siRNA沉默肾上腺髓质素基因对黑素瘤细胞增殖和凋亡的影响

Impact of small interference RNA.induced silencing of adrenomedullin gene on the growth of and apoptosis in malignant melanoma ceils

目的探讨小干扰RNA（siRNA）沉默。肾上腺髓质素（ADM）基因对黑素瘤细胞系A375增殖和凋亡的影响。方法实时荧光定量-PCR（qRT-PCR）检测干扰效果，并选出干扰效果最好的siRNA。用筛选出的最有效siRNA转染A375细胞为实验组；转染非特异序列的细胞为阴性对照组，未转染的细胞为空白对照组。免疫细胞化学法蛋白水平检测干扰效果；噻唑蓝法（M1Tr）分iiJJN定沉默ADM基因24、48h后A375细胞增殖的变化；流式细胞仪（FCM）检测干扰ADM基因48h后A375细胞的凋亡情况。结果qRT-PCR显示ADM-siRNA-2干扰效果最好。免疫细胞化学结果显示转染ADM-siRNA-2后ADM蛋白的表达显著低于未转染组和阴性对照组。MTr结果显示，转染ADM-siRNA-2的细胞24、48h吸光度A值为0．389±O．0375，0．469±0．0330，低于空白对照组（0．574±0．0733、0．685±0．0154）和阴性对照组（0．548±0．0376、0．676±0．0374），差异均有统计学意义（P值均〈0．05），空白对照组和阴性对照组比较，差异无统计学意义（P值均〉0．05）。FCM检测显示转染ADM-siRNA-2组的细胞早期凋亡率为（20．200±6．046）％，高于空白对照组（1．667±0．340）％和阴性对照组（4．587±1．294）％，差异有统计学意义（P值均〈0．05），空白对照组和阴性对照组比较，差异无统计学意义（P值均〉0．05）。结论siRNA沉默ADM基因对A375增殖有抑制作用；而对凋亡则有明显促进作用。

Objective To evaluate the impact of small interference RNA （siRNA）-induced silencing of adrenomedullin （ADM） gene on the growth of and apoptosis in a malignant melanoma cell line A375. Methods Three siRNAs targeting ADM gene were constructed and transfected into A375 cells. Real-time quantitative reverse transcription PCR （qRT-PCR） was performed to measure the expression of ADM to select the most efficient siRNA for the next experiment. A375 cells were classified into 3 groups to be transfected with the selected siRNA （test group）, non-specific sequences （negative control group）, or remain untransfected （blank control group）. Then, an immunohistochemical SP method was used to determine the expression of ADM at 24 hours, methyl thiazolyl tetrazolium （MTF） assay to detect the proliferation of A375 cells at 24 and 48 hours, flow cytometry to observe cell apoptosis at 48 hours. Results qRT-PCR showed that ADM-siRNA-2 was the most efficient. The expression of ADM was significantly lower in the test group than in the other two groups. The proliferation level （represented as the absorbance value at 490 nm） of A375 cells was 0.389 0.0375 and 0.469 ± 0.0330 in the test group at 24 and 48 hours respectively, significantly lower than that in the negative control group （0.548 ± 0.0376, 0.676 ± 0.0374, both P 〈 0.05） and blank control group （0.574 ± 0.0733, 0.685 ± 0.0154, both P 〈 0.05）. Flow cytometry revealed a statistical increase in early apoptosis rate in the test group compared with the blank control group and negative control group （（20.200 ± 6.046）% vs. （1.667±0.340）% and （4.587 ± 1.294）%, both P 〈 0.05）. No significant differences were observed in the proliferation level or early apoptosis rate between the negative control group and blank control group （both P 〈 0.05）. Conclusion The siRNA-induced silencing of ADM gene can inhibit the proliferation of, but promote the apoptosis in, A375 cells.