Label-Free Isothermal Amplification Assay for Specific and Highly Sensitive Colorimetric miRNA Detection

We describe a new method for the detection of miRNA in biological samples. This technology is based on the isothermal nicking enzyme amplification reaction and subsequent hybridization of the amplification product with gold nanoparticles and magnetic microparticles (barcode system) to achieve naked-eye colorimetric detection. This platform was used to detect a specific miRNA (miRNA-10b) associated with breast cancer, and attomolar sensitivity was demonstrated. The assay was validated in cell culture lysates from breast cancer cells and in serum from a mouse model of breast cancer

Human Equilibrative Nucleoside Transporter-1 Knockdown Tunes Cellular Mechanics through Epithelial-Mesenchymal Transition in Pancreatic Cancer Cells

<div><p>We report cell mechanical changes in response to alteration of expression of the human equilibrative nucleoside transporter-1 (hENT1), a most abundant and widely distributed plasma membrane nucleoside transporter in human cells and/or tissues. Modulation of hENT1 expression level altered the stiffness of pancreatic cancer Capan-1 and Panc 03.27 cells, which was analyzed by atomic force microscopy (AFM) and correlated to microfluidic platform. The hENT1 knockdown induced reduction of cellular stiffness in both of cells up to 70%. In addition, cellular phenotypic changes such as cell morphology, migration, and expression level of epithelial-mesenchymal transition (EMT) markers were observed after hENT1 knockdown. Cells with suppressed hENT1 became elongated, migrated faster, and had reduced E-cadherin and elevated N-cadherin compared to parental cells which are consistent with epithelial-mesenchymal transition (EMT). Those cellular phenotypic changes closely correlated with changes in cellular stiffness. This study suggests that hENT1 expression level affects cellular phenotype and cell elastic behavior can be a physical biomarker for quantify hENT1 expression and detect phenotypic shift. Furthermore, cell mechanics can be a critical tool in detecting disease progression and response to therapy.</p></div

Compare deformability of control and hENT1 knockdown cells using a microfluidic separation chip.

<p>(A) Photograph (left) of microfluidic separation chip (MS-chip) visualized by red dye inside and a representative scanning electron microscopic (right) image of the post array (diameter of pillar: 35 µm, height of pillar: 30 µm). (B) Bright field image and scheme of cells flowing in MS-chip when the cell diameter is smaller than pillar gap size (red arrow indicates cells). It is assumed here that friction is due only to shear stress (F_p: pressure force against the applied pressure (10 psi in this study), F_f: friction force). (C) The fluorescence image of one of channels among eight shows retained Panc 03.27 cells in the MS-chip after separation of Panc 03.27-ctrl (red fluorescence) and Panc 03.27-hENT1 knockdown (green fluorescence). Representative higher magnification confocal microscopic images of the MS chip (gap size from 12 µm to 6 µm) show the efficiency of separation through gaps. Size distribution of cells (D: Capan-1, G: Panc 03.27 cells). Statistical analysis of cells (E: Capan-1, H: Panc 03.27) and fraction ratio of cells (F: Capan-1, I: Panc 03.27) retained on chip. Values represent mean ± standard deviation.</p

hENT1 knockdown induces changes in EMT markers.

<p>Western blots and relative expression levels of E-cadherin (110 kDa), N-cadherin (140 kDa) in (A) Capan-1 and (C) Panc 03.27 cells after treatment with hENT1 siRNA. Columns in the histograms are the mean of two independent experiments (intensity ratio of GAPDH to E-cadherin or N-cadherin). Representative confocal images of E-cadherin (red fluorescence) and N-cadherin (green fluorescence) expressions in (B) Capan-1 and (D) Panc 03.27 cells.</p

Quantify cell roundness.

<p>(A) Representative confocal micrographs of pancreatic cells (Top panel: Capan-1, Bottom panel: Panc 03.27) showing F-actin distribution, (B) form factor (f = 4πa/p²; a: area; p: perimeter) analyzed by Image J (Capan-1 cells, ctrl: n = 37, scrl: n = 59, hENT1↓: n = 29; Panc 03.27, ctrl: n = 36, scrl: n = 28, hENT1↓:n = 28, N.S: statistically not significant).</p

Establish hENT1 knockdown pancreatic cancer cell lines.

<p>(A) Western blots of hENT1 (55 kDa) and GAPDH (37 kDa) in Capan-1 (left) and Panc 03.27 cells (right) without treatment (ctrl) or after treatment of negative siRNA (scrl) or hENT1 siRNA (hENT1↓), (B) Bar histograms show Young's modulus of control, negative siRNA transfected, and hENT1 knockdown cells. (N.S: statistically not significant).</p

Analysis of focal adhesion area.

<p>Confocal micrographs (z-stacks of basal plane, 0–1.5 µm) showing vinculin (red) and F-actin (green) in (A) Capan-1 and (B) Panc 03.27 cells. Area of focal adhesion (pixel²) is analyzed by Image J.</p

Downregulation of hENT1 promote cell migration.

<p>The scratches were introduced to monolayer (A) Capan-1 and (D) Panc 03.27 cells. Photographs were taken immediately after wound induction for 18 hours. The wound sealing areas of (B) Capan-1 and (E) Panc 03.27 cells were calculated using Image J and compared with control, scramble siRNA transfected, and hENT1 knockdown cells. Migration speed of (C) Capan-1 and (F) Panc 03.27 cells was calculated.</p

Schematic illustrations describing overall flow of this study.

<p>The hENT1 knockdown induces changes in cellular mechanics via EMT accompanied by alterations in E-cadherin and N-cadherin expression levels, cellular morphology, and motility of pancreatic cancer cells. Further, hENT1 knockdown induces decrease in cell stiffness as demonstrated on representative force separation curves obtained from Panc 03.27 cells (upper graph from a parent cell; the second graph from a hENT1 knockdown cell) using AFM.</p

Alterations in tumor perfusion after MHT treatment.

<p>Concurrent reduction in arterial and venous flow after with MHT treatment which contributed to decreased half-life AVTT. Tabulated data represents average of ∼30 ROIs per treatment group (n = 5) where * denotes p<0.03.</p