Synthetic MIR143-3p Suppresses Cell Growth in Rhabdomyosarcoma Cells by Interrupting RAS Pathways Including PAX3–FOXO1

Abstract

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma most frequently found in children. In RMS, there are two major subtypes, embryonal RMS (ERMS) and alveolar RMS (ARMS). ARMS has the worse prognosis of the two owing to the formation of the chimeric PAX3–FOXO1 gene. A novel therapeutic method is required for treating ARMS. In our previous study, we found that the ectopic expression of chemically modified MIR143-3p#12 (CM-MIR143#12), which is RNase-resistant and shows the highest anti-proliferation activity among the synthesized MIR143 derivatives that were tested, induces significant cell growth suppression by targeting KRAS, AKT, and ERK in colorectal cancer cells. The expression of MIR143-3p in RMS was dramatically downregulated compared with that of normal tissue. Ectopic expression of CM-MIR143#12 in RMS cells resulted in a significant growth inhibitory effect through the induction of apoptosis and autophagy. Interestingly, we found that CM-MIR143#12 also silenced the expression of chimeric PAX3–FOXO1 directly and, using siR-KRAS or siR-AKT, that KRAS networks regulated the expression of PAX3–FOXO1 in ARMS cells. In ERMS harboring NRAS mutation, CM-MIR143#12 silenced mutated NRAS. These findings indicate that CM-MIR143#12 efficiently perturbed the RAS signaling pathway, including the ARMS-specific KRAS/PAX3–FOXO1 networks.

Expression of oncogenic HRAS in human Rh28 and RMS-YM rhabdomyosarcoma cells leads to oncogene-induced senescence

“…It is important to note that while an excess of RAS signaling was not tolerated in Rh28 aRMS cells, deficient RAS signaling was also detrimental. This is shown by the effect of dominant negative HRAS in the Rh28 cells, and others have also recently found that impaired RAS signaling suppresses aRMS cell and xenograft growth 59 , 60 . The changes in RAS protein expression we observed in response to ectopic expression of constitutively active RAS effectors in both Rh28 and RMS-YM cells likely reflects the homeostatic feedback loops that occur in RAS signaling 61 .…”

Understanding of cell death induced by the constituents of Taxus yunnanensis wood

“…Protein samples (1–5 µg/lane) were subjected to sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins in the gel were transferred to 0.45-µm polyvinylidene fluoride membranes (Immobilon-P Membrane, EMD Millipore, MA, USA) 12 . The membranes were blocked with PVDF Blocking Reagent for Can Get Signal (TOYOBO, Osaka, Japan) for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies at a 1:1000 dilution.…”

“…The membranes were then washed 3 times with PBS containing 0.1% Tween 20, incubated further with HRP-conjugated sheep anti-mouse or donkey anti-rabbit IgG antibody (Cell Signaling Technology [CST], Inc., Danvers, MA, USA) at room temperature, and then washed 3 times with PBS containing 0.1% Tween 20. The immunoblots were visualized using the Luminata Forte Western HRP substrate (EMD Millipore) 12 . Densitometry of the immunoblots was performed using Image J (version 2.0.0-rc-69/1.52p, NIH).…”

“…To detect mRNA, cDNA was reverse transcribed from 0.5 µg of the total RNA using the PrimeScript RT Reagent Kit (Takara, Shiga, Japan) 12 . The cDNA was amplified with specific primers for FAS (Forward, 5'-GGACCCTCCTACCTCTGGTT-3'; Reverse, 5'-ACCTGGAGGACAGGGCTTAT-3'), TRAIL (Forward, 5'-ATCATGGCTATGATGGAGGT-3'; Reverse, 5'-CTGTTCATACTCTCTTCGTC-3') and ACTB (Forward, 5'- GATTCCTATGTGGGCGACGA-3'; Reverse, 5'- AGGTCTCAAACATGATCTGGGT-3') by using Universal SYBR Select Master Mix (Applied Biosystems, Thermo Fisher Scientific, MA, USA).…”

“…Relative expression levels were quantified by the ΔCt method and normalized to ACTB . To detect miRNA, 25 ng of total RNA was reverse transcribed to cDNA using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) 12 . Expression levels of MIR34a were assessed by performing TaqMan MicroRNA Assays (Applied Biosystems).…”