker.

Early diagnosis of colorectal cancer can significantly improve the overall survival rate of patients, thus selective and sensitive detection of biomarkers in serum samples is vital for early detection and dynamic monitoring of cancer. Nucleoside diphosphate kinase NM23-H2 (NDKB) is an important biomarker and therapeutic target for the diagnosis of colorectal cancer (CRC). Here, a label-free and ultrasensitive biosensor for NDKB protein markers is presented for the first time, combining the characteristic capture selectivity of molecularly imprinted polymers (MIPs) and the ultrasensitivity of surface-enhanced Raman Spectroscopy (SERS) technique. The imprinted cavity serves as the only channel for Raman reporter to approach the SERS substrate, providing highly complementary non-covalent binding sites that selectively capture the target protein based on ionic, hydrogen bonding or hydrophobic interactions. Specific recognition of the NDKB protein will perfectly fill the imprinted cavity, which makes it difficult for the Raman reporter to get close to the SERS substrate, and the Raman signal decreases significantly, while the proteins of other structural sizes can not match the imprinted cavity. Through the change of the Raman signal, the proposed biosensor can realize the ultra-sensitive detection of NDKB, and the limit of detection (LOD) is 0.82pg/mL. Compared with the traditional immunoassay technology, this combined approach with the advantages of low cost, fast response, high sensitivity and selectivity, provides clinical application potential for the early diagnosis of CRC.

The CIS association of CD47 with integrin Mac-1 regulates macrophage responses by stabilizing the extended integrin conformation

CD47 is a ubiquitously expressed cell surface integrin-associated protein. Recently, we have demonstrated that integrin Mac-1 (αMβ2, CD11b/CD18, CR3), the major adhesion receptor on the surface of myeloid cells, can be coprecipitated with CD47. However, the molecular basis for the CD47-Mac-1 interaction and its functional consequences remain unclear. Here, we demonstrated that CD47 regulates macrophage functions directly interacting with Mac-1. In particular, adhesion, spreading, migration, phagocytosis, and fusion of CD47-deficient macrophages were significantly decreased. We validated the functional link between CD47 and Mac-1 by co-immunoprecipitation analysis using various Mac-1-expressing cells. In HEK293 cells expressing individual αM and β2 integrin subunits, CD47 was found to bind both subunits. Interestingly, a higher amount of CD47 was recovered with the free β2 subunit than in the complex with the whole integrin. Furthermore, activating Mac-1-expressing HEK293 cells with PMA, Mn2+, and activating antibody MEM48 increased the amount of CD47 in complex with Mac-1, suggesting CD47 has a greater affinity for the extended integrin conformation. Notably, on the surface of cells lacking CD47, fewer Mac-1 molecules could convert into an extended conformation in response to activation. Additionally, we identified the binding site in CD47 for Mac-1 in its constituent IgV domain. The complementary binding sites for CD47 in Mac-1 were localized in integrin epidermal growth factor-like domains 3 and 4 of the β2 and calf-1 and calf-2 domains of the α subunits. These results indicate that Mac-1 forms a lateral complex with CD47, which regulates essential macrophage functions by stabilizing the extended integrin conformation.

In Silico Identification of Cassava Genome-Encoded MicroRNAs with Predicted Potential for Targeting the ICMV-Kerala Begomoviral Pathogen of Cassava.

Cassava mosaic disease (CMD) is caused by several divergent species belonging to the genus Begomovirus (Geminiviridae) transmitted by the whitefly Bemisia tabaci cryptic species group. In India and other parts of Asia, the Indian cassava mosaic virus-Kerala (ICMV-Ker) is an emergent begomovirus of cassava causing damage that results in reduced yield loss and tuber quality. Double-stranded RNA-mediated interference (RNAi) is an evolutionary conserved mechanism in eukaryotes and highly effective, innate defense system to inhibit plant viral replication and/or translation. The objective of this study was to identify and characterize cassava genome-encoded microRNAs (mes-miRNA) that are predicted to target ICMV-Ker ssDNA-encoded mRNAs, based on four in silico algorithms: miRanda, RNA22, Tapirhybrid, and psRNA. The goal is to deploy the predicted miRNAs to trigger RNAi and develop cassava plants with resistance to ICMV-Ker. Experimentally validated mature cassava miRNA sequences (n = 175) were downloaded from the miRBase biological database and aligned with the ICMV-Ker genome. The miRNAs were evaluated for base-pairing with the cassava miRNA seed regions and to complementary binding sites within target viral mRNAs. Among the 175 locus-derived mes-miRNAs evaluated, one cassava miRNA homolog, mes-miR1446a, was identified to have a predicted miRNA target binding site, at position 2053 of the ICMV-Ker genome. To predict whether the cassava miRNA might bind predicted ICMV-Ker mRNA target(s) that could disrupt viral infection of cassava plants, a cassava locus-derived miRNA-mRNA regulatory network was constructed using Circos software. The in silico-predicted cassava locus-derived mes-miRNA-mRNA network corroborated interactions between cassava mature miRNAs and the ICMV-Ker genome that warrant in vivo analysis, which could lead to the development of ICMV-Ker resistant cassava plants.

METTL3-mediated long non-coding RNA MIR99AHG methylation targets miR-4660 to promote bone marrow mesenchymal stem cell osteogenic differentiation

ABSTRACT Whether long non-coding RNA Mir-99a-Let-7c Cluster Host Gene (LncRNA MIR99AHG) is involved in osteoporosis (OP) remains vague, so we hereby center on its implication. Old C57BL/6J mice were injected with the silencing lentivirus of MIR99AHG and subjected to microCT analysis and immunohistochemistry on osteogenic cells. The osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) with or without transfection was determined by alkaline phosphatase (ALP) and Alizarin Red S staining. Total N(6)-methyladenosine (m6A) on the bone marrow mesenchymal stem cells (BMSCs) was quantified. The potential methylation site and the complementary binding sites with candidate microRNA (miR) were predicted via bioinformatic analyses, with the latter being confirmed via dual-luciferase reporter, RNA immunoprecipitation and RNA pull-down assays. Quantitative real-time PCR and Western blot were used for quantification assays. MIR99AHG was decreased during the osteogenic differentiation of BMSCs, where increased Osterix (OSX), Collagen, Type I, Alpha 1 (Col1A1), Osteocalcin (OCN) and RUNX Family Transcription Factor 2 (RUNX2) as well as more color-stained areas were found. Also, silencing MIR99AHG relieved the OP in mice and reduced the loss of osteogenic cells. M6A methylation in undifferentiated BMSCs was low and MIR99AHG overexpression abolished the effects of overexpressed METTL3 on promoting osteogenic differentiation. MiR-4660, which was downregulated in BMSCs without differentiation but increased during osteogenic differentiation, could bind with MIR99AHG. Furthermore, miR-4660 promoted osteogenic differentiation and reversed the effects of overexpressed MIR99AHG. The present study demonstrated that METTL3-mediated LncRNA MIR99AHG methylation enhanced the osteogenic differentiation of BMSCs via targeting miR-4660.

Imprinted polymer beads featuring both predefined multiple-point interaction and accessible binding sites for precise recognition of 2′-deoxyadenosine

“Tailor-made” formation of complementary binding sites via lock-and-key molecular binding mechanism is critical for selective adsorption of target molecules in environmental and chemical engineering fields. However, the innovative design of molecularly imprinted polymers (MIPs) remains a challenging task. Here, photo-crosslinked functional polymer building blocks consisting of monomeric methacrylic acid (MAA), 1-(vinylbenzyl)thymidine (VBT) and post-crosslinked monomeric cinnamoyloxyethyl methacrylate (CEMA) were firstly synthesized. MIPs beads (PC-MIPs) were then prepared by emulsion droplet microreactor, and finally their precise recognition of 2'-deoxyadenosine (dA) was investigated by batch mode adsorption. The prepared PC-MIPs microspheres have a large number of voids on the surface with interconnected cavities inside, and the bubble-like framework inside may be caused by the full encapsulation of bubbles from the low boiling point isolated phase. Due to the high density of accessible imprinted sites and strong interaction with dA, the maximum monolayer adsorption capacity of PC-MIPs was calculated to be 134.5 μmol g−1 for dA, surpassing most of the reported dA-imprinted sorbents. In addition, unique advantages of the post-crosslinking strategy combined with the emulsion template method exhibited good selectivity (imprinting factor higher than 2.71) and high stability, as well as excellent recyclability toward dA. Moreover, 82.4 % of dA was extracted by PC-MIPs from spiked human urine samples. Therefore, our work demonstrated an alternate strategy to design polymers bearing precise molecular recognition capability, and provided a new perspective for dA enrichment from biological samples.

Identification of the hsa_circ_0039466/miR-96-5p/FOXO1 regulatory network in hepatocellular carcinoma by whole-transcriptome analysis.

BackgroundCircular RNAs (circRNAs) are important for the process of cancer initiation and progression. However, the role of circRNAs in hepatocellular carcinoma (HCC) remains incompletely understood. Therefore, we further explored the expression network of circRNAs in HCC.MethodsWhole-transcriptome microarrays of HCC and paired normal liver tissues were obtained from the Gene Expression Omnibus (GEO) database. The structures of tumor-associated circRNAs were acquired by the Cancer-Specific CircRNA Database (CSCD). StarBase, circBank, and R packages (miRNAtap and multiMiR) were used to predict miRNA targets of circRNAs and downstream molecules of miRNAs. Expression relationships between RNA-RNA interactions were evaluated by data from The Cancer Genome Atlas (TCGA) and GEO databases. ClusterProfiler and DOSE R packages were used for pathway enrichment to explore the biological functions of potential target genes. Finally, a possible circRNA-miRNA-mRNA regulatory network was established based on the competing endogenous RNA (ceRNA) hypothesis.ResultsThe differentially expressed circRNAs (DECs) were matched with cancer-specific circRNAs in the CSCD database and a screening analysis was performed to obtain 5 cancer-specific circRNAs. A total of 329 possible target miRNAs for 5 cancer-specific circRNAs were predicted by the circBank database, and intersection analysis with differentially expressed miRNAs (DEmiRNAs) revealed that miR-6746-3p and miR-96-5p were the two most suitable miRNAs targets for our selected circRNAs. Further expression verification and prediction of base complementary paired binding sites demonstrated the hsa_circ_0039466/miR-96-5p axis as a crucial pathway in HCC. Next, we found that FOXO1 and LEPR were two potential downstream molecules of the hsa_circ_0039466/miR-96-5p axis through target gene prediction analysis, differential expression analysis, and intersection analysis. The pathway enrichment results suggested that the hsa_circ_0039466/miR-96-5p axis affects the progression and outcome of HCC through the insulin resistance pathway. Finally, through multi-data crossover analysis and data analysis of HCC samples further confirmed the existence of the hsa_circ_0039466/miR-96-5p/FOXO1 ceRNA regulatory network and that the axis was closely related to clinical stage.Conclusionshsa_circ_0039466 facilitates the expression of FOXO1 by sponging miR-96-5p, and ultimately inhibits tumor progression. These results provide a theoretical basis for further understanding of the gene expression network of HCC.

Insights Into Platelet-Derived MicroRNAs in Cardiovascular and Oncologic Diseases: Potential Predictor and Therapeutic Target.

Non-communicable diseases (NCDs), represented by cardiovascular diseases and cancer, have been the leading cause of death globally. Improvements in mortality from cardiovascular (CV) diseases (decrease of 14%/100,000, United States) or cancers (increase 7.5%/100,000, United States) seem unsatisfactory during the past two decades, and so the search for innovative and accurate biomarkers of early diagnosis and prevention, and novel treatment strategies is a valuable clinical and economic endeavor. Both tumors and cardiovascular system are rich in angiological systems that maintain material exchange, signal transduction and distant regulation. This pattern determines that they are strongly influenced by circulating substances, such as glycolipid metabolism, inflammatory homeostasis and cyclic non-coding RNA and so forth. Platelets, a group of small anucleated cells, inherit many mature proteins, mRNAs, and non-coding RNAs from their parent megakaryocytes during gradual formation and manifest important roles in inflammation, angiogenesis, atherosclerosis, stroke, myocardial infarction, diabetes, cancer, and many other diseases apart from its classical function in hemostasis. MicroRNAs (miRNAs) are a class of non-coding RNAs containing ∼22 nucleotides that participate in many key cellular processes by pairing with mRNAs at partially complementary binding sites for post-transcriptional regulation of gene expression. Platelets contain fully functional miRNA processors in their microvesicles and are able to transport their miRNAs to neighboring cells and regulate their gene expression. Therefore, the importance of platelet-derived miRNAs for the human health is of increasing interest. Here, we will elaborate systematically the roles of platelet-derived miRNAs in cardiovascular disease and cancer in the hope of providing clinicians with new ideas for early diagnosis and therapeutic strategies.

1468-P: Effect of Kcnq1ot1 on Influencing the Function of Pancreatic ß-Cells Targeting miR-92a-1-5p

Objective: To study the role and mechanism of Kcnq1ot1 by targeting to regulate miR-92a-1-5p to effect insulin secretion in pancreatic β cells. Methods: The qRT-PCR was used to detect the expression of Kcnq1ot1 in different tissues of C57BL/6J mice, Min6 with PA intervention, islets of T2D model mice and the serum of T2D patients. The biological information website was used to predict that Kcnq1ot1 had complementary binding sites with miR-92a-1-5p, which was verified by the luciferase reporting gene system. The si-Kcnq1ot1, miR-92a-1-5p mimics, miR-92a-1-5p inhibitors and their respective controls were respectively transfected into Min6, and the effect of Ins1, Ins2, Insr, Nkx6.1, Irs1 and Ngn3 were detected by qRT-PCR, the Western blot method was used to detect the expression of insulin and Insr, and the GSIS experiment was used to detect its effect on insulin secretion. Results: The expression of Kcnq1ot1 in pancreas was significantly higher than that in other tissues. Compared with the control group, the expression of Kcnq1ot1 in T2D patients' serum, Min6 after PA treatment and the islets of T2D model mice were lower. And the expression of Kcnq1ot1 in serum is negatively correlated with FBG or HbA1c. Low expression of Kcnq1ot1 led to remarkable reduction of insulin secretion in Min6 cells. Down-regulation of Kcnq1ot1 or overexpression of miR-92a-1-5p reduced the mRNA expression levels of Ins1, Ins2, Insr, Nkx6.1, Irs1 and Ngn3, and the protein levels of insulin and Insr. We screened miR-92a-1-5p as targets of Kcnq1ot1, and their interaction was validated with dual luciferase reporter gene experiment. After knocking down Kcnq1ot1, the expression of miR-92a-1-5p was found to increase. Further rescue experiments showed that down-regulation of miR-92a-1-5p could reverse the effect of low Kcnq1ot1 expression on the function of pancreatic β-cells. Conclusion: Kcnq1ot1 may regulate the function of pancreatic β cells by targeting to negatively regulate miR-92a-1-5p. Disclosure Y.Chen: None. Y.Li: None. W.Xu: None. B.Lin: None. X.Mai: None. Y.Peng: None. D.Chen: None. Y.Su: None. W.Li: n/a. Funding Science &amp; Technology Project of Guangzhou（202102010175）

Puerarin promotes apoptosis and senescence of bladder cancer cells

• Puerarin inhibits the proliferation and induces apoptosis and senescence of bladder cancer cells. • Puerarin exerts anticancer effects by up-regulating miR-139-5p in bladder cancer cells. • CCNB1 is the target gene of miR-139-5p. • Puerarin down-regulates CCNB1 in bladder cancer cells. • Puerarin down-regulates CCNB1 and PI3K/AKT pathways through miR-139-5p. This study aims to explore the mechanism of puerarin regulating the proliferation, apoptosis and senescence of T24 and EJ cells via miR-139-5p/CCNB1 and PI3K/AKT pathways. Initially, Cell Counting Kit ( CCK)-8, flow cytometry, β-galactosidase detection kit, and RT-qPCR are used to assess the effect of puerarin on the proliferation apoptosis and senescence of bladder cancer cells by regulating miR-139-5p, respectively. Then, potential complementary binding sites between miR-139-5p and the 3′ untranslated region (3′ UTR) of the CCNB1 gene are obtained by the bioinformatics software, starBase database. Further, a dual-luciferase reporter system is used to assess the targeted relationship of miR-139-5p to CCNB1. CCK-8, flow cytometry, and β-galactosidase detection kit were used to detect the influence of CCNB1 on the proliferation, apoptosis, and senescence of bladder cancer cells. RT-qPCR and Western blotting are used to determine the regulation of CCNB1 and PI3K/AKT pathways by miR-139-5p. Finally, the subcutaneous tumor formation in nude mice and immunohistochemical experiments are performed to assess the effect of puerarin on the proliferation of bladder cancer cells in vivo . Puerarin induced proliferation inhibition, apoptosis, and senescence of bladder cancer cells in vitro . Puerarin down-regulated the expression of CCNB1, p-PI3K, and p-AKT proteins by overexpressing miR-139-5p, thereby exerting a tumor suppressor effect in bladder cancer. Compared with the control group, the volume and weight of subcutaneous tumors of nude mice in the puerarin administration group are reduced. Moreover, the ki-67 positive staining rate and the expression level of CCNB1, p-PI3K, and p-AKT protein are reduced. The experimental results showed that the anti-tumor effect of puerarin against bladder cancer might be achieved by inhibiting CCNB1 and PI3K/AKT pathway via up-regulating miR-139-5p.