Real-time Imaging Research Of Lung Cancer Cells Using MiR-155 Molecular Beacon Fluorescence Loaded Into Chitosan Nanoparticle

Background and ObjectiveLung cancer is the most common cancer in the world with the total 5-year survival rate of less than 15% and threaten seriously on human health. Operative approaches for early diagnosis of lung cancers could improve significantly total 5-year survival.New strategies based on cancer stem cell(CSC)theory were promising. CD133 was widely considered as classical CSCs marker and had been successfully used to isolate and identify CSCs in various solid cancers, including glioma, colon cancer, renal cancer, liver cancer and pancreatic cancer. Recently, Eramo A et al proved CD133 as new lung cancer stem cells(LCSCs)marker, which provided opportunities for design of early diagnosis strategies of lung cancer targeting to lung cancer stem cells. In addition, A body of researches suggested miRNAs playing an important role in maintenance of biological characteristics of lung epithelium stem cells (LSCs) and in occurrence and development of lung cancer were potent molecular signatures for early diagnosis of lung cancer. Accordingly, we presumed that new strategies for early diagnosis of lung cancer using dual targets including lung cancer stem cells and miRNAs were more potent.Therefore,we investigated CD133(+)LCSCs related-miRNAs differentially expressing in CD133(+)LCSCs through Real-time PCR in this study.Sensitive Real-time imaging of specific markers in intact cells were operative approaches for early diagnosis of lung cancers.Molecular beacon(MB)fluorescence designed according to fluorescence resonance energy transfer(FRET)was a sensitive and specific approach showing many advantages in Real-time imaging detection of expression of specific targets and studying interactions between biological molecules in intact living cells compared to conventional approaches such as Real-time PCR and fluorescence in situ hybridization(FISH). But few was reported about Real-time imaging detection of expression of miRNAs using molecular beacons so far.There were many difficulties applying molecular beacons in living cells similar to antisense oligonucleotides, including biostability and transfection efficiency. Some studies indicated that it was feasible to improve biostability of molecular beacons using protective carriers and prevent sequenstration in nucleus. Chitosan nanoparticle with superordinary properties of DNA protection and decaying release was promising molecular beacon carrier among various candidates. However, little was known if chitosan nanoparticle could be used as molecular beacons carrier so far.For thess proposes, has-miR-155 differentially over-expressing in isolating CD133(+)LCSCs was selected to designed and synthetized specific molecular beacon(miR-155 MB). miR-155 molecular beacon nanoparticle complexes were further prepared for Real-time imaging detection of expression of miR-155 gene in living SPC-A1 lung cancer cells, which settled foundation for Real-time imaging detection of expression of specific miRNAs in CD133(+)LCSCs.Methods1.CD133(+)LCSCs were identified in NSCLC tissues with different pathological types using CD133 monoclonal antibody, and were further enriched from NSCLC tissue specimens using immunomagnetic bead sorting. Expression of several lung cancer- related-miRNAs (hsa-miR-155, hsa-miR-205a, hsa-miR-191, hsa-miR-21, hsa-miR-210,hsa-miR-17-5p and hsa-miR-106a)According to reported results were further investigated in enriched CD133(+)LCSCs using Real-time PCR approach.2.Specific miR-155 molecular beacon targeting mature miR-155 and its precursors was designed and synthetized according to sequence of mature has-miR-155,and random sequence molecular beacon(RS MB)not perfectly complementary to any endogenous sequences was also synthetized as negative control. Specificity and thermodynamic of molecular beacons were evaluated by solution hybridization assay. Expression of miR-155 gene in fixed H446,SPC-A1 lung cancer cells and frozen NSCLC tissue sections was investigated using miR-155 molecular beacon and further verified through Real-time PCR approach. PC-3 prostatic carcinoma cells and Vero cells were used as control.3.Molecular beacon nanoparticle complexes were prepared using incubation procedure. Biostability of Molecular beacon nanoparticle complexes were investigated by DNase I protection assay and incubation assay with cell lysates. Transfection and Real-time imaging detection using molecular beacon nanoparticle complex was further performed in living SPC-A1 lung cancer cells and observed through laser confocal microscope. Random sequence molecular beacon and Vero cells were also used as negative controls to evaluate specificity of fluorescence signal generated in cells.Results1.Immunofluorescence analysis performed on NSCLC patient-derived tumor sections indicated the existence of extremely low percentage of CD133(+)LCSCs ranging from 0.11% to 1.2% in all 12 of NSCLC specimens analyzed and absence of CD133(+)cells within control lung tissues derived from normal tissue samples surrounding the tumor of the same patient. CD133(+)LCSCs were further enriched CD133(+)LCSCs from NSCLC tissue specimens using immunomagnetic bead sorting. Mature hsa-miR-17-5p and hsa-miR-155 differentially over-expressed in enriched CD133(+)LCSCs with about 1.44 and 1.13 fold increase respectively compare to CD133(-) cells by Real-time PCR.2. MiR-155 molecular beacon and RS molecular beacon showed perfect specificity and thermodynamic stability by solution hybridization assay. Strong red fluorescent signal was observed mainly in the cytoplasm of fixed H446, SPC-A1 lung cancer cells and NSCLC tissues with extremely low background similar to passive control PC-3 prostatic carcinoma cells after incubating with miR-155 molecular beacon. In contrast, no significant fluorescent signal was generated in cells of negative groups including H446 and SPC-A1 lung cancer cells and NSCLC tissue sections incubating with negative control random sequence molecular beacon and Vero cells incubating with miR-155 molecular beacon. Specific binding of miR-155 molecular beacon with its targets in lung cancer cells was further verified by Real-time PCR.3. Molecular beacon nanoparticle complexes were prepared using incubation procedure. Complete condense of molecular beacons with chitosan nanoparticle was determined at the WCS-NP/WODN ratio of above five by agarose gel electrophoresis and fluorescence retard array and physiochemical properties analysis indicate the complex was suitable for further transfection.DNase I protection assay indicated that molecular beacon nanoparticle complexes were protected from enzyme degradation. About 2- to 3-fold fluorescent enhancement was detected in naked molecular beacons solution compared to molecular beacons background after incubating with cytosolic fractions of cell lysates for 2 hours. In contrast, about 9-fold fluorescent enhancement was detected in naked molecular beacons solution compared to molecular beacons background after incubating with nuclear fractions of cell lysate at the same conditions,indicating nuclear protein influence significantly on conformation of molecular beacons produsing strong non-specific fluorescence. Molecular beacon chitosan nanoparticle complexes were further incubated with nuclear fractions of cell lysates for 2 hours at the same conditions and no significant fluorescent enhancement happened, implying chitosan nanoparticle prevented molecular beacon from binding with proteins. However, about 5- to 6-fold fluorescent enhancement was detected in molecular beacon chitosan nanoparticle complexes solution after incubating until 24 hours, indicating decayed release potential of chitosan nanoparticle. Strong fluorescence was observed in about 40~50% of mainly cytoplasm of transfected SPC-A1 lung cancer cells after further transfecting with miR-155 molecular beacon chitosan nanoparticle at the WCS-NP/WODN ratio of six, showing a high transfection efficiency and specificity. Weaker fluorescent signal in the nucleus of SPC-A1 cells compare in cytoplasm implied that using chitosan nanoparticle as carrier of molecular beacon was a feasible approach to control nuclear sequestration and decrease nuclear non-specific fluorescence.4. Researches on Real-time imaging detection of expression of miR-155 gene in xenograft tumors are in process.Conclusions1.Extremely low percentage of CD133(+)LCSCs ranging from 0.11% to 1.2% existed in NSCLC tissues. CD133(+)LCSCs were enriched from NSCLC tissues using immunomagnetic bead sorting. Mature hsa-miR-17-5p and hsa-miR-155 differentially over-expressed with about 1.44 and 1.13 fold increase respectively compare to CD133(-) cells by Real-time PCR.2. Specific miR-155 molecular beacon was successfully designed and synthetized, and was further used to detect expression of miR-155 gene in fixed lung cancer cells.3. Specific miR-155 molecular beacon incorporated into with chitosan nanoparticle was successfully deliver into living SPC-A1 lung cancer cells for Real-time imaging detection of expression of miR-155 gene, which set foundation for Real-time imaging researches of several specific miRNAs in living CD133(+)LCSCs based on molecular beacon fluorescence approach.