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Construction of Lentiviral Vector for miR-217 Overexpression and Knockdown and Its Effect on CML

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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 × 109 and 2.61 × 109 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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Data Availability

The data that support the findings of this study are available from the corresponding author on reasonable request.

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Funding

This work was supported by the major scientific and technological innovation projects of the Shandong Province of China (Grant No. 2019JZZY011018) and the Yantai Science and Technology Plan Project of China (Grant Nos. 2019MSGY133 and 2019YD004).

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Author notes

  1. Xin Yang, Huihui Jiang have equally contributed to this study.

Authors and Affiliations

  1. The Clinical Laboratory, Yantai Yuhuangding Hospital, 20 Yuhuangding East Road, Yantai, 264000, Shandong, China

    Xin Yang & Chengming Sun

  2. Clinical Laboratory, Qingdao Central Hospital, The Second Affiliated Hospital of Qingdao University, Qingdao, 266000, Shandong, China

    Huihui Jiang

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  1. Xin Yang
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All authors contributed to data analysis, drafting, and revising the article, gave final approval of the version to be published, and agreed to be accountable for all aspects of the work.

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Correspondence to Chengming Sun.

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Yang, X., Jiang, H. & Sun, C. Construction of Lentiviral Vector for miR-217 Overexpression and Knockdown and Its Effect on CML. Mol Biotechnol 65, 1253–1262 (2023). https://doi.org/10.1007/s12033-022-00615-9

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