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Abstract
Introduction/Background*Uterine leiomyosarcoma (LMS) is notorious for its poor prognosis. New therapeutics strategy for LMS is mandatory. Kinesin family member4A (KIF4A) is a member of the kinesin4 subfamily and plays an important role in cell division. In recent years, KIF4A is revealed to plays significant roles in some cancers with tumor proliferation. The aim of this study is to investigate the role of KIF4A in LMS.
Methodology To identify novel biomarkers of LMS, we performed shotgun proteomics of one normal human uterine smooth muscle cell line (UtSMC) and three LMS cell lines, SK-LMS, SKN and SK-UT1, using isobaric tags for relative and absolute quantitation (iTRAQ). Cell variability was evaluated by MST-8 assay in original LMS cells and KIF4A suppressed cells with siRNA in vitro. For evaluation of proliferation in vivo, we established KIF4A knockdown cell lines using shRNA and injected them subcutaneously to 6weeks ICR nude mice and evaluated changes in tumor size over time. To elucidate the mechanism, we performed cell cycle analysis by fluorescence-activated cell sorting (FACS) and western blotting.
Result(s)*A total of 2084 proteins were identified using iTRAQ. KIF4A was identified as a protein with more than twice the expression level of normal smooth muscle cells. By western blotting, all three LMS cell line had expressed KIF4A. KIF4A downregulation significantly suppressed the growth of those cell lines in vitro (-37.2±4.73% in SK-LMS, -87.7±4.02% in SKN and -28.1±3.00% in SK-UT1, p<0.05). By FACS, the percentage of G2/M phase cells was significantly increased in KIF4A suppressed SK-LMS and SK-UT1 and by western blotting, p-cdc2 was upregulated in KIF4A suppressed cells (figure 1). It was suggested that KIF4A downregulation induced G2/M arrest by inhibiting dephosphorylation of cdc2. Also in vivo, downregulation of KIF4A was found to significantly suppress tumor size (1186±118mm3 vs 461±84mm3 in SK-LMS and 1704±441mm3 vs 514±230mm3 in SK-UT1, p<0.05, figure 2).
The expression of proteinsinvolved in G2/M checkpoint
The effect of suppressing growth in vivo
Conclusion*We identified a novel expressed protein, KIF4A, which can be a therapeutic target for uterine leiomyosarcoma.