RNA干扰硒蛋白V基因对人恶性黑色素瘤细胞A375生物学行为的影响（英文）

发布时间：2018-11-20 08:45

【摘要】：目的体外观察sh RNA靶向干扰硒蛋白V(SELV)基因对人恶性黑色素瘤A375细胞生物学行为的影响。方法构建以SELV为靶基因的sh RNA表达载体,将其转染至体外培养的A375细胞中,并利用嘌呤霉素筛选出稳定转染细胞株。采用实时荧光定量反转录PCR(q RT-PCR)和Western blotting法分别检测转染细胞SELV的m RNA和蛋白表达水平;采用MTT法检测转染后细胞的增殖能力,并利用流式细胞术检测转染细胞的细胞周期和早期凋亡率。采用q RT-PCR和ELISA法对转染细胞的SELV功能相关因子含锚蛋白重复序列-细胞因子信号抑制物盒蛋白家族9(ASB9)和O-乙酰氨基葡萄糖转移酶(OGT)的表达情况进行检测。结果成功构建靶向SELV的sh RNA重组质粒,并获得稳定转染的A375细胞。转染后SELV的表达显著降低(P0.05),细胞增殖和细胞周期未发生明显变化,但细胞早期凋亡率显著增加(P0.05)。而转染细胞的ASB9和OGT表达量也明显增加(P0.05)。结论靶向SELV的sh RNA重组质粒能够下调SELV在A375细胞中的表达,提高细胞早期凋亡率,并导致ASB9和OGT的表达升高。
[Abstract]:Objective to investigate the effect of sh RNA targeted interference selenoprotein V (SELV) gene on the biological behavior of human malignant melanoma cell line A375. Methods the sh RNA expression vector targeting SELV gene was constructed and transfected into A375 cells in vitro. The stable transfection cell line was screened by purine mycin. The expression levels of m RNA and protein in transfected SELV cells were detected by real-time quantitative reverse transcription PCR (q RT-PCR) and Western blotting method respectively. The proliferative ability of transfected cells was detected by MTT assay, and the cell cycle and early apoptosis rate of transfected cells were detected by flow cytometry. Q RT-PCR and ELISA methods were used to detect the expression of ASB9 and Oacetylglucosaminyltransferase (OGT) (OGT) in SELV functional related factor containing anchor protein repeats (ASB9) and Oacetylglucosaminyltransferase (OGT) in transfected cells. Results the sh RNA recombinant plasmid targeting SELV was successfully constructed and stable transfected A375 cells were obtained. After transfection, the expression of SELV decreased significantly (P0.05), cell proliferation and cell cycle did not change significantly, but the rate of early apoptosis increased significantly (P0.05). The expression of ASB9 and OGT in transfected cells was also significantly increased (P0.05). Conclusion sh RNA recombinant plasmid targeting SELV can down-regulate the expression of SELV in A375 cells, increase the rate of early apoptosis and increase the expression of ASB9 and OGT.
【作者单位】：上海交通大学公共卫生学院;深圳市慢性病防治中心;
【基金】：supported financially by grants from the National Natural Science Foundation of China (No. 81172669, 81202239 and 81102123)
【分类号】：R739.5

【共引文献】