Wu Liangcai, Zhu Lifei, Li Yanchang, Zheng Zhixin, Lin Xi, Yang Chaoying
1Department of Dermatology, The Sixth Affiliated Hospital, Sun Yat-sen University, No. 26, Yuancun ErHeng Road, Guangzhou, 510655 China.
2Department of Pharmacology, Medical College, Jinan University, Guangzhou, 510632 China.
Cancer Cell Int. 2020 Jan 10;20:12. doi: 10.1186/s12935-019-1087-4. eCollection 2020.
Melanoma is the most aggressive type of skin cancer with high mortality rate and poor prognosis. lncRNA MEG3, a tumor suppressor, is closely related to the development of various cancers. However, the role of lncRNA MEG3 in melanoma has seldom been studied.
RT-PCR was used to examine the expressions of lncRNA MEG3 and E-cadherin in melanoma patients and cell lines. Then, the biological functions of lncRNA MEG3 and E-cadherin were demonstrated by transfecting lncRNA MEG3-siRNA, lncRNA MEG3-overexpression, E-cadherin-siRNA and E-cadherin-overexpression plasmids in melanoma cell lines. Moreover, CCK8 assay and colony formation assay were utilized to assess the cell proliferation; Transwell assay was performed to evaluate the cell invasive ability; and tumor xenografts in nude mice were applied to test the tumor generation. Additionally, the target interactions among lncRNA MEG3, miR-21 and E-cadherin were determined by dual luciferase reporter assay. Finally, RT-PCR and WB were further conducted to verify the regulatory roles among lncRNA MEG3, miR-21 and E-cadherin.
The clinical data showed that lncRNA MEG3 and E-cadherin expressions were both declined in carcinoma tissues as compared with their para-carcinoma tissues. Moreover, lncRNA MEG3 and E-cadherin expressions in B16 cells were also higher than those in A375 and A2058 cells. Subsequently, based on the differently expressed lncRNA MEG3 and E-cadherin in these human melanoma cell lines, we chose B16, A375 and A2058 cells for the following experiments. The results demonstrated that lncRNA MEG3 could suppress the tumor growth, tumor metastasis and formation; and meanwhile E-cadherin had the same effects on tumor growth, tumor metastasis and formation. Furthermore, the analysis of Kaplan-Meier curves also confirmed that there was a positive correlation between lncRNA MEG3 and E-cadherin. Ultimately, dual luciferase assays were further used to verify that lncRNA MEG3 could directly target miR-21 which could directly target E-cadherin in turn. Additionally, the data of RT-PCR and WB revealed that knockdown of lncRNA MEG3 in B16 cells inhibited miR-21 expression and promoted E-cadherin expression, but overexpression of lncRNA MEG3 in A375 and A2058 cells presented completely opposite results.
Our findings indicated that lncRNA MEG3 might inhibit the tumor growth, tumor metastasis and formation of melanoma by modulating miR-21/E-cadherin axis.
黑色素瘤是最具侵袭性的皮肤癌类型，死亡率高且预后差。lncRNA MEG3作为一种肿瘤抑制因子，与多种癌症的发生发展密切相关。然而，lncRNA MEG3在黑色素瘤中的作用鲜有研究。
采用RT-PCR检测黑色素瘤患者及细胞系中lncRNA MEG3和E-钙黏蛋白的表达。然后，通过在黑色素瘤细胞系中转染lncRNA MEG3-siRNA、lncRNA MEG3过表达载体、E-钙黏蛋白-siRNA和E-钙黏蛋白过表达质粒，验证lncRNA MEG3和E-钙黏蛋白的生物学功能。此外，利用CCK8法和集落形成试验评估细胞增殖；通过Transwell试验评估细胞侵袭能力；将肿瘤细胞接种到裸鼠体内以检测肿瘤生成情况。另外，采用双荧光素酶报告基因试验确定lncRNA MEG3、miR-21和E-钙黏蛋白之间的靶向相互作用。最后，通过RT-PCR和WB进一步验证lncRNA MEG3、miR-21和E-钙黏蛋白之间的调控作用。
临床数据显示，与癌旁组织相比，lncRNA MEG3和E-钙黏蛋白在癌组织中的表达均下降。此外，B16细胞中lncRNA MEG3和E-钙黏蛋白的表达也高于A375和A2058细胞。随后，基于这些人黑色素瘤细胞系中lncRNA MEG3和E-钙黏蛋白的差异表达，我们选择B16、A375和A2058细胞进行后续实验。结果表明，lncRNA MEG3可抑制肿瘤生长、转移和形成；同时E-钙黏蛋白对肿瘤生长、转移和形成也有相同作用。此外，Kaplan-Meier曲线分析也证实lncRNA MEG3和E-钙黏蛋白之间呈正相关。最终，双荧光素酶试验进一步验证lncRNA MEG3可直接靶向miR-21，而miR-21又可直接靶向E-钙黏蛋白。此外，RT-PCR和WB数据显示，敲低B16细胞中的lncRNA MEG3可抑制miR-21表达并促进E-钙黏蛋白表达，但在A375和A2058细胞中过表达lncRNA MEG3则呈现完全相反的结果。
我们的研究结果表明，lncRNA MEG3可能通过调节miR-21/E-钙黏蛋白轴抑制黑色素瘤的肿瘤生长、转移和形成。
