Circ_002770调控miR-331-3p影响黑色素瘤的上皮-间充质转化和侵袭

Circ_002770 accelerates epithelial-mesenchymal transition and invasion of malignant melanoma through sponging microRNA-331-3p

目的探讨Circ_002770调控微小RNA(miR)-331-3p对黑色素瘤上皮-间充质转化(EMT)和侵袭的影响。方法选取32例黑色素瘤患者的癌组织及癌旁组织,采用实时荧光定量聚合酶链反应(RT-qPCR)检测黑色素瘤组织和细胞中Circ_002770和miR-331-3p的表达水平;双荧光素酶报告实验检测miR-331-3p与Circ_002770之间的靶向关系;采用蛋白质印迹法(Western blot)检测EMT程度;Transwell小室法检测细胞侵袭。组间差异采用单因素方差分析进行统计分析。结果黑色素瘤组织(3.01±0.21)中Circ_00277的表达显著高于癌旁组织(1.00±0.06),差异有统计学意义(t=72.113,P<0.05);MM细胞中Circ_00277的表达显著高于正常皮肤上皮细胞,差异有统计学意义(t=98.021,P<0.05)。黑色素瘤组织(0.41±0.16)中miR-331-3p的表达显著低于癌旁组织(1.00±0.05),差异有统计学意义(t=36.892,P<0.05);MM细胞中miR-331-3p的表达显著低于正常皮肤上皮细胞,差异有统计学意义(t=187.239,P<0.05)。Circ_002770发挥分子海绵的作用抑制miR-331-3p表达;miR-331-3p与circ_002770野生型荧光载体共转染后A357细胞活性(0.46±0.21)显著低于对照组(1.00±0.23),差异有统计学意义(t=23.823,P<0.05)。而miR-331-3p与circ_002770突变型荧光载体共转染后A357细胞活性无明显变化(1.01±0.40比1.05±0.31)。低表达Circ_002770或过表达miR-331-3p均抑制黑色素瘤细胞的侵袭与EMT(P<0.05)。结论Circ_002770可吸附miR-331-3p促进黑色素瘤细胞的EMT和侵袭,进而影响黑色素瘤的进展。

Objective To investigate the effects and molecular mechanism of circ_002770 on epithelial-mesenchymal transition(EMT)and invasion of malignant melanoma(MM)cells via absorbing microRNA(miR)-331-3p.Methods The cancerous tissues and adjacent tissues from 32 cases of MM were adopted in this study.The expression of circ_002770 and miR-331-3p in MM tissues and cells was detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).Dual luciferase reporter experiment and RNA immunoprecipitation(RIP)assay were used to confirm the targeting relationship between circ_002770 and miR-331-3p.The Transwell assay and Western blotting were used to test the cell invasion ability and EMT respectively.Histopathological examination of each group was done by using one-way analysis of variance(ANOWA)test to assess the differences between the groups.Results The expression of Circ_002770 in MM tissues(3.01±0.21)was significantly higher than that in adjacent tissues(1.00±0.06),and the difference was statistically significant(t=72.113,P<0.05).The expression of Circ_002770 in MM tissues was significantly higher than in normal skin epithelial cells,and the difference was statistically significant(t=98.021,P<0.05).The expression of miR-331-3p in MM tissues(0.41±0.16)was significantly lower than that in adjacent tissues(1.00±0.05),and the difference was statistically significant(t=36.892,P<0.05).The expression of miR-331-3p in MM cells was significantly lower than in normal skin epithelial cells(t=187.239,P<0.05).Circ_002770 acted as a molecular sponge to inhibit the expression of miR-331-3p.After co-transfection of miR-331-3p and Circ_002770 wild-type fluorescent vector,the viability of A357 cells(0.46±0.21)was significantly lower than that of the control group(1.00±0.23),and the difference was statistically significant(t=23.823,P<0.05).However,there was no significant change in the viability of A357 cells after co-transfection of miR-331-3p and Circ_002770 mutant fluorescent vector(1.01±0.40 vs.1.05±0.31).Knockdown of circ_0007142 or overexpression of miR-647 inhibited the invasion and EMT of MM cells(P<0.05).Conclusion Circ_002770 upregulates miR-647,which subsequently accelerates the EMT and invasion of MM cells and promotes the progression of MM.