Effects of downregulating circKIF4A on proliferation,invasion and stem cell-like properties of thyroid cancer cells and its mechanism
Objective To explore the effects of downregulating circKIF4A on proliferation,invasion and stem cell-like properties of thyroid cancer cells and its potential regulatory mechanisms.Methods Expression levels of circKIF4A in thyroid cancer cell lines,stem cell lines,and 30 paired samples of thyroid cancer and adjacent non-tumor tissues were measured using quantitative real-time PCR(qRT-PCR).Thyroid cancer cells were transfected with siRNA targeting circKIF4A to downregulate its expression,with siRNA transfections serving as controls.The effects of circKIF4A knock-down on cell proliferation,invasiveness,and stem cell-like properties were assessed using CCK-8 assays,Transwell as-says,and spheroid formation tests.Bioinformatics tools CircInteractome and TargetScan were utilized to predict miRNA that might bind to circKIF4A and their downstream target genes.Further,dual-luciferase reporter assays,qRT-PCR,and Western blotting were employed to verify the interactions between these molecules.Results Compared to normal thyroid epithelial cells,circKIF4A expression was significantly elevated in thyroid cancer tissues and cell lines.Knockdown of circKIF4A significantly reduced the proliferative and invasive capabilities,and the spheroid-forming ability of thyroid can-cer cell lines TPC-1 and KAT-5(P＜0.05).Nuclear-cytoplasmic fractionation experiments revealed that circKIF4A was predominantly located in the cytoplasm.miR-335-5p was upregulated in TPC-1 and KAT-5 cell lines with circKIF4A knockdown(P＜0.05).In cells co-transfected with miR-335-5p and wild-type circKIF4A,luciferase activity was signifi-cantly reduced compared to those co-transfected with wild-type circKIF4A and miR-NC(P＜0.05).Expression of KIF4A mRNA and protein was downregulated in cells transfected with miR-335-5p compared to those transfected with miR-NC(P＜0.05).Conversely,addition of a miR-335-5p inhibitor in cells transfected with si-circKIF4A led to an upregulation of KIF4A mRNA and protein expression(P＜0.05).Conclusions circKIF4A can enhance the proliferation,invasion,and sphere-forming ability of thyroid cancer cells by indirectly upregulating the expression of KIF4A protein through the regula-tion of miR-335-5p.
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