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

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 24hours,methyl thiazolyl tetrazolium (MTT) 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. Key words: RNA, small interfering;  Adrenomedullin;  Melanoma;  Cell line, tumor;  Flow cytometry