Specific survivin dual fluorescence resonance energy transfer molecular beacons for detection of human bladder cancer cells

Abstract

Aim: Survivin molecular beacons can be used to detect bladder cancer cells in urine samples non-invasively. The aim of this study is to improve the specificity of detection of bladder cancer cells using survivin dual fluorescence resonance energy transfer molecular beacons (FRET MBs) that have fluorophores forming one donor-acceptor pair.

Methods: Survivin-targeting dual fluorescence resonance energy transfer molecular beacons with unique target sequences were designed, which had no overlap with the other genes in the apoptosis inhibitor protein family. Human bladder cancer cell lines 5637, 253J and T24, as well as the exfoliated cells in the urine of healthy adults and patients with bladder cancer were examined. Images of cells were taken using a laser scanning confocal fluorescence microscope. For assays using dual FRET MBs, the excitation wavelength was 488 nm, and the emission detection wavelengths were 520±20 nm and 560±20 nm, respectively.

Results: The human bladder cancer cell lines and exfoliated cells in the urine of patients with bladder cancer incubated with the survivin dual FRET MBs exhibited strong fluorescence signals. In contrast, no fluorescence was detected in the survivin-negative human dermal fibroblasts-adult (HDF-a) cells or exfoliated cells in the urine of healthy adults incubated with the survivin dual FRET MBs.

Conclusion: The results suggest that the survivin dual FRET MBs may be used as a specific and non-invasive method for early detection and follow-up of patients with bladder cancer.

Figure 1

A schematic illustration showing the working mechanism of an MB and a dual FRET MB. (A) The MB adopts a stem-loop structure and thus holds the fluorophore (green) and quencher (black) in close proximity. As a result, the fluorescence emission of the fluorophore is strongly suppressed (in the absence of a target). The target sequence hybridizes with the loop domain of the MB and forces the stem helix to open, whereupon fluorescence is restored because of the spatial separation of the fluorophore from the quencher. (B) Hybridization of donor and acceptor MBs to adjacent regions on the same mRNA target results in FRET between donor fluorophores (FAM, excitation at 490 nm and emission at 520 nm) and acceptor fluorophores (Cy3, excitation at 550 nm and emission at 570 nm) upon donor excitation (490 nm). Because only the donor fluorophore is directly excited, the emission detected from the acceptor fluorophore is due to FRET.

Figure 2

(A) Solution studies of probe-target hybridization of dual FRET MBs. Fluorescence emission spectra of the Cy3 donor and Cy5 acceptor. The green curve was generated from the MB donor in the presence of the target (515 nm excitation, Cy3 second peak excitation). The red curve was generated from the MB acceptor in the presence of the target (645 nm excitation, Cy5 excitation). The blue curve was generated from both donor and acceptor MBs in the presence of the target (515 nm excitation). The gray curve was generated from the MB donor in the absence of the target (515 nm excitation). The purple curve was generated from both donor and acceptor MBs in the absence of the target (515 nm excitation). The pink curve was generated from random dual MBs in the presence of the survivin target (515 nm excitation). The wine-colored curve was generated from PBS. (B) Solution studies of probe-target hybridization of dual FRET MBs. Fluorescence emission spectra of the FAM donor and the Cy3 acceptor under FAM excitation at 490 nm. The green curve was generated from the MB donor in the presence of the target. The red curve was generated from the MB acceptor in the presence of the target. The blue curve was generated from both donor and acceptor MBs in the presence of the target. (C) Western blot analyses of survivin protein expression in BCa cell lines (5637, T24, and 253J) and the normal cell line, HDF-a. GAPDH was the loading control.

Figure 3

Survivin mRNA detection in BCa cells incubated with survivin dual FRET MBs. (A) At the same excitation wavelength (488 nm), laser scanning confocal fluorescence microscopy images of 5637 cells incubated with the different MBs and HDF-a cells incubated with survivin dual FRET MBs. The fluorescence emission wavelengths were 520±20 nm and 560±20 nm, respectively (magnification×200). (B) The fluorescence microscopy images of 5637 cells incubated with different MBs and HDF-a cells incubated with survivin dual FRET MBs. The excitation filter range was 470-490 nm and the emission filter range was 580 nm long-pass IF (magnification×200). (C) The signal-to-background ratio of laser scanning confocal fluorescence microscopy images of 5637 cells and HDF-a cells. At an emission wavelength of 560±20 nm, the 5637 cells incubated with dual FRET MBs gave a much better signal-to-background ratio than other groups (^bP<0.05). (D) The signal-to-background ratios of fluorescence microscopy images of 5637 cells and HDF-a cells. The 5637 cells incubated with FRET MBs gave a much better signal-to-background ratio than other groups (^bP<0.05).

Figure 4

Survivin mRNA detection in BCa cell lines and exfoliated cells in urine from patients with BCa and healthy adults using fluorescence microscopy. Significant fluorescence signal was detected in 253J and T24 cells, and exfoliated cells from the urine of patients with BCa but not in exfoliated cells from the urine of healthy adults. L=light field, F=red fluorescence field (magnification×200).