Mechanism of miRNA-19a targeting the SMO gene regulates the hedgehog signalling pathway and affects the biological characteristics of myeloma cells
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摘要:
目的 探索miRNA-19a对多发性骨髓瘤细胞增殖、凋亡和转移的影响，及对SMO蛋白和Hedgehog信号通路的影响。 方法 人骨髓瘤细胞系U266B1、LP-1、RPMI 8226和NCI-H929为研究模型，首先检测4种细胞中SMO蛋白的背景表达，然后使用U266B1细胞作为模型，设计了miRNA-19a inhibitor，以此建立miRNA-19a敲低组、对照质粒组和空载组。探究miRNA-19a敲减后细胞中SMO蛋白含量以及细胞增殖、凋亡和转移的变化。采用MTT法检测细胞毒性，流式细胞术检测细胞凋亡情况，Transwell试验检测细胞侵袭能力。 结果 SMO蛋白在U266B1、LP-1、RPMI 8226和NCI-H929细胞系中表达量分别为0.925±0.057、0.889±0.067、0.904±0.075、0.507±0.048，其中以NCI-H929细胞系中表达量较低(P < 0.05)。MiRNA-19a敲低组、对照质粒组和空载组miRNA-19a含量分别为0.325±0.058、1.158±0.128、1.172±0.131，miRNA-19a敲低组miRNA-19a含量显著降低(P < 0.05)。对照质粒组、miRNA-19a敲低组和空载组SMO蛋白表达量分别为0.787±0.104、0.354±0.068、0.807±0.106，miRNA-19a敲低组SMO蛋白含量明显降低(P < 0.05)。MTT实验显示，miRNA-19a敲低组细胞增殖OD值在24~72 h内明显低于空载组和对照质粒组(均P < 0.05)。凋亡实验显示，miRNA-19a敲低组细胞凋亡率明显高于空载组和对照质粒组(均P < 0.05)。Transwell实验显示，miRNA-19a敲低组侵袭细胞数明显低于其他2组(均P < 0.05)。 结论 miRNA-19a/SMO蛋白可能是骨髓瘤细胞系U266B1中控制细胞恶性增长的关键信号，可以作为多发性骨髓瘤治疗的潜在靶点。 -
关键词:
- 多发性骨髓瘤 /
- 微RNA-19a /
- Hedgehog信号通路 /
- SMO蛋白
Abstract:Objective To explore the effects of miRNA-19a on the proliferation, apoptosis and metastasis of multiple myeloma cells, as well as on smoothened frizzled class receptor(SMO) protein and the hedgehog signalling pathway. Methods Human myeloma cell lines U266b1, LP-1, RPMI 8226 and NCI-H929 were used as the research models. Firstly, the background expression of SMO protein in four kinds of cells was detected. Using U266b1 cells as the model, the miRNA-19a inhibitor was designed, and the miRNA-19a knockdown group, control plasmid group and empty group were established. Changes in the SMO protein content, cell proliferation, apoptosis and metastasis were explored after miRNA-19a knockdown. Cytotoxicity was detected by MTT assay, apoptosis was detected by flow cytometry and cell invasion was detected by Transwell test. Results The expression levels of SMO protein in the U266b1, LP-1, RPMI 8226 and NCI-H929 cell lines were 0.925±0.057, 0.889±0.067, 0.904±0.075, and 0.507±0.048, respectively, whereas the expression level of SMO protein in the NCI-H929 cell line was low (P < 0.05). The miRNA-19a contents in the miRNA-19a knockdown group, control plasmid group and empty group were 0.325±0.058, 1.158±0.128 and 1.172±0.131, respectively. The miRNA-19a contents in the miRNA-19a knockdown group decreased significantly (P < 0.05). The expression levels of SMO in the control plasmid group, miRNA-19a knockdown group and empty group were 0.787±0.104, 0.354±0.068 and 0.807±0.106, respectively. The content of SMO protein in the miRNA-19a knockdown group decreased significantly (P < 0.05). MTT assay showed that the OD value of cell proliferation in the miRNA-19a knockdown group was significantly lower than that in the empty group and control plasmid group within 24-72 h (all P < 0.05). Apoptosis experiments showed that the apoptosis rate of the miRNA-19a knockdown group was significantly higher than that of the empty group and control plasmid group (all P < 0.05). Transwell experiments showed that the number of invasive cells in the miRNA-19a knockdown group was significantly lower than that in the two other groups (all P < 0.05). Conclusion miRNA-19a/SMO protein may be the key signal to control the malignant growth of cells in myeloma cell line U266b1. It can be used as a potential target for the treatment of multiple myelomas. -
图 1 4种细胞系中SMO蛋白的表达情况
注：与NCI-H929比较，aP < 0.05。
Figure 1. Expression of SMO protein in four cell lines
图 2 转染后各组细胞miRNA-19a mRNA的表达情况
注：与miRNA-19a敲低组比较，aP < 0.05。
Figure 2. Expression of miRNA- 19a mRNA in each group after transfection
图 3 敲减miRNA-19a后U266B1细胞系中各组细胞SMO蛋白表达量比较
注：与miRNA-19a敲低组比较，aP < 0.05。
Figure 3. Expression of SMO protein in U266B1 cell line after knockdown of miRNA- 19a
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