Aptamer Recognition Induced Target-Bridged Strategy for Proteins Detection Based on Magnetic Chitosan and Silver/Chitosan Nanoparticles Using Surface-Enhanced Raman Spectroscopy

Poor selectivity and biocompability remain problems in applying surface-enhanced Raman spectroscopy (SERS) for direct detection of proteins due to similar spectra of most proteins and overlapping Raman bands in complex mixtures. To solve these problems, an aptamer recognition induced target-bridged strategy based on magnetic chitosan (MCS) and silver/chitosan nanoparticles (Ag@CS NPs) using SERS was developed for detection of protein benefiting from specific affinity of aptamers and biocompatibility of chitosan (CS). In this process, one aptamer (or antibody) modified MCS worked as capture probes through the affinity binding site of protein. The other aptamer modified Raman report molecules encapsulated Ag@CS NPs were used as SERS sensing probes based on the other binding site of protein. The sandwich complexes of aptamer (antibody)/protein/aptamer were separated easily with a magnet from biological samples, and the concentration of protein was indirectly reflected by the intensity variation of SERS signal of Raman report molecules. To explore the universality of the strategy, three different kinds of proteins including thrombin, platelet derived growth factor BB (PDGF BB) and immunoglobulin E (lgE) were investigated. The major advantages of this aptamer recognition induced target-bridged strategy are convenient operation with a magnet, stable signal expressing resulting from preventing loss of report molecules with the help of CS shell, and the avoidance of slow diffusion-limited kinetics problems occurring on a solid substrate. To demonstrate the feasibility of the proposed strategy, the method was applied to detection of PDGF BB in clinical samples. The limit of detection (LOD) of PDGF BB was estimated to be 3.2 pg/mL. The results obtained from human serum of healthy persons and cancer patients using the proposed strategy showed good agreement with that of the ELISA method but with wider linear range, more convenient operation, and lower cost. The proposed strategy holds great potential in highly sensitive and selective analysis of target proteins in complex biological samples

Upregulation of MircoRNA-370 Induces Proliferation in Human Prostate Cancer Cells by Downregulating the Transcription Factor FOXO1

<div><p>Forkhead box protein O1 (FOXO1), a key member of the FOXO family of transcription factors, acts as a tumor suppressor and has been associated with various key cellular functions, including cell growth, differentiation, apoptosis and angiogenesis. Therefore, it is puzzling why FOXO protein expression is downregulated in cancer cells. MicroRNAs, non-coding 20∼22 nucleotide single-stranded RNAs, result in translational repression or degradation and gene silencing of their target genes, and significantly contribute to the regulation of gene expression. In the current study, we report that miR-370 expression was significantly upregulated in five prostate cancer cell lines, compared to normal prostatic epithelial (PrEC) cells. Ectopic expression of miR-370 induced proliferation and increased the anchorage-independent growth and colony formation ability of DU145 and LNCaP prostate cancer cells, while inhibition of miR-370 reduced proliferation, anchorage-independent growth and colony formation ability. Furthermore, upregulation of miR-370 promoted the entry of DU145 and LNCaP prostate cancer cells into the G1/S cell cycle transition, which was associated with downregulation of the cyclin-dependent kinase (CDK) inhibitors, <em>p27^Kip1</em> and <em>p21^Cip1</em>, and upregulation of the cell-cycle regulator cyclin D1 mRNA. Additionally, we demonstrated that miR-370 can downregulate expression of FOXO1 by directly targeting the <em>FOXO1</em> 3′-untranslated region. Taken together, our results suggest that miR-370 plays an important role in the proliferation of human prostate cancer cells, by directly suppressing the tumor suppressor FOXO1.</p> </div

MiR-370 induces proliferation by promoting the G1-S transition. A

<p>, Flow cytometric analysis of PC3 and DU145 cells transfected with miR-370 mimic or negative control (NC) 48 hours after transfection. <b>B,</b> Representative micrographs (left) and quantification (right) of the BrdU incorporation assay in PC3 and DU145 cells transfected with miR-370 or NC 48 hours after transfection. <b>C,</b> Western blotting analysis indicating decreased expression of p21^Cip1, p27^Kip1 and increased expression of cyclin D1 and phosphorylated Rb (p-Rb) in PC3 and DU145 cells transfected with miR-370 or NC 48 hours after transfection. α-tubulin was used as a loading control. <b>D,</b> Real time PCR analysis of <i>p21^Cip1, p27^Kip1</i> and cyclin D1 mRNA in PC3 and DU145 cells transfected with miR-370 or NC 48 hours after transfection. <i>GAPDH</i> was used as a loading control. Bars represent the mean ± SD values of three independent experiments; *<i>P</i><0.05.</p

MiR-370-induced prostate cancer cell proliferation is mediated by FOXO1. A,

<p>Real-time PCR analysis of <i>p21^Cip1, p27^Kip1</i> and cyclin D1 mRNA expression in the indicated cells. <i>GAPDH</i> was used as a loading control 48 hours after transfection. <b>B,</b> Luciferase activity assay of the indicated cells transfected with a FOXO1 reporter 48 hours after transfection. <b>C,</b> MTT assay of prostate cancer cells transfected with miR-370 mimic, co-transfected with miR-370 and FOXO1, or co-transfected with miR-370 and FOXO1-3′-UTR.</p

Inhibition of miR-370 suppresses the growth of prostate cancer cells. A,

<p>Real-time PCR analysis of <i>p21^Cip1, p27^Kip1</i> and cyclin D1 mRNA in PC3 and DU145 cells transfected with miR-370 inhibitor or negative control (NC) 48 hours after transfection. <i>GAPDH</i> was used as a loading control. <b>B,</b> Flow cytometric analysis of PC3 and DU145 cells transfected with miR-370 inhibitor or NC 48 hours after transfection. <b>C,</b> MTT assays revealed that inhibition of miR-370 reduced cell growth. <b>D,</b> Representative micrographs (left) and quantification of colonies containing more than 50 cells (middle) or colonies larger than 0.1 mm (right) in the anchorage-independent growth assays. Bars represent the mean ± SD values of three independent experiments; *<i>P</i><0.05.</p

MiR-370 downregulates FOXO1 by directly targeting the <i>FOXO1</i> 3′UTR.

<p><b>A</b>, Sequence of the <i>FOXO1</i> 3′UTR miR-370 binding seed region and mutation of the <i>FOXO1</i> 3′-UTR seed region to create FOXO1-mu. <b>B,</b> Western blotting analysis of FOXO1 expression in miR-370 or negative control (NC)-transfected PC3 and DU145 cells 48 hours after transfection. <b>C,</b> Relative FOXO1 reporter activities in miR-370 or NC-transfected cells 48 hours after transfection. <b>D,</b> Western blotting analysis of GFP reporter gene expression in miR-370 or NC-transfected cells 48 hours after transfection. <b>E,</b> Relative luciferase activity of PC3 or DU145 prostate cancer cells co-transfected with increasing amounts of miR-370 mimic oligonucleotides (20, 50 nM), and the pGL3 control reporter, pGL3-FOXO1-3′UTR reporter, or pGL3-FOXO1-3′UTR-mu reporter, 48 hours after transfection, respectively. Bars represent the mean ± SD values of three independent experiments; *<i>P</i><0.05.</p

Upregulation of miR-370 promotes the proliferation of prostate cancer cells.

<p><b>A</b>, Real-time PCR analysis of miR-370 expression in normal prostate epithelial cells (PrECs; shown as N1 and N2) and prostate cancer cell lines including PC3, DU145, 22Rv1 and LNCaP cells <b>B</b>, MTT assays indicating that the proliferation of miR-370-transfected cells increased, compared to negative control (NC)-transfected cells. <b>C,</b> Colony formation assay of miR-370 overexpressing cells; representative micrographs (left) and quantification (right) of crystal violet stained cell colonies. <b>D,</b> Quantification of Ki-67 positive cells in PC3 and DU145 cells transfected with miR-370 mimic or negative control (NC) 48 hours after transfection. <b>E,</b> Upregulation of miR-370 promoted prostate cancer cell tumorigenicity; representative micrographs (left) and quantification of colonies containing more than 50 cells (middle) or colonies larger than 0.1 mm (right) in the anchorage-independent growth assay. Bars represent the mean ± SD values of three independent experiments; *<i>P</i><0.05.</p

Inhibition of miR-370 activates the FOXO1 pathway. A,

<p>Relative luciferase activity assay of PC3 and DU145 cells co-transfected with the pGL3-FOXO1-3′UTR plasmid and increasing amounts (20, 50 nM) of miR-370 mimic- or miR-370 inhibitor-oligonucleotides, 48 hours after transfection, respectively. <b>B,</b> Western blotting analysis of FOXO1 expression in miR-370 or negative control (NC)-transfected PC3 and DU145 cells 48 hours after transfection. <b>C,</b> Luciferase activity assay of PC3 and DU145 cells transfected with the pGL3-FOXO1-3′UTR plasmid and increasing amounts (20, 50 nM) of miR-370 inhibitor-oligonucleotides 48 hours after transfection. Bars represent the mean ± SD values of three independent experiments; *<i>P</i><0.05.</p