Magnetic Nanoparticle Conjugated With MiR-155Molecular Beacon For The Diagnosis Of NSCLC

Background and objective: Lung cancer is the leading cause of cancer-related mortalityworldwide, and its total5-year survival rate is only about15%. NSCLC accounts for80%oflung cancer. Lack of effective methods for early diagnosis has greatly impacted on theprognosis and survival rate of cancer. Our groups always do some research on how to findearly diagnosis methods of lung cancer from the initiation of lung cancer. Cancer stem cells ortumor initiating cells possess strong capacity of self-renewal and multiplex differentiationpotential, and they are considered to be the main responsible for tumorigenesis, resistance totumor therapy, metastasis and recurrence. Besides a good potential treatment target, cancerstem cells can provide a new clue for early diagnosis of cancer. microRNAs (miRNAs) playimportant roles in the process of cancer occurrence and development. Detection of relatedmiRNAs in NSCLC stem cells is an important strategy for early diagnosis of lung cancer.Molecular beacon is a highly sensitive and specific detection tool, and it is designed on thebasis of fluorescence resonance energy transfer principles. It can be used in the study ofnucleic acid-the nucleic acid and visualization in living cells and then can provides thetechnical support for tumor diagnosis and in vivo imaging. However magnetic nanoparticle isan ideal molecular beacon carrier that can be used at the cell and organ level. At nanoscalemagnetic nanoparticles possess good controllability. Magnetic nanoparticles coated withPLGA have good biological compatibility and low toxicity. PEG modified magneticnanoparticles have a good long cyclical stability. NSCLC model is used in this study andNSCLC stem cells were sorted. The expression of mina of NSCLC was detected usingmolecular beacon and nanotechnology in vivo. It provides a new method for early diagnosisof lung cancer.Methods: CD133+CD338+lung cancer stem cells were sorted from A549cellsapplying immune magnetic bead isolation system. Then the stem cells were identified. The expression of related miRNAs was detected in lung cancer stem cells. MiR-155molecularbeacon and random sequence molecular beacon were designed and synthesized.Liquid-phase hybridization experiment was applied to test its specificity. Prostate cancercells (PC-3) was used as a positive control. In NSCLC living cells miR-155expression wasdetected via laser co focal microscopy and was verified by quantitative real time RT-PCR.Lung cancer engraft model was established in nude mice. MiR-155imaging in engrafttumor was observed using animal imaging system for in vivo imaging after local injectionsof miR-155molecular beacon. Complex of magnetic nanoparticles conjugated withmolecular beacons was prepared by chemical adsorption method. miR-155expression inNSCLC cells was detected after complex of magnetic nanoparticles conjugated withmolecular beacons transfection using laser confocal microscope. miR-155imaging inxenograft tumor was observed using in vivo imaging system after local injections and tailvein injection of complex of magnetic nanoparticles conjugated with molecular beacons.Results:1. CD133+CD338+NSCLC cancer stem cells were obtained using immune magneticbead isolation system. The proliferation ability of NSCLC stem cells is stronger than normallung cancer cells. CD338and CD133were expressed in NSCLC stem cells. The expressionsof some genes related to lung cancer stem cells (CD133, CD326, OCT-4, CD338, Nestin)were at least2folds higher than those A549cells.1×104CD133+CD338+NSCLC stem cellscan induce tumor in nude mice. The expression of miR-155in NSCLC stem cells was2.5folds higher than that in A549cells.2. Molecule beacons that were designed and synthesized had a high specificity vialiquid-phase hybridization. Strong green fluorescence in cytoplasm of A549cells andSPC-A1cells was detected after adding miR-155molecule beacons. The background is verylow. No obvious fluorescent signal was observed after adding random sequence molecularbeacons. The results of miR-155expression using quantitative real time RT-PCR wereconsistent with using laser confocal microscopy. Green fluorescent signal was very strongin xenografted tumors injected with miR-155molecular beacons. No positive fluorescencesignal was detected in xenografted tumors injected with random sequence molecular beacons.3. Synthetic complex of magnetic nanoparticles conjugated with molecule beaconswere uniform in size via transmission electron microscope. Green fluorescent signal inNSCLC cells was observed after complex of magnetic nanoparticles conjugated withmolecular beacons transfection using laser confocal microscope. Green or red fluorescentsignal was very strong in xenografted tumors after injecting complex of magneticnanoparticles conjugated with molecule beacons.Conclusion:1. CD133+CD338+NSCLC stem cells were obtained using immune magnetic beadisolation system. The related experiments verified stemness of CD133+CD338+NSCLCstem cells. High expression of miR-155was detected in CD133+CD338+NSCLC stem cellsby quantitative real time RT-PCR.2. Molecule beacons that were designed and synthesized had high specificity. Theycan be used to detect miR-155expression in living lung cancer cells and xenograftedtumors.3. Synthetic complex of magnetic nanoparticles conjugated with molecule beaconscould detect miR-155expression in living lung cancer cells and xenografted tumors. Thismethod will shed new insight into the early diagnosis of lung cancer.