The Research Of REST Gene On The Abilities Of Proliferation, Invasion And Migration Of Small Cell Lung Cancer

Objective:Lung cancer is one of the most common malignant tumors in China,ranking first in the incidence and mortality of malignant tumors.Among them,male patients are the most common,while female patients are second only to breast cancer.According to histological characteristics,lung cancer mainly includes two types:non-small cell lung cancer?NSCLC?and small cell lung cancer?SCLC?.Among them,small cell lung cancer?SCLC?has the characteristics of high malignancy,short multiplication time and extensive metastasis.New genes related to small cell lung cancer are screened and their pathogenesis and metastasis mechanism are explored.Potential targeted therapy genes are particularly important for the treatment of small cell lung cancer.REST/NRSF was discovered and named by Mandel and Anderson Laboratories in1995,namely,repressor element1-silencing transcription factor,inhibitor l-silencing transcription factor,neuron-restrictive silencer factor and neuron-restrictive silencer factor.In the initial study,REST/NRSF is a negative regulator of neuron formation,and its binding with target genes plays an important role in cell differentiation.Thereafter,it was found that REST/NRSF target genes were expressed in different degrees in both nerve and non-nerve tissues.In non-neuronal cells,REST/NRSF could inhibit the expression of nerve genes through histone deacetylation,chromatin remodeling and methylation,and participate in the transcriptional regulation of target genes.In recent years,researchers have found that abnormal expression of REST/NRSF is associated with various diseases,including cerebral ischemia,neurodegenerative diseases,epilepsy and tumors.Among them,the relationship between REST/NRSF and tumors has been studied most deeply.These studies show that REST/NRSF can participate in the occurrence and development of tumors as tumor suppressor and tumor promoter in different types of tumors.In this experiment,we constructed a lentivirus vector of REST gene by transfecting the virus into small cell lung cancer cell line H446 to study the effect of REST gene on the proliferation,invasion and metastasis of small cell lung cancer.Methods:Human small cell lung cancer cells NCI-H446 were cultured in 1640 complete medium containing 20%fetal bovine serum at 37?and 5%CO₂,and human normal lung epithelial cells BEAS-2B were cultured in 1640 complete medium containing 10%fetal bovine serum at 37?and 5%CO₂.The lentiviral vector expressing REST was constructed to produce lentiviruses and infect NCI-H446 cells.The experimental group and the empty vector control group were set up.Western blot was used to detect the expression of REST protein in human small cell lung cancer cell line NCI-H446,human normal lung epithelial cell BEAS-2B,NCI-H446 cells transfected with lentivirus over-expressing REST vector and NCI-H446 cells transfected with lentivirus empty vector.CCK-8 method,scratch test and Transwell invasion test were used to detect the proliferation,migration and invasion ability of cells in experimental group and control group.Statistical analysis:SPSS 20.0 statistical software was used for data analysis,Image J for image processing,GraphPad Prism5 for experimental results mapping and statistical analysis,P<0.05 was considered to have statistical significance.Results:Green fluorescence could be seen in the cells of small cell lung cancer transfected with lentiviral vector,so it was confirmed that the target vector had been successfully transfected into the cells and expressed.Western blot assay showed that REST protein was highly expressed in NCI-H446 cells compared with the control group.REST protein expression in BEAS-2B cells was higher than that in NCI-H446 cells;REST protein expression in lentivirus-transfected REST group was significantly higher than that in non-transfected H446 cells;REST protein expression in lentivirus-transfected REST group was significantly higher than that in lentivirus-empty vector group;REST protein expression in lentivirus-transfected REST group was higher than that in lentivirus-transfected REST Higher.CCK-8 experiment showed that the proliferation ability of small cell lung cancer cells in experimental group was significantly inhibited compared with the same kind of cells in control group.Scratch test showed that the migration rate of small cell lung cancer cells in the experimental group was significantly slower than that in the control group.Transwell invasion experiment showed that the number of cell penetrating membrane in experimental group was significantly less than that in empty carrier control group.Conclusion:1.REST gene is expressed at a low level in human small cell lung cancer NCI-H446 cell line,but higher in human normal lung epithelial cell BEAS-2B cell line,suggesting that the decreased expression of REST may be involved in the occurrence and progression of small cell lung cancer.REST gene plays an anti-oncogene role in small cell lung cancer.2.REST gene lentiviral expression vector transfected into human small cell lung cancer NCI-H446 cells can increase the expression of REST gene,inhibit cell growth,reduce its proliferation,invasion and migration ability.