Peptide Nucleic Acid Targeting Inhibit Mitochondrial Fusion Of Lung Cancer Stem Cells To Reverse The Cisplatin Resistance

Nearly, the incidence and mortality of lung cancer showed a trend of increasing year by year and has become a serious threat to people’s health as one of malignant tumors. Among them, the incidence of lung adenocarcinoma increased rapidly and has replaced the lung squamouscarcinoma as the most commom pathological types. Although chemotherapy is one of the most important means of lung cancer treatment, the effect of chemotherapy is still very limited.Now it is generally believed that the existence of lung cancer stem cells is the root that lead to lung cancer chemotherapy resistance, recurrence and metastasis.Mitochondria play a key role in cell apoptosis, they show continuous dynamic change in morphology and cell distribution. The mitochondria can meet the need of the energy in cell regions, maintain the stability of mt DNA and protect the intact of organelles structures by regulating the division and fusion of mitochondrial inner and outer membrance. The study found that cancer stem cells had more mitochondrial fusion than the other cancer cells. Mitochondrial fusion in cancer stem cells maybe helpful to tumor antiapoptotic and may become the real targets in clinical. Therefore, it has important clinical significance that targeted inhibit mitochondrial fusion of lung cancer stem cells to reverse lung adenocarcinoma cell cisplatin resistance and improve the survival rate. Whereas, it is still on vacancy about mitochondrial fusion phenomenon research of cancer stem cells. Therefore, this experiment was designed to observe the mitochondrial fusion phenomenon of lung cancer stem cells and compare apoptosis rate of lung cancer stem cells after using peptide nucleic acid targeted inhibit mitochondrial fusion. So that provide experimental basis for targeting therapy of lung cancer stem cells. Objective:1. To observe the expression of mitochondria fusion related proteins in lung cancer stem cells.2. To investigate the mitochondrial and energy metabolism-related features of CSCs.3. To explore the influence of peptide nucleic acid targeted inhibit mitochondria fusion of lung cancer stem cells.Methods: A549 cell spheres were formed through serum-free culture. PT-PCR and Western blot technique respectively detect the m RNA and protein expression of Mitofusin1, Mitofusin2, Opa1 and Atad3 a in A549 cell spheres and A549 cells. Flow cytometry instrument detect mitochondria membrane potential of A549 cells and A549 cell spheres; observing the distribution of mitochondria in A549 cell spheres and A549 cells by Laser Confocal Microscop(LSCM); Solid phase peptide synthesis peptide nucleic acid- three benzene phosphorus compounds. LSCM observate the transfection and identification of peptide nucleic acid- three benzene phosphorus compounds in lung cancer stem cells. RT-PCR and Western blot detecte the mt DNA gene expression lung cancer stem cells and western blot detecte the expression of mitochondrial fusion related protein Mfn1, Mfn2, Opa1 and Atad3 a after the transfection and interference of three benzene phosphorus compound; ATP Bioluminescence Assay Kit was used to analyze ATP content in A549 cell spheres and A549 cell after the transfection and interference of three benzene phosphorus compound; Flow cytometry test apoptosic rate and mitochondrial membrane potential in all groups.Result: The m RNA and protein expression of mitochondria fusion related protein Mfn1, Mfn2, Opa1 and Atad3 a in A549 cell spheres increased significantly; Mitochondrial distribution in A549 cell spheres around the nuclear and mitochondrial membrane potential in the A549 cell spheres are higher; After peptide nucleic acid-three benzene phosphorus compounds acted on mitochondria fusion in A549 cell spheres and A549 cell, the expression of mitochondrial fusion related protein Mfn1, Mfn2, Opa1 decreased,while the expression of Atad3 a is no change, at the same time the ATP content were all reduced, the cell apoptosis rate increased significantly and the mitochondrial membrane potential decreased obviously.Conclusion: 1.The m RNA and protein expression of mitochondria fusion related protein Mfn1, Mfn2, Opa1 and Atad3 a in A549 cell spheres increased significantly than in A549 cells, peptide nucleic acid- three benzene phosphorus compounds targeting inhibit mitochondrial fusion enhances the sensitivity of cisplatin chemotherapy. 2.Peptide nucleic acid- three benzene phosphorus compounds accumulation in A549 lung cancer cell spheres as their high ?ψm, closed gene expression of mt DNA double chain promoter common sequence, interfere with the energy metabolism, inhibition of mitochondrial fusion, inducing cell apoptosis, enhance A549 lung cancer cell spheres cisplatin chemotherapy sensitivity.