Effects Of Nifeviroc Combined With CCR5 Gene Silencing Or Overexpressing On Proliferation Of Lung Cancer A549 Cells

BackgroundLung cancer is the most common malignant tumor in the world,with 1.8 million new cases and 1 million deaths every year.As a worldwide public health problem,its high fatality rate has been paid much attention in the world medical organization.Lung cancer is divided into two subtypes according to its pathology and histology,non-small cell lung cancer(NSCLC,including squamous cell,large cell and adenocarcinoma)and small cell lung cancer(SCLC).Among them,non-small cell lung cancer accounts for 80% ~ 85% of lung cancer,and the 5-year survival rate is less than 15%.The clinical manifestations of NSCLC are relatively complex.The early symptom phenotype of NSCLC has no outstanding distinguishing indicators,and the patients even have no abnormal detection.Therefore,early diagnosis is not easy in clinical practice.About 50% of patients have shown signs of distant metastasis at the early stage of diagnosis.For NSCLC patients with distant metastasis,comprehensive treatment mainly based on chemotherapy and supplemented by other methods is adopted.Although it can play a positive role in the survival period of advanced NSCLC patients,the chemotherapy has poor targeting in the process of killing tumor cells,causing heavy damage to normal cells in the body.Especially great damage to white blood cells,metabolic organs liver,kidney in the metabolic process will also be subjected to a great degree of pressure.Especially great damage to white blood cells,metabolic organs liver,kidney in the metabolic process will also be subjected to a great degree of pressure.In addition,the prognosis of most patients was not ideal,and the Overall Survival rate(OS)was generally low.In recent years,with the development of medical molecular biology technology,molecular targeted therapy has become an important treatment method in advanced NSCLC and has been included in the NCCN(National Comprehensive Cancer Network)guidelines.New molecular-targeted drugs targeting NSCLC are widely favored by researchers.Through specific targeted interventions,the survival benefits of NSCLC patients are significantly improved,and the combination with other therapies or adjuvant therapy opens a new chapter in the treatment of NSCLC.Nifeviroc,a small molecule compound CCR5 antagonist,can inhibit viral fusion and entry by interfering with the combination of virus and CCR5,thus effectively inhibiting HIV-1 infection.Its anti-tumor effect has been found in clinical research and development of anti-HIV drugs.Modern medical research has shown that the occurrence and development of tumors are often accompanied by abnormal changes in cell biological behavior.Clinical studies have shown that the CC Chemokine(c-c motif)receptor 5(CCR5)is abnormally expressed in NSCLC patients and has the potential to be a predictor of poor prognosis.High expression of CCR5 affects tumor microenvironment and tumor cells by binding to its ligand,suggesting that CCR5 may have tumor promoting function.Therefore,small molecule antagonists targeting CCR5 are expected to play an important role in the treatment of NSCLC.ObjectiveBased on previous research results,we used computer-assisted drug design in this study to further the docking mode of Nifeviroc and CCR5 protein receptors.To investigate the role of CCR5 gene knockdown and overexpression in the proliferation and cell cycle of A549 cells,and jointly explore the effects on NSCLC proliferation and apoptosis with Nifeviroc.And further analysis confirmed that CCR5 may be an important target for NSCLC treatment.It provides theoretical support for the preliminary exploration and development of molecular targeted drugs against NSCLC.Contents1.Research on CADD of CCR5 antagonist: with CCR5 as the target,the docking mode of small molecule antagonist Nifeviroc and CCR5 protein receptor was explored through the Libdock module in Discovery Studio2018 software and surflex-dock module in sybly-2.1 software.2.CCR5 silent and overexpressed stable cell lines were constructed by lentivirus vector and transfection efficiency was measured.The transfected A549 cells were treated with CCR5 overexpression and shRNA interfering lentiviral vectors.Real-time PCR and Western Blot were used to detect the transfection efficiency of overexpressed and silenced A549 cells from gene and protein levels,respectively.3.Effect of Nifeviroc combined with overexpression and silencing of CCR5 gene on malignant proliferation of A549 NSCLC cells.CCK8 assay,cell clone formation assay,EdU assay,Cell cycle detection by flow cytometry PI staining,and Western Blot were used to compare the proliferation inhibition of overexpressed,silent A549 cells treated with Nifeviroc.In vitro experiments were conducted to explore the targets of Nifeviroc.4.Nifeviroc combined with overexpression and silencing of CCR5 gene on A549 cell apoptosis of NSCLC.Apoptosis was observed by Hoechest staining;changes in apoptotic proteins were detected by Western Blot;Annexin V-APC / 7-AAD staining by flow cytometry was used to compare the apoptosis of the control group with that of overexpressed and silent strains.Results1.The docking score of the small molecule antagonist Nifeviroc with the CCR5 protein receptor was higher than that of the positive control Maraviroc.Nifeviroc can form a stable structure in the form of hydrogen bonds with TRP-86 and GLU-283 amino acid residues in the CCR5 protein receptor.This shows that the structure is stable and the docking is good.2.Lentiviral vector was successfully transfected to construct a silent,over-expressed A549 stable transfected cell line.The CCR5 nucleic acid and protein expression levels were reduced or increased to varying degrees.3.Small molecule antagonist Nifeviroc can make A549 cells antagonize CCR5:(1)decreased survival rate(CCK8 test);(2)decreased independent viability(plate clone formation test);(3)reduced number of cells that undergo DNA synthesis(EDU detection);(4)Achieve cell cycle arrest(flow cytometry / WB analysis),thereby inhibiting A549 cell proliferation.4.Hoechest staining observation,cell flow cytometry,and related pro-apoptotic protein WB analysis have preliminary confirmed that the small molecule antagonist Nifeviroc can promote cell apoptosis by antagonizing CCR5.