横纹肌肉瘤中融合基因阳性和阴性细胞功能学及差异miRNA生物信息学分析

Functional and differential miRNA bioinformatics analysis offusion gene positive and negative cells in rhabdomyosarcoma

目的比较横纹肌肉瘤(RMS)融合基因阳性和阴性细胞的生物学行为,结合生物信息学分析差异miRNA,并在RMS细胞中验证筛选差异miRNA的表达。方法分别利用CCK-8、Transwell、流式细胞术检测RMS中融合基因阳性RH30细胞和融合基因阴性RD及PLA-802细胞的增殖、侵袭、迁移和凋亡能力;利用GEO2R分析融合基因阳性和阴性细胞的差异miRNA;利用Sangerbox绘制差异miRNA火山图,运用qRT-PCR验证筛选的差异miRNA。结果RD细胞的增殖能力高于RH30细胞和PLA-802细胞(P<0.05)。RH30细胞的侵袭、迁移和抗凋亡能力高于PLA-802细胞和RD细胞(P<0.05)。火山图结果显示上调miRNA(红色)多于下调miRNA(绿色)(P<0.05)。根据|log2FC|>3,P<0.05筛选得到12个差异miRNA(上调miRNA:miR-1、let-7d-5p和let-7b-5p,下调miRNA:miR-196a-5p、miR-455-3p、miR-21-5p、miR-193a-3p、miR-29b-3p、miR-29a-3p、miR-100-5p、miR-222-3p和miR-221-3p),qRT-PCR检测12个差异miR-NA的表达,其结果与生物信息学分析结果一致。结论RMS融合基因阳性细胞的侵袭、迁移、抗凋亡能力高于融合基因阴性细胞,融合基因阳性与阴性组间差异miRNA为RMS研究提供新的方向。

Objective To compare the biological behavior between rhabdomyosarcoma(RMS) fusion gene positive and negative cells, analyze differential miRNAs of RMS fusion gene positive and negative using bioinformatics, and verify the expression of differential miRNAs. Methods The viability, invasion, migration and apoptotic capacity of fusion gene positive RH30 cells and fusion gene negative RD and PLA-802 cells in RMS were detected by CCK-8, Transwell, and flow cytometry, respectively. GEO2 R was used to analyze the differential miRNA of the fusion gene positive and negative cells. Sangerbox was used to analyze differential miRNAs and made Volcano map. qRT-PCR was used to verify the selected differential miRNAs. Results The proliferation ability of RD cells was higher than that of RH30 cells and PLA-802 cells(P<0.05). The invasion, migration and anti-apoptotic ability of RH30 cells were higher than those of PLA-802 cells and RD cells(P<0.05). Volcano plot results showed that the number of up-regulated miRNAs was more than down-regulated miRNAs(P<0.05). According to |log2 FC|>3, P<0.05, 12 differential miRNAs were screened(up-regulated miRNAs contained miR-1, let-7 d-5 p and let-7 b-5 p, down-regulated miRNAs contained miR-196 a-5 p, miR-455-3 p, miR-21-5 p, miR-193 a-3 p, miR-29 b-3 p, miR-29 a-3 p, miR-100-5 p, miR-222-3 p and miR-221-3 p), and qRT-PCR results were consistent with bioinformatics analysis. Conclusion The ability of invasion, migration and resistant apoptosis in RMS fusion gene-positive cells is higher than that of fusion gene-negative cells. The differentially expressed miRNAs between fusion gene positive and negative groups can provide new directions for RMS research.