恶性黑色素瘤组织AURKA表达对细胞增殖和迁移的影响

Effects of AURKA expression on cell proliferation and migration in malignant melanoma tissue

目的探讨极光激酶A(AURKA)在恶性黑色素瘤组织中的表达以及对黑色素瘤细胞增殖、凋亡以及迁移的影响。方法收集2018-01-01-2021-01-01在宁夏医科大学总医院病理科储存的30例原位及转移性黑色素瘤组织,免疫组织化学法检测AURKA在正常皮肤组织、原位以及转移性黑色素瘤组织中的表达水平。分别选用来源于同一患者的原位黑色素瘤细胞WM115及转移性黑色素瘤细胞WM266-4,通过小干扰RNA(siRNA)干扰AURKA表达,将细胞分为阴性对照组(si-NC)和AURKA干扰组(si-AURKA#1、si-AURKA#2和si-AURKA#3)。进一步应用CCK8、划痕以及侵袭实验检测AURKA敲低后对细胞增殖、迁移和侵袭的影响,同时流式细胞术检测其对2种细胞凋亡和周期的影响。采用单因素方差分析法比较各组间差异,2组间比较采用t检验。结果AURKA在皮肤恶性黑色素瘤组织中呈现高表达。转染si-AURKA后WM115细胞si-NC、si-AURKA#1、si-AURKA#2、si-AURKA#3各组AURKA蛋白相对表达量分别为0.911±0.031、0.515±0.117、0.575±0.095和0.512±0.059,差异有统计学意义,F=16.09,P<0.01;WM266-4细胞si-NC、si-AURKA#1、si-AURKA#2、si-AURKA#3各组AURKA蛋白相对表达量分别为0.912±0.071、0.481±0.077、0.494±0.077和0.456±0.097,差异有统计学意义,F=31.19,P<0.01;其中转染si-AURKA#3组WM115以及WM266-4细胞AURKA的表达较si-NC组均降低,差异有统计学意义,均P<0.01。CCK8结果表明,敲低AURKA基因明显抑制了原位与转移性黑色素瘤细胞的增殖,差异有统计学意义,F_(WM115)=24.88,P_(WM115)<0.05;F_(WM266-4)=83.12,P_(WM266-4)<0.01。划痕实验表明,si-AURKA组划痕愈合率小于si-NC和Control组,差异有统计学意义,F_(WM115)=37.99,P_(WM115)<0.001;F_(WM266-4)=26.24,P_(WM266-4)<0.05。侵袭实验表明,si-AURKA处理后(WM115)、WM266-4细胞侵袭数明显少于si-NC和Control组,差异有统计学意义,F_(WM115)=16.20,P_(WM115)<0.01;F_(WM266-4)=6.97,P_(WM266-4)<0.05。细胞凋亡结果表明,WM115以及WM266-4细胞凋亡率si-AURKA组较Control和si-NC组明显增多,差异有统计学意义,F_(WM115)=117.30,F_(WM266-4)=37.77,均P<0.01。细胞周期实验检测表明,si-AURKA处理之后WM115、WM266-4细胞G_(2)/M期的细胞百分比较Control和si-NC组增多,差异有统计学意义,F_(WM115)=718.60,F_(WM266-4)=19.78,均P<0.01。结论AURKA在恶性黑色素瘤组织中高表达,敲低AURKA表达明显抑制原位以及转移性黑色素瘤细胞的增殖和迁移并阻滞细胞周期,提示其可能作为潜在评估指标,参与黑色素瘤的发生发展和转移。

Objective To investigate the expression of Aurora kinase A(AURKA)in malignant melanoma tissues and its role in the proliferation,migration and apoptosis of WM115 and WM266-4 cells.Methods All 30 cases of in situ and metastatic melanoma tissues stored in the Pathology Department of the General Hospital of Ningxia Medical University from 2018-01-01 to 2021-01-01 were collected.The expression levels of AURKA in normal skin tissues,in situ and metastatic melanoma tissues were detected by immunohistochemistry.In situ melanoma cell WM115 and metastatic melanoma cell WM266-4 from the same patient were selected to interfere with the expression of AURKA by small interfering RNA(siRNA).The cells were divided into negative control group(si-NC)and AURKA interference group(si-AURKA#1,siAURKA#2,si-AURKA#3).The effects of AURKAknockdown on cell proliferation,migration and invasion were further detected by CCK8,scratch and invasion experiments.At the same time,the effects on apoptosis and cell cycle were detected by flow cytometry.One-way analysis of variance was used to compare the differences between multiple groups,and the t-test was used for comparison between the two groups.Results AURKA was highly expressed in malignant melanoma tissues.After transfection of si-AURKA,the relative expression levels of AURKA protein in WM115cells si-NC,siAURKA#1,si-AURKA#2,si-AURKA#3in each group were 0.911±0.031,0.515±0.117,0.575±0.095and0.512±0.059,respectively.The difference was statistically significant,F=16.09,P<0.01;the relative expression of AURKA protein in WM266-4cells si-NC,si-AURKA#1,si-AURKA#2,si-AURKA#3in each group was 0.912±0.071,0.481±0.077,0.494±0.077and 0.456±0.097,the difference was statistically significant,F=31.19,P<0.01;the expression of AURKA in WM115and WM266-4cells in the transfected si-AURKA#3group was lower than that in the si-NC group,with a significant difference.all P<0.01.The CCK8experiment results showed that down-regulation of AURKAexpression significantly reduced the proliferation of WM115and WM266cells(F_(WM115)=24.88,P_(WM115)<0.05;F_(WM266-4)=83.12,P_(WM266-4)<0.01).The scratch experiment results showed that the scratch healing rate of the si-AURKA group was lower than that of the si-NC group and the control group,and the difference was statistically significant(F_(WM115)=37.99,P_(WM115)<0.001;F_(WM266-4)=26.24,P_(WM266-4)<0.05).Invasion assay showed that the number of WM115and WM266-4cell invasions after si-AURKA treatment was significantly less than that of the si-NC group and the control group,and the difference was statistically significant(F_(WM115)=16.20,P_(WM115)<0.01;F_(WM266-4)=6.97,P_(WM266-4)<0.05).The apoptosis results showed that the apoptosis rate of WM115and WM266-4cells in the si-AURKA group was significantly higher than that in the control group and the si-NC group,and the difference was statistically significant(F_(WM115)=117.30,P_(WM115)<0.01;F_(WM266-4)=37.77,P_(WM266-4)<0.01).Cell cycle test showed that the percentage of cells in G_(2)/M phase of WM115and WM266-4cells after si-AURKA treatment was higher than that of control group and si-NC group,the difference was statistically significant(F_(WM115)=718.60,P_(WM115)<0.01;F_(WM266-4)=19.78,P_(WM266-4)<0.01).Conclusions AURKA is highly expressed in malignant melanoma tissues.Knockdown of AURKAexpression could cause inhibition of cell proliferation and migration and cell cycle arrest of in situ and metastatic melanoma cells,suggesting that it may be used as a potential evaluation indicator to participate in the occurrence,development and metastasis of melanoma.