ABSTRACT
Background and purpose:MicroRNA(miRNA) is a class of small non-coding RNA playing an important regulatory role in many tumors. This study investigated which miRNA might negatively regulate the expression of Aurora-B in osteosarcoma cells, and to lay the foundation for the further investigation of the effort and regulation of Aurora-B in osteosarcoma malignant phenotype.Methods:Bioinformatics prediction software (http://www.targetscan.org) and luciferase assays were used to investigate which miRNA might target to modulate the Aurora-B. Real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) and Western blot assay were used to further verify which miRNA could negative regulate the expression ofAurora-B gene.Results:Bioinformatics prediction showed let-7 family have the possibility to modulate the expression of Aurora-B; Luciferase assays showed thatAurora-B might be the target gene of let-7a/b/c/d/e/f/g/i; RTFQ-PCR and Western blot analysis testiifed that both the expression levels of Aurora-B mRNA and Aurora-B protein were signiifcantly decreased in Let-7a/g/i up-regulated U2-OS and HOS cells, compared to the cells in the negative control group; but in Let-7b/c/d/e/f up-regulated U2-OS and HOS cells, the expression levels of Aurora-B mRNA and Aurora-B protein have no signiifcant difference, compared to the cells in the negative control group.Conclusion:Let-7a/g/i may downregulate the expression of Aurora-B in human osteosarcoma cells.
ABSTRACT
Objective To investigate the effects of down-regulating phosphorylated human epidermal growth factor receptor 2 (HER2) on the proliferation and metastasis in human osteosarcoma cells (U2-OS) in vitro. Methods Various concentrations of HER2 phosphorylation inhibitor lapatinib ditosylate (5, 10, 20, 30 and 40 μmol/L) were adopted to deal with U2-OS. MTT assay was performed to evaluate the cell proliferation during various times (24, 48 and 72 h), and the IC50 value in 24 h was calculated. The value of 10μmol/L (IC50=22.15μmol/L) was chosen to deal with U2-OS cells. The expres-sion level of phosphorylated HER2 (p-HER2) was measured by Western blot assay. The cell migration and invasion abilities were detected by Wound healing and Transwell invasion assays. Results The cell proliferation of U2-OS was significantly inhibited by HER2 phosphorylation inhibitor lapatinib ditosylate in a concentration- and time-dependent manner. During 24 hours, the p-HER2 level was significantly lower in lapatinib ditosylate group than that of negative control group (0.093± 0.033 vs 0.306±0.033), the cell migration rate was significantly lower in lapatinib ditosylate group than that of negative con-trol group (32.70%±3.00%and 94.52%±4.76%), and the trans-membrane cells were significantly lower than those of nega-tive control group (37/HP±5/HP and 85/HP±10/HP), respectively. Conclusion The down-regulating p-HER2 in U2-OS could efficiently inhibit the cell proliferation, migration and invasion in vitro. HER2 has the potential to become a molecular target for anti-osteosarcoma metastasis.
ABSTRACT
Objective High expression of the valosin-containing protein ( VCP) gene can enhance the metastasis of osteosar-coma via the AKT/PI3K/NF-KappaB/MMP-9 signaling pathway, but the molecular mechanisms underlying the up-regulation of VCP in osteosarcoma cells remains unknown .This study aimed to determine whether miRNA-129-5p can regulate the VCP expression and its targets in human osteosarcoma cells . Methods The microRNA target-predicting software TargetScanhuman 6.2 ( http://www.tar-getscan.org/) was used to predict the possible targets of miRNA-129-5p on the VCP gene.Then, two recombinant gene report vectors containing the wild VCP gene 3′UTR ( psi-VCP vector ) and mutant VCP gene 3′UTR ( psi-VCPmut vector ) were constructed , se-quenced, and identified.The human osteosarcoma U2-OS cells were co-transfected with miRNA-129-5p mimic and psi-VCP vector or psi-VCPmut vector, respectively.A non-specificity mimic transfection served as negative control , and the luciferase activity was detec-ted in each group. Results The software prediction showed only one conserved function site of miRNA-129-5p on the VCP gene 3′UTR163-169 bp.Luciferase activity was significantly lower in the psi-VCP vector +miRNA-129-5p transfection group (15.529 ± 1.902) than in the VCP control group (21.781 ±0.854), VCP mutation experimental group (19.978 ±1.377), and VCP mutation control group (21.952 ±1.516) (P<0.05), with no remarkable difference between the VCP mutation control and VCP control groups (P=0.276). Conclusion miRNA-129-5p can probably regulate the targets of the VCP gene in human osteosarcoma U 2-OS cells.