title: [The establishment of 5637 bladder cancer cell line with stable overexpression of miR-449c].
abstract: Objective To establish a human bladder cancer cell line that stably overexpresses miR-449c. Methods The DNA fragments encoding pre-miR-449c were amplified from genomic DNA templates of HEK293 cells using high-fidelity PCR and cloned into FtetUGW-T vector. The recombinant plasmid was verified by sequencing and applied for lentivirus packaging. Human bladder cancer 5637 cells were infected with the lentivirus and the infected cells after selection with puromycin were observed using an inverted fluorescent microscope for enhanced green fluorescent protein (EGFP) expression, real-time quantitative PCR for miR-449c expression and Western blotting for the expression of its downstream target C-myc. Results The FtetUGW-T/miR-449C lentivirus was successfully established. The human 5637 bladder cancer cells infected with the lentivirus expressed EGFP and overexpressed miR-449c. The expression of c-myc in the cells that stably overexpressed miR-449c was significantly reduced compared with control cells. Conclusion 5637 bladder cancer cell line that stably overexpresses miR-449c is successfully established.
authors.affiliation: PLA Institute for Urinary Surgery Research, First Affiliated Hospital, Third Military Medical University, Chongqing 400038, China.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Wan
authors.affiliation: Department of Cell Biology, College of Basic Medical Sciences, Chongqing 400038, China.
authors.forename: Yaqin
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Department of Cell Biology, College of Basic Medical Sciences, Chongqing 400038, China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Cell Biology, College of Basic Medical Sciences, Chongqing 400038, China.
authors.forename: Huanhuan
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Cell Biology, College of Basic Medical Sciences, Chongqing 400038, China.
authors.forename: Jiazhong
authors.initials: J
authors.lastname: Shi
authors.affiliation: Department of Cell Biology, College of Basic Medical Sciences, Chongqing 400038, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Yang
authors.affiliation: PLA Institute for Urinary Surgery Research, First Affiliated Hospital, Third Military Medical University, Chongqing 400038, China. *Corresponding author, E-mail:zhiwen@tmmu.edu.cn.
authors.forename: Zhiwen
authors.initials: Z
authors.lastname: Chen
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D008856:Microscopy, Fluorescence; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology
chemical_list: C544530:MIRN449 microRNA, human; C489427:MYC protein, human; D035683:MicroRNAs; D016271:Proto-Oncogene Proteins c-myc; D049452:Green Fluorescent Proteins