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目的验证黑色素瘤患者与正常人血清中miR-21水平差异,明确其在黑色素瘤细胞凋亡中的作用,为探索黑色素瘤潜在的治疗靶标提供理论基础。方法分别采集黑色素瘤患者与正常人血清样本,提取microRNA后利用qRT-PCR分析miR-21在血清中的水平,同时采用ELISA法检测其靶基因PDCD4在血清中的含量。构建miR-21稳定抑制的黑色素瘤细胞株,进一步分析其对PDCD4表达的影响。采用流式细胞术分析miR-21对黑色素瘤细胞凋亡的影响。结果 miR-21在黑色素瘤患者血清中的表达是阴性对照组的(4.62±2.42)倍,其靶基因PDCD4表达是阴性对照组的(0.36±0.21)倍,差异均具有统计学意义(均P<0.01)。miR-21在黑色素瘤细胞中表达被抑制时,PDCD4在A-375和SK-MEL-1细胞中的表达分别是阴性对照组的(1.69±0.39)和(2.20±1.06)倍,细胞凋亡水平分别是空白质粒对照组的(2.82±0.24)、(2.26±0.23)倍,差异均具有统计学意义(均P<0.01)。结论 miR-21在黑色素瘤患者血清中异常升高,推测其能够抑制凋亡蛋白相关蛋白PDCD4的表达,进一步抑制黑色素瘤细胞凋亡,提示miR-21可能作为黑色素瘤治疗的潜在靶分子。
Objective To verify the difference of miR-21 levels in serum of melanoma patients and normal persons, and clarify its role in melanoma cell apoptosis so as to provide a theoretical basis for exploring potential therapeutic targets of melanoma. Methods Serum samples were collected from patients with melanoma and normal controls. The microRNAs were extracted and the levels of miR-21 in serum were detected by qRT-PCR. The levels of PDCD4 in serum were detected by ELISA. Construction of stable miR-21-suppressed melanoma cell lines, and further analysis of PDCD4 expression. The effect of miR-21 on the apoptosis of melanoma cells was analyzed by flow cytometry. Results The expression of miR-21 in the serum of melanoma patients was (4.62 ± 2.42) times of the negative control group, and the target gene PDCD4 expression was (0.36 ± 0.21) times of the negative control group, the difference was statistically significant (P <0.01). The expression of PDCD4 in A-375 and SK-MEL-1 cells was (1.69 ± 0.39) and (2.20 ± 1.06) folds of the negative control group, respectively, when the expression of miR-21 was suppressed in melanoma cells. (2.82 ± 0.24) and (2.26 ± 0.23) times respectively in blank plasmid control group, the difference was statistically significant (both P <0.01). Conclusion miR-21 is abnormally elevated in the serum of melanoma patients. It is speculated that miR-21 inhibits the expression of PDCD4 and further inhibits the apoptosis of melanoma cells, suggesting miR-21 may serve as a potential target for melanoma therapy.