目的：探讨长链非编码RNA CDKN2B反义RNA1（long non-coding RNA CDKN2B-antisense RNA 1, lncRNA CDKN2B-AS1）通过靶向miR-7-5p影响黑色素瘤B16-F10细胞的恶性生物学行为的影响。方法：选用黑色素瘤B16-F10细胞，构建shRNA CD‐KN2B-AS1载体并转染到B16-F10细胞，实验分为对照组、sh-CDKN2B-AS1 组、miR-7-5p mimics 组、miR-7-5p inhibitor组。用RT-PCR检测转染后B16-F10细胞中CDKN2B-AS1表达水平，用克隆形成实验和MTT法检测克隆形成数及细胞的增殖能力，用划痕愈合实验和Transwell实验检测细胞的迁移和侵袭能力。用荧光素酶报告基因实验检证CDKN2B-AS1和miR-7-5p相互间靶向关系。用RT-PCR和Western blotting检测转染miR-7-5p mimics、inhibitor后B16-F10细胞中miR-7-5p和Ki67、cleaved caspase-3、上皮钙黏蛋白（E-cadherin）、神经钙黏蛋白（N-cadherin）、Twist1蛋白的表达水平。结果：与对照组比较，sh-CDKN2B-AS1组 B16-F10细胞中CDKN2B-AS1表达水平显著下调（P<0.01），细胞的增殖、迁移及侵袭能力显著下降（均P<0.01）。荧光素酶报告基因实验证明 CDKN2B-AS1 与 miR-7-5p 存在靶向作用关系。sh-CDKN2B-AS1 组与 miR-7-5p mimics 组细胞中 miR-7-5p 和cleaved caspase-3、E-cadherin水平均明显上调（均P<0.05），Ki67、N-cadherin、Twist1水平明显下调（均P<0.05）。结论：CDKN2BAS1通过靶向miR-7-5p促进黑色素瘤发展，干扰CDKN2B-AS1可抑制黑色素瘤B16-F10细胞的恶性生物学行为。

Objective: To investigate the effect of long non-coding RNA CDKN2B antisense RNA 1 (CDKN2B-AS1) on malignant biological behaviors of melanoma B16-F10 cells by targeting miR-7-5p. Methods: Melanoma B16-F10 cells were chosen for this study.shRNA CDKN2B-AS1 vector was constructed and transfected into B16-F10 cells. The experimental cells were divided into control group, sh-CDKN2B-AS1 group, miR-7-5p mimic group and miR-7-5p inhibitor group. The expression level of CDKN2B-AS1 mRNA in the transfected B16-F10 cells was detected by RT-PCR; the number of clone formation and the proliferation ability of the cells were detected by Clone formation assay and MTT assay; and the migration and invasion ability of the cells were detected by Scratch-healing assay and Transwell assay. The targeting relationship between CDKN2B-AS1 and miR-7-5p was detected by Luciferase reporter gene assay. The mRNA expression of miR-7-5p and protein expressions of Ki67, cleaved caspase-3, E-cadherin, N-cadherin and Twist1 in B16-F10 cells after transfection with miR-7-5p mimics/inhibitor were detected by RT-PCR and Western blotting, respectively. Results:Compared with the control group, the expression level of CDKN2B-AS1 mRNA in B16-F10 cells of sh-CDKN2B-AS1 group was significantly decreased (P<0.01); the proliferation, migration and invasion ability of cells were significantly decreased (all P<0.01).Luciferase reporter gene assay showed that CDKN2B-AS1 directly targeted miR-7-5p. The mRNA expression of miR-7-5p, and protein expressions of cleaved caspase-3 and E-cadherin in sh-CDKN2B-AS1 group and miR-7-5p mimic group were significantly up-regulated (all P<0.05), while the protein expressions of Ki67, N-cadherin, and Twist1 were significantly down-regulated (all P<0.05). Conclusion:CDKN2B-AS1 targets miR-7-5p to promote the development of melanoma, and interfering with CDKN2B-AS1 can inhibit the malig‐nant biological behaviors of melanoma B16-F10 cells.

国家自然科学基金资助项目（No.81560697）