1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were purchased from Sahn Chemical Technology (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). TRIzol® reagent and a 100-bp DNA marker were purchased from Invitrogen (Carlsbad, CA, USA). Reverse transcription-polymerase chain reaction (RT-PCR) kit, Taq DNA polymerase and dNTPs were obtained from Takara Bio (Otsu, Japan). Primers were obtained from Invitrogen Trading (Shanghai, China). Goat anti-MMP-2 polyclonal antibody (sc-6838) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). HRP-conjugated AffiniPure rabbit anti-goat IgG (H+L) (SA-00001-4), mouse anti-β-actin monoclonal antibody (66009-1-Ig), and HRP-conjugated AffiniPure goat anti-mouse IgG (H+L) (SA-00001-1) were purchased from Proteintech (Rosemont, IL, USA). Polyvinylidene fluoride membrane, N,N′-methylenebis(acrylamide), sodium dodecyl sulfate, ammonium sulfate, N,N,N′N′- tetramethylethylenediamine, glycine, Whatman filter paper, non-fat milk powder, phenylmethanesulfonyl fluoride, trypsin, trishydroxymethyl-aminomethane (Tris), and Coomassie Brilliant Blue G-250 were purchased from Merck (Darmstadt, Germany). Recombinant MMP-2 protein was purchased from SinoBiological Inc. (Beijing, China). Human MMP-1 protein was purchased from PeproTech (Rocky Hill, NJ, USA). 4-Aminophenyl mercuric acetate (APMA) was obtained from InnoChem Technology (Beijing, China). All other chemicals were of analytic grade and used as received. Stock solutions (2.0×10⁻² M) of Fe³⁺, K⁺, Na⁺, Mg²⁺, Ca²⁺, Cu²⁺, Fe²⁺, Zn²⁺, HCO₃⁻, NO₃⁻, ClO₄⁻, F⁻, Br⁻, and amino acids such as L-tryptophan, L-serine, and L-aspartic acid were prepared in aqueous solutions. Stock solutions of 7-(diethylamino)-2-oxo- 2H-chromene-3-succinimidyl ester and MMP-2 probe (20 μM) for spectral measurements were prepared in dimethyl sulfoxide (DMSO):H₂O (1:1,000, v/v) solution. A stock solution of the MMP-2 probe for fluorescence imaging in cells was prepared in DMSO. TCNB buffer was comprised of 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl and 0.05% (v/v) Brij® 35 (with pH 7.5).

¹H nuclear magnetic resonance (NMR) and ¹³C NMR spectra were measured using an Ascend™ 400 spectrometer (Bruker, Billerica, MA, USA) and chemical shifts were reported as ppm, with tetramethylsilane as the internal standard. Mass spectrometric data were obtained using a Microtof-QIII™ mass spectrometer (Bruker). UV-vis absorption spectra were recorded using a UV2550 spectrophotometer (Shimadzu, Kyoto, Japan). Fluorescence spectra were measured using a FS5 fluorescence spectrometer (Edinburgh Instruments, Livingston, UK). Other instruments used were as follows: FV10SW confocal laser scanning microscope (Olympus, Tokyo, Japan), JY92-II ultrasonic cell crusher (Ningbo Xinzhi Biological Polytron Technologies, Ningbo, China), CO₂ incubator (model no. 5215-2; Shellab, Cornelius, OR, USA), StepOne™ real-time fluorescence quantitative PCR (qPCR) instrument (Applied Biosystems, Foster City, CA, USA), TE-70 semi-dry transfer film (GE Healthcare Life Sciences, Little Chalfont, UK), electrophoresis apparatus (model no. DYCP-31C; Liuyi Biotechnology, Beijing, China), and BioPhotometer® D30 nucleic acid protein analyzer (Eppendorf, Hamburg, Germany).

CaSki, SiHa, C33A and HeLa cervical cancer cell lines were obtained from the China Infrastruture of Cell Line Resources (catalog nos. 3142C0001000000829, 3142C0001000000089, 3142C0001000000132 and 3142C0001000000009, respectively). The cells were cultured in DMEM high glucose medium supplemented with 10% (v/v) FBS, 100 U/ml penicillin, and 100 U/ml streptomycin. Cells were cultured at 37°C in a humidified incubator with 5% CO₂.

Initially, 75 μl of MMP-2 (160 mM) and 75 μl of APMA (5 mM) were mixed and reacted in an oscillator at 37°C for 2 h to activate MMP-2. Then, 150 μl of activated MMP-2 (80 mM) was added to 150 μl of the MMP-2 probe (20 μM) and mixed in the oscillator at 37°C for 180 min. The test solutions were placed back into the oscillator at 37°C immediately after each measurement.

The total volume of each PCR reaction was 20 μl, which included 10 μl 2X SYBR® Premix Ex Taq™ II, 0.8 μl forward primer (10 μM), 0.8 μl reverse primer (10 μM), 0.4 μl ROX reference dye, 2 μl cDNA, and 6 μl ddH₂O. The reaction conditions were as follows: 95°C for 30 sec, then 40 cycles of 95°C for 5 sec and 60°C for 30 sec.

Total RNA was isolated from cells using TRIzol® purification. After denaturing RNA at 94°C for 5 min, 500 ng of RNA was transcribed into cDNA. Next, cDNA was amplified using the primers and target fragments. The amplification was performed using a thermocycler for 32 cycles according to the following program: 30 sec at 94°C, 30 sec at 58°C, and 30 sec at 72°C. Final extension was performed at 72°C for 10 min.

Firstly, 40 μg of total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 10–12% (v/v) gradient gel. Subsequently, proteins were transferred to polyvinylidene fluoride membranes and incubated with blocking buffer [phosphate- buffered saline (PBS) containing 5% (w/v) non-fat skim milk] for 2 h at room temperature. Then, the membranes were incubated with primary antibodies (goat anti-MMP-2) with gentle shaking overnight at 4°C, washed twice with PBS for 5 min, and further incubated with secondary antibodies for 2 h at room temperature. Finally, MMP-2 was detected using a chemiluminescence reaction. Band staining intensity was measured using BandScan 5.0 software. Protein expression levels were normalized to β-actin protein.

The MMP-2 fluorescence probe (2 μM) was dissolved in PBS. CaSki, SiHa, C33A and HeLa cells were inoculated into confocal culture dishes. After 24 h, the living cells, which were in the exponential phase of growth, were incubated with MMP-2 fluorescence probe for 120 min at 37°C. Then, the cells were washed 3 times with PBS and observed using confocal laser scanning microscopy.

The collected data were analyzed using SPSS 19.0 software (IBM, Armonk, NY, USA) by one-way analysis of variance. Multiple comparisons among groups were performed using Fisher's least significant difference test. A P-value of <0.05 was considered to indicate a statistically significant result.

The procedures for the synthesis of the MMP-2 probe are shown in Fig. 1. Coumarin-Osu was used as a fluorescent chromophore and Dabcyl-Osu was used as a quencher for the preparation of the fluorescent MMP-2 probe. Coumarin-Osu was prepared conveniently according to previous literature (34–36). The UV-vis absorption spectrum of Coumarin-Osu (10 μM) in DMSO:H₂O (1:1,000, v/v) solution exhibited a broad coumarin-based n-n* transition band around 445 nm. The fluorescence spectrum of the same solution showed a broad emission peak around 480 nm with strong green fluorescence. In contrast, Dabcyl-Osu, which shows maximum absorption at 480 nm, is greatly effective in restraining the blue to green emission spectra of various fluorescent dyes and has often been used as a quencher in other studies. The emission spectrum of Coumarin-Osu and the absorption spectrum of Dabcyl-Osu overlapped enough to provide a basis for the synthesis of a probe. Thus, a coumarin-based fluorescence FRET probe was prepared by coupling the fluorophore (Coumarin-Osu) with the quencher (Dabcyl-Osu) through a polypeptide chain (GPLGVRGKGG) that can be cleaved by MMP-2. The constructed probe was characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF) and high-pressure liquid chromatography (HPLC). MALDI-TOF: calculated for [M₊H]⁺: 1391.72, found: 1392.09. Purity (HPLC): 98.0533%. In the normal biological environment, FRET between the fluorophore and the quencher caused effective fluorescence quenching. However, upon cleavage of the polypeptide chain (GPLGVRGKGG) between G and V of the core MMP-2 substrate sequence PLGVR, the fluorophore and quencher were separated (Fig. 2) (37,38). Thus, the fluorescence of coumarin was recovered.

To explore the practicality of the designed probe for the measurement of MMP-2 in living systems, the dynamic fluorescence spectra of the probe (10 μM, DMSO:TCNB buffer = 1:1,000, v/v) which was incubated with MMP-2 were measured over time. As shown in Fig. 3, the fluorescence intensity of the probe itself was very weak in the beginning. However, it increased obviously with emission at 480 nm as time went on and then leveled off after ~2 h. The fluorescence intensity of the probe increased by ~10-fold (39). Selective experiments of the probe for the target molecule were carried out at lower concentrations for practical application. When other biologically relevant species such as MMP-1 (10 nM), K⁺, Mg²⁺, Na⁺, Fe³⁺, Cu²⁺, Zn²⁺, Ca²⁺, L-tryptophan, L-serine, L-aspartic acid, HCO₃⁻, NO₃⁻, ClO₄⁻, F⁻ and Br⁻ (all 10 mM) were incubated with the probe (20 nM) at 37°C for 2 h, no obvious changes in the fluorescence spectra were observed (Fig. 4). For practical application, it was necessary to investigate the limit of detection (LOD) and the detection range of the probe targeting MMP-2 by means of calibrating the fluorescence assay (40,41). The fluorescence titration of the probe (10 μM) upon the addition of activated MMP-2 (0.0, 0.5, 1.0, 2.0, 3.0, 4.5, 5.5, 7.0, 8.5 and 10 ng/ml) in TCNB buffer solution was performed (Fig. 5). Each test was conducted after the probe was incubated with MMP-2 for 2 h. The linear regression of the fluorescence intensity (F/F₀, at 480 nm) vs. the concentration of MMP-2 is shown in Fig. 5 inset. The probe featured an LOD for MMP-2 up to 0.05 ng/ml with the emission intensity increasing ~23%. Moreover, the fluorescence intensity was linearly correlated with the concentration of MMP-2 in the range of 0.05–10 ng/ml. The lower limit of LOD hinted a higher sensitivity to detect MMP-2, indicating the possibility of potent application for the early diagnosis of cervical carcinoma through collection of exfoliated cells from the vagina, and following regular cell culture technique to obtain massive cervical carcinoma cells. The cells can be used for MMP-2 fluorescence analysis via a fluorescent reader. Definitely a reliable control is required based on pathological criteria, which will be the focus of subsequent research by us.

qPCR was used to detect the expression of MMP-2 mRNA in the 4 cervical cancer cell lines for preliminary screening. Total RNA was isolated from cells using TRIzol® purification and first-strand cDNA was synthesized using a qPCR kit. Specific primers were as follows: MMP-2 sense, 5′-TGGGGCCTCTCCTGACATTGA-3′ and antisense, 5′-CACAGTCCGCCAAATGAACCG-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sense, 5′-AGAAGGCTGGGGCTCATTTG-3′ and antisense, 5′-AGGGGCCATCCACAGTCTTC-3′. The target fragments were 157 and 258 bp, respectively. The expression of MMP-2 was detected by fluorescence qPCR using cDNA as a template. The amplification plot and melting curve showed a single amplification product without non-specific amplification and without miscellaneous melting curve peaks. The results indicated that MMP-2 expression in cervical cancer cell lines was ordered as CaSki > SiHa > C33A > HeLa (Fig. 6). Each reaction was performed in triplicate and analyzed individually. The results were calculated using the 2^−ΔΔCt relative quantification method and normalized using HeLa cells as a reference control and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal standard (Table I).

RT-PCR was used to examine the expression of MMP-2 mRNA in the 4 cervical cancer cell lines. Specific primers were as follows: MMP-2 sense, 5′-TGGGGCCTCTCCTGACATTGA-3′ and antisense, 5′-CACAGT CCGCCAAAT GAACCG-3′, 157 bp; GAPDH sense, 5′-AATCCCATCACCATCTTCCA-3′ and antisense, 5′-CCTGCTTCACCACCTTCTTG-3′, 580 bp. PCR fragments were separated by electrophoresis on a 1.5% (w/v) agarose gel with GAPDH as an internal control. The results indicated that the relative MMP-2 gene expression levels in the 4 cervical cancer cell lines were ordered as follows: CaSki > SiHa > C33A > HeLa (Fig. 7). The PCR products were analyzed and data calculations were performed using GeneTools gel analysis software (Syngene, Cambridge, UK), and GAPDH was used as an internal control for data normalization (Table II).

Western blotting was used to investigate the expression of MMP-2 protein in the 4 cervical cancer cell lines. Western blot results revealed that the expression of MMP-2 protein in the 4 cervical cancer cell lines was ordered as follows: CaSki > SiHa > C33A > HeLa (Fig. 8 and Table III).

The mRNA and protein expression of the MMP-2 gene in 4 cervical cancer cell lines was examined by qPCR, RT-PCR and western blotting. The results illustrated that the expression of the MMP-2 gene differed among the 4 tested cell lines, which provided a basis for the cell imaging experiments. After the 4 cervical cancer cell lines were incubated individually with the fluorescence probe for 120 min at 37°C, fluorescence images were acquired using confocal laser scanning microscopy. The fluorescence intensities were in the following order: CaSki > SiHa > C33A > HeLa, which were consistent with the experimental results of qPCR, RT-PCR and western blotting (Fig. 9).

In conclusion, a coumarin-based FRET probe targeting MMP-2 was successfully designed, synthesized and used to measure MMP-2 expression in 4 cervical cancer cell lines. The cell lines showed green fluorescence with intensities in the order of CaSki > SiHa > C33A > HeLa. These results were consistent with those of qPCR, RT-PCR and western blotting. Taken together, the findings indicate that the fluorescence probe accurately measures MMP-2 expression in living cervical cancer cells. In vivo fluorescence imaging experiments and clinical trials are underway.