| Lung cancer is a highly heterogeneous malignancy which is a worldwide highly health-threatening issue.The WHO statistics show that nearly 165,000 people develop lung cancer every year,and accounting for nearly a quarter of all cancer related deaths.Among them,the lung adenocarcinoma take possession of the largest proportion in non-small cell lung cancer.Being different from lung squamous carcinoma which is highly associated with smoking,lung adenocarcinoma is highly incidental in non-smokers as well.SET,as a proto-oncogene that changes in the transcription and expression levels of various tumor cells,plays a key role in regulating many physiological processes in the organism.such as mitosis,RNA transcription,protein translation,chromosome duplication,etc.Bioinformatics methods,such as a variety of target gene prediction software were employed for combined prediction,found that mi R-21,miR-23 a and miR-199 b and many other kinds of miRNA target SET gene,the Pri-miRNA21/23 restructuring lentivirus vector were subsequently built,and successfully expressed in L-02 liver cells.But whether the miRNA can regulate biological activities on lung cancer cells through targeting SET gene has not yet been reported,our research aim at further studies from multiple perspectives of the mechanism that microRNAs regulate lung adenocarcinoma development through targeting SET.Methods: 1.Three kinds of miRNAs interact with SET had been screened in our previous research study,whose corresponding primers were currently designed and synthesised.Five normal pulmonary cells/lung cancer cells were cultivated to logarithmic phase,total cells RNA were extracted,following reverse transcription and real-time fluorescent quantitative PCR detection,and consequently figure out maximum differently expressed micrornas between the tumor cells and normal cells,thus making matting for the next step in-depth study.2.By applying bioinformatics methods,our study confirmed the relationship between miR-21-5p and SET 3'UTR,setting a solid foundation for the construction of corresponding vector for dual luciferase reporting system,which was later transfected into A549 cells,relative luciferase activity were estimated to further comfirm the relationship between miR-21-5p and SET.3.Build miR-21-5p stable interference lentivirus vector,transfection cell line A549 cells after get stable interference,extraction of total RNA,real-time fluorescent quantitative PCR,and from the level of the miRNA transfection virus validation.4.The biological effects of miR-21-5p inhibition on lung adenocarcinoma cell A549 were investigated by Western blot,cell lines,Transwell experiment and CCK8 experiment.The results of the above experiments can preliminarily explain the mechanism that miRNA regulate the development of lung adenocarcinoma by targeting SET.Results: The results of real-time fluorescence quantitative PCR showed that mir-21-5p expression in A549 was significantly higher than that in pulmonary epithelial cells BEAS-2B,and human embryonic lung cell WI-38(p<0.001).The results showed that mir-21-5p targeted SET3 'UTR.The construction of TUDE21 lentiviral vector is completed before successfully transfected to A549 cells and construct mir-21-5p stable interfered cell lines(p<0.001).The results of Western blot showed that the TAF1-alpha-subunit expression was absent in the normal A549 cells,while the TAF1-alpha-subunit in the A549 cells with stable interfered mir-21-5p has been restored to normal expression level,which is significantly upregulated by comparing with the control A549(p<0.05).The experimental results suggested that the proliferation(p<0.01),invasion(p<0.001)and migration(p<0.001)ability of mir-21-5p stable interfered A549 cells were significantly lower than that of normal A549 cells.Conclusion: Combined with all the above results,we believe that mir-21-5p can regulate the proliferation,invasion and migration of lung adenocarcinoma cells by targeting the TAF1-alpha-subunit of the targeted SET gene to some extent. |
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