The therapeutic value of the SphK1-targeting microRNA-3677 in human osteosarcoma cells

Abstract

Sphingosine kinase 1 (SphK1) is a potential therapeutic target for human osteosarcoma (OS). SphK1-targeting microRNAs (miRNAs) could have important therapeutic value for OS. We discovered that micorRNA-3677 (miR-3677) is a SphK1-targeting miRNA, inhibiting OS cell progression. The results of RNA-Pull down assay confirmed direct binding between biotinylated-miR-3677 and SphK1 mRNA in primary human OS cells. In established and primary human OS cells forced overexpression of miR-3677, by a lentiviral construct, decreased SphK1 3'-UTR (untranslated region) activity and downregulated SphK1 expression. Both were however enhanced with miR-3677 inhibition in OS cells. Function studies demonstrated that OS cell growth, proliferation and migration were inhibited with miR-3677 overexpression, but augmented with miR-3677 inhibition. MiR-3677 overexpressioninduced anti-OS cell activity was reversed with re-expression of the 3'-UTR-depleted SphK1. Additionally, in SphK1 knockout OS cells (by CRISPR/Cas9 strategy), altering miR-3677 expression failed to further alter cell functions. Finally, we show that miR-3677 expression was significantly downregulated in primary human OS tissues, correlating with SphK1 mRNA upregulation. We conclude that targeting SphK1 by miR-3677 inhibits human OS cell progression.

MicroRNA-6862 inhibition elevates sphingosine kinase 1 and protects neuronal cells from MPP+-induced apoptosis

“…We further hypothesized that SphK1 knockout should mimic miR-6862 overexpression-induced actions. Therefore, a lentiCRISPR-GFP-SphK1-KO construct (from Dr. Yao at Nanjing Medical University [ 25 ]) was transduced to SH-SY5Y cells. Stable cells were established (koSphK1 cells, see Methods).…”

“…Briefly, SphK1 3’-UTR containing the putative binding sites of miR-6862(at position 113-120) was provided by Dr. Yao [ 25 ]. It was inserted into a firefly luciferase reporter vector pGL4.13 (luc2/SV40) [ 25 ].…”

“…This will cause the degradation and/or translation inhibition of targeted genes [ 23 , 24 ]. Several SphK1-targeting miRNAs have been identified, and many of them are in cancer cells [ 25 – 27 ]. Here we identifiedmicroRNA-6862 (miR-6862) as a novel SphK1-targeting miRNA.…”

Long noncoding RNA small nucleolar RNA host gene 15 deteriorates liver cancer via microRNA‐18b‐5p/LIM‐only 4 axis

“…In line with the guidebook of Pierce TM magnetic RNA protein pull‐down kit (Millipore, Bedford, MA), RNA pull down assay was implemented 17,18 . SMMC‐7721 cells were transfected with 50 nmol/L biotin‐labeled miR‐18b‐5p wild type (WT) or miR‐18b‐5p mutant (MUT).…”

“…In line with the guidebook of Pierce TM magnetic RNA protein pull-down kit (Millipore, Bedford, MA), RNA pull down assay was implemented. 17,18 SMMC-7721 cells were transfected with 50 nmol/L biotin-labeled miR-18b-5p wild type (WT) or miR-18b-5p mutant (MUT). At 24 hr post transfection, cells were rinsed with PBS and incubated with a specific cell lysate (Ambion, Austin, TX) and with magnetic beads coated with streptomycin (Sigma-Aldrich, St. Louis, MO).…”