微小RNA-363-3p靶向调控PTEN及对黑色素瘤细胞侵袭迁移的影响  

Targeted regulation of PTEN by microRNA-363-3p and its effect on invasion and migration of melanoma cells

作　　者：纪新尊 程小珍 孙洋[1] 纪定文 JI Xinzun;CHENG Xiaozhen;SUN Yang;JI Dingwen(Department of General Surgery,Sanya People's Hospital,Sanya 5720000,China)

机构地区：[1]三亚市人民医院普外科,海南三亚572000 [2]海口市人民医院肿瘤内科,570208 [3]三亚市人民医院肿瘤外科,572000

出　　处：《临床肿瘤学杂志》2020年第9期783-789,共7页Chinese Clinical Oncology

摘　　要：目的探讨微小RNA-363-3p(miR-363-3p)对10号染色体缺失的磷酸酶及张力蛋白同源物(PTEN)的靶向调控作用及黑色素瘤细胞侵袭迁移的影响。方法采用Tumor-miRNA-Pathway网站在线分析miR-363-3p在皮肤黑色素瘤组织中的水平及参与调控的信号通路情况;荧光定量PCR(qPCR)检测正常人皮肤黑色素细胞PIG1和人恶性黑色素瘤细胞A375的miR-363-3p水平。脂质体法向A375细胞转染miR-363-3p抑制剂(Inhibitor组)和阴性无关序列(NC组)并设未转染的细胞为对照组,qPCR检测转染48 h后的miR-363-3p水平以评估转染效率,划痕实验和Transwell小室实验检测划痕愈合率和穿膜细胞数,TargetScan7.1在线预测miR-363-3p与PTEN的靶向结合序列并通过荧光素酶报告基因验证两者的靶向关系,qPCR和Western blotting检测PTEN、基质金属蛋白酶(MMP)-3、磷酸化磷脂酰肌醇-3激酶(p-PI3K)和磷酸化丝氨酸/苏氨酸蛋白激酶1(p-Akt1)水平。结果在线分析显示皮肤黑色素瘤组织的miR-363-3p水平高于正常皮肤组织,且可调控包括PI3K/Akt在内的37条信号通路。A375细胞的miR-363-3p水平高于PIG1细胞(P<0.05);Inhibitor组的miR-363-3p水平、划痕愈合率和穿膜细胞数分别为0.311±0.082、(13.241±2.603)%和(81.190±6.663)个,均低于对照组的1.002±0.745、(84.068±4.168)%和(242.501±26.536)个及NC组的0.937±0.524、(81.267±5.719)%和(265.592±36.027)个(P<0.05);与对照组和NC组相比,Inhibitor组的PTEN水平升高,而MMP-3、p-PI3K和p-Akt1水平降低(P<0.05);PTEN野生型质粒与miR-363-3p模拟物共转染后,荧光素酶活性降低(P<0.05),而其余共转染组合荧光素酶活性变化不显著(P>0.05)。结论黑色素瘤组织和细胞中,miR-363-3p均为过表达模式,且下调其水平可抑制迁移侵袭能力,可能通过靶向PTEN及激活PI3K/Akt信号通路来发挥促癌作用。MiR-363-3p/PTEN轴有望成为黑色素瘤诊治的潜在靶点。Objective To investigate the effect of microRNA-363-3p(miR-363-3p)on the targeted regulation of phosphatase and tensin homolog deleted on chromosome 10(PTEN)as well as the invasion and migration of melanoma cells.Methods MiR-363-3p level in cutaneous melanoma and the involved signal pathways were analyzed online by using Tumor-miRNA-Pathway website.Real-time quantitative PCR(qPCR)was used to detect miR-363-3p levels in human malignant melanoma A375 cells and skin melanocytes PIG1.A375 cells were transfected with miR-363-3p inhibitor(Inhibitor group)and negative unrelated sequence(NC group)by liposome method,and untransfected cells were set as the Control group.The miR-363-3p level at 48 h after transfection was detected by qPCR as to evaluate the transfection efficiency.The wound healing rate and number of cells penetrating the membrane were detected by scratch test and Transwell chamber test.The target binding sequence of miR-363-3p and PTEN was predicted online by TargetScan 7.1,and the target relationship between miR-363-3p and PTEN was verified by double luciferase reporter gene.Levels of PTEN,matrix metalloproteinase(MMP)-3,phosphorylated phosphatidylinositol-3 kinase(p-PI3K)and phosphorylated serine threonine protein kinase 1(p-Akt1)were measured by qPCR and western blotting.Results Online analysis showed that miR-363-3p level in melanoma tissue was higher than that in normal skin tissue,and it could regulate 37 signaling pathways including PI3K/Akt.The miR-363-3p level in A375 cells was higher than that in PIG1 cells(P<0.05).MiR-363-3p level,wound healing rate and number of penetrating cells in Inhibitor group were 0.311±0.082,(13.241±2.603)%and 81.190±6.663,lower than 1.002±0.745,(84.068±4.168)%and 242.501±26.536 in Control group and 0.937±0.524,(81.267±5.719)%and 265.592±36.027 in NC group(P<0.05).Compared with Control group and NC group,PTEN level in Inhibitor group was increased,but levels of MMP-3,p-PI3K and p-Akt1 were decreased(P<0.05).The luciferase activity of wild-type PTEN plasmid co-tr

关 键 词：黑色素瘤 微小RNA-363-3p 10号染色体缺失的磷酸酶及张力蛋白同源物 侵袭迁移 

分 类 号：R739.5[医药卫生—肿瘤]