Construction of Lentiviral Vector for miR-217 Overexpression and Knockdown and Its Effect on CML
MOLECULAR BIOTECHNOLOGY(2022)
Abstract
We attempted to construct a myeloid leukemia cell strain for stable overexpression and knock-down of miR-217 and explored the possible mechanism underlying miR-217 in chronic myeloid leukemia (CML). MiR-217 overexpression and the knock-down lentiviral vector with puromycin resistance were constructed and packaged within recombinant lentivirus. Stably transfected K562 cells were obtained through puromycin screening, and the qPCR assay detected the relative expression of the target gene. The proliferation, apoptosis, and methylation level of PER2 within cultured cells were detected using the CCK-8 assay, flow cytometry, and TaqMan real‑time fluorescence quantitative methylation-specific PCR. qPCR and Western blot detected the expression of miR-217-related genes within the constructed K562 cell model. Colony PCR and sequencing proved that recombinant lentivirus expression vectors pSE16 and pSE17 were correctly constructed. The lentivirus titer was 2.95 × 10 9 and 2.61 × 10 9 IU/mL. The miR-217 expression level was high in pSE5316-K562 cells, and that of the miR-217 sponge was high in pSE5317-K562 cells. Overexpressed miR-217 could inhibit the K562 cell proliferation and induce apoptosis. Inhibition of miR-217 enhanced the expression of DNMT3A, decreased the PER2 expression, and elevated the degree of PER2 methylation. The miR-217 overexpression and knock-down of the K562 cell line were successfully constructed, providing a tool for further exploring the miR-217 mechanism in CML. DNMT3A could be the molecular target of miR-217 by regulating PER2 gene methylation and getting involved with the occurrence and development of CML.
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Key words
MiR-217,CML,Lentiviral,PER2,DNMT3A
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