微小RNA-19b-3p靶向作用卷曲螺旋结构域6对黑色素瘤细胞增殖、侵袭及小鼠肿瘤体积的影响

Effect of miR-19b-3p targeting coiled-coil domain containing 6 on melanoma cell proliferation,invasion and tumor volume in mice

目的探讨微小RNA(miRNA,miR)-19b-3p靶向调控卷曲螺旋结构域6(CCDC6)在黑色素瘤细胞增殖、侵袭及小鼠肿瘤体积的机制.方法收集黑色素瘤组织及健康人群痣组织各56例.实时定量聚合酶链反应(Real-time PCR)测定miR-19b-3p与CCDC6的相对表达量,细胞增殖与毒性(CCK-8)检测WM35细胞增殖能力,细胞侵袭实验检测WM35细胞侵袭能力,蛋白质印迹法(Western blot)检测CCDC6及磷酸化蛋白激酶B(p-Akt)蛋白水平,荧光素酶报告分析验证miR-19b-3p与CCDC6关系,裸鼠肿瘤模型建立以探究miR-19b-3p对肿瘤体积的影响.应用SPSS 19.0统计软件分析,计数资料组间比较采用x2检验.两两比较和多组间比较分别采用t检验和单因素方差分析,相关分析采用Pearson法.结果与良性痣组织(1.43±0.38)比较,黑色素瘤组织(2.89±0.46)中miR-19b-3p水平明显增加(t=6.823,P<0.05),且miR-19b-3p与Ⅲ/Ⅳ期(x2=12.087,P<0.05)及发生远处转移(x2=12.600,P<0.05)显著相关.与空白对照组[miR-19b-3p:1.16±0.28;细胞侵袭数:(26.35±4.73)个/HF]和miR对照组[miR-19b-3p:1.22 ±0.30;细胞侵袭数:(28.16±4.80)个/HF]比较,miR-19b-3p模拟物组[miR-19b-3p:4.06±0.28,t=3.977,江4.003,P<0.05;细胞侵袭数:(49.64±5.11)个/HF,t =3.981,t=4.500,P<0.05] miR-19b-3p表达水平及细胞侵袭能力均显著增高(P<0.05),而miR-19b-3p抑制剂组[miR-19b-3p:0.41±0.08,=3.771,t=3.579,P<0.05;细胞侵袭数:(14.47 ±0.96)个/HF,t=3.669,t=4.004,P<0.05] miR-19b-3p表达水平及细胞侵袭能力均显著降低.CCK-8检测结果显示,在24、48、72 h时,miR-19b-3p模拟物组细胞的增殖能力显著增强,miR-19b-3p抑制剂组增殖能力显著下降(F=4.026、4.609、7.446,P<0.05).与miR对照组比较[体积:(467.34±36.75) mm^3;CCDC6:1.00±0.12;miR-19b-3p:2.17±0.42],miR-19b-3p模拟物组小鼠黑色素瘤组织的体积[(1 058.66±66.17) mm^3]显著增加,CCDC6表达显著降低(0.42±0.08),miR-19b-3p水平显著增加(4.01±0.66),差异均有统计学意义(t=22.836,t =8.129,t=10.663,P<0.05).结论 miR-19b-3p可促进黑色素瘤的侵袭与增殖,其机制与调控下游CCDC6及p-Akt密切相关.

Objective To investigate the mechanism of microRNA (miRNA, miR)-19b-3p targeted regulation of coiled-coil domain containing 6 (CCDC6) in melanoma cell proliferation, invasion and mouse tumor volume. Methods Fifty-six cases of melanoma tissue and fifty-six healthy people were collected. The relative expression of miR-19b-3p and CCDC6 was detected by real-time quantitative polymerase chain reaction (Real-time PCR). The proliferation of WM35 cells was detected by cell counting kit-8 (CCK-8). The invasion ability of WM35 cells was detected by cell invasion assay. The levels of CCDC6 and p-protein kinase B (Akt) protein were detected by Western blotting. The relationship between miR-19b-3p and CCDC6 was verified by luciferase report analysis. The nude mouse tumor model was established to explore the effect of miR-19b-3p on tumor volume. Results Compared with benign sputum tissue (1.43±0.38), miR-19b-3p levels in melanoma tissues (2.89±0.46) were significantly increased (t=6.823, P<0.05), and miR-19b-3p was significantly associated with stage Ⅲ/Ⅳ(χ2=12.087, P<0.05) and distant metastasis (χ2=12.600, P<0.05). Compared with the blank control group [miR-19b-3p: 1.16±0.28;Cell invasion:(26.35±4.73)/HF] and the miR control group [miR-19b-3p: 1.22±0.30;Cell invasion:(28.16±4.80)/HF], the miR-19b-3p expression level and cell invasion ability of miR-19b-3p mimetic group [miR-19b-3p: 4.06±0.28, t=3.977, t=4.003, P<0.05;Cell invasion:(49.64±5.11)/HF, t=3.981, t=4.500, P<0.05] were significantly increased (P<0.05), while the miR-19b-3p expression level and cell invasion ability were significantly decreased in the miR-19b-3p inhibitor group [miR-19b-3p: 0.41±0.08, t=3.771, t=3.579, P<0.05;Cell invasion:(14.47±0.96)/HF, t=3.669, t=4.004, P<0.05], P<0.05. The results of CCK-8 assay showed that the proliferation of miR-19b-3p mimetic cells was significantly increased, while the proliferation was significantly decreased in miR-19b-3p inhibitor group at 24, 48 and 72 h (F=4.026, 4.609, 7.446, P<0.05). Compared with the miR control group [volume:(467.34±36.75) mm^3;CCDC6: 1.00±0.12;miR-19b-3p: 2.17±0.42], the volume of melanoma tissue in the miR-19b-3p mimic group [(1 058.66±66.17) mm^3] was significantly increased, the expression of CCDC6 was significantly decreased (0.42±0.08), and the level of miR-19b-3p was significantly increased (4.01±0.66), t=22.836, 8.129, 10.663, P<0.05. Conclusion MiR-19b-3p can promote the invasion and proliferation of melanoma cells, and its mechanism is closely related to the regulation of downstream CCDC6 and p-Akt.