ABSTRACT
The lentiviral vector was used for construction of a recombinant mediating RNA interference (RNAi) against Beclin1 gene in this study. Recombinant vector plasmid was transfected into non small cell lung cancer (NSCLC) A549 cells by liposome. PCR results showed that three amplified positive fragments were inserted into pRNAT-U6. 2/Lenti vectors. DNA sequencing results showed that the three recombinant lentivirus plasmids, pRNAT-U6. 2/Lenti-si356, pRNAT-U6. 2/Lenti-si423 and pRNAT-U6. 2/ Lenti-si684 were constructed successfully. After transfection with liposome, RT-PCR and Western blot analysis confirmed that the expression of Beclin1 mRNA and protein was inhibited in the three recombinant lentivirus plasmids transfected groups, and gene silencing efficacy was 35.56%, 89.22% and 66.78%, respectively. The results demonstrated that the lentiviral vectors of RNAi targeting Beclin1 gene were successfully constructed, and NSCLC A549 stable cell line with Beclin1 gene knockdown was established. This study finally provided a new cell model to explore the biological behavior of the Beclin1 gene in NSCLC A549 cells.
Subject(s)
Humans , Apoptosis Regulatory Proteins , Genetics , Autophagy , Genetics , Base Sequence , Beclin-1 , Carcinoma, Non-Small-Cell Lung , Genetics , Pathology , Cell Line, Tumor , Genetic Vectors , Genetics , Lentivirus , Genetics , Lung Neoplasms , Genetics , Pathology , Membrane Proteins , Genetics , Molecular Sequence Data , RNA Interference , RNA, Small Interfering , Genetics , TransfectionABSTRACT
Objective To establish human lung adenocarcinoma multidrug resistance cell lines in vitro,observe their biological characteristics,and investigate the mRNA expressions of DNA pol?,mdr 1,mrp1,GST-?,lrp and topo Ⅱ genes.Methods Paclitaxel-resistant cell lines(A549/TXL20) were established in vitro by exposure to stepwise increased concentrations of the drug in a cell culture medium.Biological morphology and cell cycles were analyzed by morphometry and flow cytometry.The chemoresistance indexes of cells were measured by methyl tetrazolium assay.Evaluation of growth and in vitro drug sensitivity were performed.RT-PCR was employed to analyze the mRNA expressions of the DNA pol?,mdr 1,mrp1,GST-?,lrp,and topo Ⅱ genes.Results ① Compared with parent cells,the resistant sublines had a lower confluent density.They were smaller and mixed with giant cells in different sizes and with different numbers of nucleoli,and the growth property of A549/TXL20 did not change significantly compared with A549 cell lines.② The resistant cells,A549/TXL20,were 19.3 times more resistant to paclitaxel and 67.4 times more resistant to cisplatin than the parent cells,and also demonstrated cross-resistance to mitomycin,vinblastine,and 5-fluouracil(5-FU). ③ Compared with the A549 celllines,an unreasonably higher level of drug resistance and lower drug concentration was detected in A549/TXL20 cells after exposure to the drug in the culture medium.④ The mRNA expression level of DNA pol?,mdr1,GST-?,mrp1 andlrp genes in A549/TXL20 cells was significantly higher than that in A549 cell lines(P