The Preliminary Study On The Effect Of Blocking The Expression Of MtDNA On Lung Cancer Cell/lung Cancer Stem Cell By Antisense Peptide Nucleic Acid

Lung cancer is one of the common carcinoma. Due to bad habit of life, environment pollution, etc,the mobility of lung cancer is increasing(465 percent increasing in China).The mobility and mortality of Lung cancer is already becoming first in carcinoma[1].However, the effect of clinic synthetic treatment to lung cancer is not satisfied, the five-year survival is less than fifteen percent[2]. Therefore, finding new targets and methods of treatment is urgent.Mitochondrion is the most important organelle providing energy, support and join in kinds of important function in cells, such as mediate metabolism, keep steady state of ion, proliferation and apoptosis of cells. As only one of ex-nucleus hereditied material in eukaryon, mitochondrial DNA(mtDNA) code thirteen polypeptides related to energy metabolism. The damage and transcriptional disturbance of mitochondrial will reduce the expression of polypeptides, decent the efficiency of transmit of electron chain, open the Permeability transition poreand induce to apoptosis. The change of structure and function of mitochondrion can interfere the growth, energy metabolism and proliferation of tumor cells.Besides, the apoptosis and necrosis of cells will also be primed[3]. Accordingly, target to metabolism of lung cancer cell is new direction to explore. If we can apply anti-sense technology and anti-sense matter, specifically the transcription of closed lung cancer cell mtDNA promoter may result in mitochondrial protein synthesis system disorder, which led to mitochondrial electron transport chain of barrier, thereby affecting the survival of lung cancer cells and normal function of maintained.This study 1. To observe and compare mitochondrion membrane potential in kinds of lung cancer cells and normal human epithelial cells, and confirm the mitochondrion membrane potential of lung cancer cell was higher than normal human bronchial epithelial cell. 2.To build an anti-sense peptide nucleic acid (Anti-sense peptide nucleic acid, asPNA, asPNA) - triphenyl phosphate complex, the use of electronic shift lipophilic cationic substances (delocalized lipophilic cations, DLCs) - triphenyl phosphorus, can be selective accumulation of lung cancer cells mitochondria in high mitochondrial membrane potential, carrying mtDNA heavy chain transcription for the promoter region of asPNA, inhibition transcription of lung cancer mtDNA, to observed in lung cancer cellular energy production, apoptosis / necrosis and other biological characteristics.Course of the study, we found that mitochondrial membrane potential exist significant heterogeneity in lung cancer cell, and this heterogeneity has a link with the degree of lung cancer cell differentiation, means lung cancer stem cell subsets was the high mitochondrial membrane potential than the differentiation of lung cancer cells. 3. we have the separation and identification of lung cancer stem cells, and observations biological properties of sub-groups after the asPNA-triphenylphosphine complex transfection of lung cancer stem cells.Mtheods:1. We were detected and compare different lung cancer cell line (SPC-A1, H446) and normal small airway epithelial cells (Human Bronchus Epithelium, HBE) of the mitochondrial membrane potential with flow cytometry and confocal microscopy.2. Designed and synthesized asPNA-Triphenyl phosphate complex using solid-phase synthesis, high-performance liquid chromatography and mass spectrometry for the identification, observe the transfection to SPC-A1cell under Confocal laser scanning microscope.The expression of mtDNA heavy chain gene encoding ATP synthase 8 (ATPase8) after complex transfected SPC-A1 cells examined by RT-PCR, to detection ATP synthesis changes by using luminometer. The cell cycle and apoptosis / necrosis of the transfected cells were also assessed by using flow cytometry respectively.3. To observe the effects of different tumor cell lines (A549, SPC-A1, H446, SGC, Hela) cloning of heterogeneity, and detection the cells of different clones of mitochondrial membrane potential by laser confocal microscopy. Immunofluorescence assay stem cell marker expression in SPC-A1 cells clones; observe the distribution of SP cells and the distribution of mitochondria in different clones by laser confocal microscopy.4. We were cultured lung cancer stem cell ball in SPC-A1with an improved serum-free stem cell culture method; immunofluorescence assay CD133 expression of lung cancer stem cell ball; detection of nude mice tumorigenicity for lung cancer stem cells; comparison lung cancer stem cell ball and differentiation of SPC-A1 cells of the mitochondrial membrane potential with flow cytometry.5. To observe the effect of the asPNA-triphenyl phosphate complex transfected lung cancer stem cells by using laser confocal microscopy, and detection of nude mice tumorigenicity for asPNA-triphenyl phosphate complex transfected lung cancer stem cells.6. Statistical analysis: The results are expressed as means±SE, that among the groups using t tests to SPSS12.0 software for analysis. P <0.05 for significant difference.Results:1. The mitochondrion membrane potential of lung cancer cell was higher than normal human asPNA-triphenyl phosphate complex epithelial cell.The flow cytometry measurment showed that the mitochondrion membrane potential,which was reflected by fluorescence intensity,either SPC-A1(266.9±24.4, P < 0.01) or H446(194.4±17.0,P < 0.01) was higher than HBE (138.1±19.7) ). The laser scanning confocal fluorescence microscope measurment showed that the mitochondrion membrane potential,which was reflected by fluorescence intensity,either SPC-A1(1.90±0.06,P < 0.01) or H446(1.91±0.03,P< 0.01) was higher than HBE (1.72±0.02).2. The quality of asPNA-triphenyl phosphate complex d meeted the criterion.The high pressure liquid chromatography measurement of asPNA-triphenyl phosphate complex compound showed a single spike in 22.1 min ,which was as same as the standard preparation .The designed molecular weight of asPNA-triphenyl phosphate complex is 3979.9. After the addition of electric charge,the mass chromatographic analysis was performed,which showed that the molecular weitht of the compound was 3982.2, which showed that the quality of asPNA-triphenyl phosphate complex meeted the criterion.3. The asPNA-triphenyl phosphate complex were transduced into mitochondrion of SPC-A1 cell and induced the expected biological effects.The laser scanning confocal fluorescence microscope measurment showed that asPNA-triphenyl phosphate complex were located in cytoplasm.The morphous and the spatial distribution were as same as mitochondrion,which indicated that asPNA-triphenyl phosphate complex can be transduced into SPC-A1 mitochondrion. The RT-PCR analysis showed that the transcriptional level of ATPase8 of asPNA-triphenyl phosphate complex transduced SPC-A1 cells.The flow cytometry analysis showed that the apoptosis rate of asPNA-triphenyl phosphate complex transduced SPC-A1 cells was increased (11.83 %±0.66 % vs 2.56 %±0.71 % ) asPNA-triphenyl phosphate complex can be transduced into SPC-A1 mitochondrion.The results showed that ATP synthesis of asPNA-triphenyl phosphate complex transduced SPC-A1 cells was decreased (1.2049±0.1122 nmol/mg vs .1983±0.1570 nmol/mg P< 0.01).4. The mitochondrion membrane potential of tumor cells showed heterogeneity,which maybe associated with cell differentiation.The measurements of laser scanning confocal fluorescence microscope in defferent clone types tumor cell lines showed that the mitochondrion membrane potential of compacted clone type tumor cells were higher than loose clone type(compacted clone type of A549:854.5±71.96,loose clone type of A549:618.9±62.2,P< 0.01;compacted clone type of SPC-A1:2029.8±45.9,loose clone type of SPC-A1:1016.6±104.5,P< 0.01;compacted clone type of H446:1175.6±115.3,loose clone type of H446:848.2±93.8,P< 0.01;compacted clone type of SGC:1498.9±169.2,loose clone type of SGC:912.3±67.1,P< 0.01;compacted clone type of Hela:835.9±98.6,loose clone type of Hela:531.2±44.8,P< 0.01).The compacted clone type of lung cancer cells characterized some stem cell property,including the expression of some the stem cell marker and the near nucelus distribution of mitochondrion.5. The stem cell balls was sucessfully cultured and identified preliminarily.The lung cancer stem cell balls was sucessfully cultured with modified serum-free medium and identified by the stem cell marker CD133.The stem cell balls appeared a stronger tumorigenic capability.6. he mitochondrion membrane potential of lung cancer stem cell balls was higher than SPC-A1 cells.The flow cytometry measurment showed that the mitochondrion membrane potential of lung cancer stem cell balls was higher than SPC-A1 (84.45±12.54 vs 53.53±3.35,P<0.01 ).7. The asPNA-triphenyl phosphate complex compound can be transduced into mitochondrion of lung cancer stem cells and influence the tumorigenic capability.The laser scanning confocal fluorescence microscope detection showed that asPNA-triphenyl phosphate complex were located in cytoplasm.The morphous and the spatial distribution were as same as mitochondrion.The fluorescence was near nucelus distribution ,which was accordance with stem cell mitochondrion.These indicated that asPNA-triphenyl phosphate complex compound can be transduced into lung cancer stem cell mitochondrion.Conclusions:1. The mitochondrion membrane potential of lung cancer cell was higher than normal human asPNA-triphenyl phosphate complex epithelial cell.2. The asPNA-triphenyl phosphate complex can be transduced by high membrane potential into lung cancer cell mitochondrion, and inhibited the expression of mitochondrion coding genes and induced apoptosis by the interfering of cell energy metabolism.3. The mitochondrion membrane potential of lung cancer cell colony showed heterogeneity,which was negative correlation with cell differentiation.4. The asPNA-triphenyl phosphate complex can be transduced by high membrane potential into lung cancer stem cell mitochondrion,and inhibited the tumorigenic capability.