Establishment of BEAS-2B stable cell lines with lentiviral-based hsa-miR-100-3p inhibitor

Objective To establish the human bronchial epithelial (BEAS-2B ) stable cell lines with lentiviral-based hsa-miR-100-3p inhibitor and lay a foundation for the study of miR-100. Methods The reverse complementary sequence was designed based on the mature sequence of hsa-miR-100-3p. The purpose gene was amplified by polymerase chain reaction (PCR) and inserted directly into lentiviral plasmid vector GV280 after the double enzyme digestion, generatting GV280-hsa-miR-100-3p-inhibitor lentiviral expression vector. Then human embryo kidney epithelial cell (HEK-293) T cells was transfected with recombinant lentiviral plasmid along with two packaging vectors to produce virus particles. BEAS-2B cells were infected with the optimal titer of virus particles to obtain stable infected cell lines. At last, the infection efficiency was observed by fluorescence inverted microscope and the relative expression of miR-100 in BEAS-2B cells after transfecting with recombinant lentivirus was determined using reverse transcription-polymerase chain reaction (RT-PCR). Results DNA sequencing demonstrated that the lentiviral vector was made successfully. The target BEAS-2B cells were efficiently infected. More importantly, the expression of miR-100 in BEAS-2B cells was reduced significantly, measured by RT-PCR. Conclusions We successfully established the BEAS-2B stable cell lines with lentiviral-based hsa-miR-100-3p inhibitor to reduce the endogenous expression of miR-100 in BEAS-2B cells.