丝氨酸羟甲基转移酶2对人皮肤黑色素瘤细胞增殖的影响

Effects of serine-hydroxymethyltransferase 2 on cell proliferation of skin cutaneous melanoma cells

目的研究丝氨酸羟甲基转移酶2(SHMT2)对人皮肤黑色素瘤细胞增殖的影响及作用机制。方法通过GEPIA在线数据库分析人皮肤黑色素瘤中SHMT2的表达情况及其与β-catenin、c-Myc和Cyclin D1表达的相关性。通过嘌呤霉素进行压力筛选获取稳定表达SHMT2的人皮肤黑色素瘤A-375-SHMT2细胞株,采用细胞计数、CCK-8法、流式细胞术分别检测SHMT2对细胞增殖和细胞周期的影响,Western blot检测β-catenin及其下游分子Cyclin D1和c-Myc蛋白的表达。萤火虫荧光素酶报告系统实验(TOP/FOP-Flash luciferase reporter assay)检测wnt/β-catenin信号通路的活性。在稳定表达SHMT2的A-375-SHMT2细胞中使用XAV-939抑制wnt/β-catenin信号通路活性后,观察细胞增殖的变化。结果GEPIA在线数据库分析的结果显示,SHMT2在人皮肤黑色素瘤患者肿瘤组织中高表达(P<0.05),SHMT2表达与β-catenin、c-Myc和Cyclin D1呈正相关(P<0.05)。成功筛选获得稳定表达SHMT2的A-375-SHMT2细胞系及对照细胞系A-375-GFP。与A-375-GFP组相比,A-375-SHMT2组细胞的增殖能力更强(P<0.05),处于细胞周期G0/G1期的细胞比例降低(P<0.05),S期和G2/M期的细胞比例增高(P<0.05)。Western blot和双荧光素酶TOP/FOP实验的结果显示SHMT2增强wnt/β-catenin信号通路活性(P<0.05),并上调β-catenin、c-Myc和Cyclin D1的表达(P<0.05)。在A-375-SHMT2细胞中使用XAV-939抑制wnt信号通路后可以逆转SHMT2对细胞增殖的促进作用及对Cyclin D1和c-Myc的上调效应(P<0.05)。结论SHMT2通过激活wnt/β-catenin信号转导通路活性促进人皮肤黑色素瘤细胞的体外增殖。

Objective To investigate the effect and its mechanism of serine hydroxymethyltransferase 2(SHMT2)on the cell proliferation of skin cutaneous melanoma cell lines A-375.Methods The expression of SHMT2 in human skin cutaneous melanoma and its correlation withβ-catenin,c-Myc and Cyclin D1 were analyzed based on GEPIA online database.The human skin cutaneous melanoma cell line A-375-SHMT2 stably overexpressing SHMT2 was screened by puromycin.Cell proliferation and cell cycle were detected by cell growth curve,CCK-8 assay and flow cytometry.The protein levels ofβ-catenin,Cyclin D1 and c-Myc were detected by Western blot.The activity of wnt/β-catenin signaling pathway was detected by TOP/FOP-Flash luciferase reporter assay.After XAV-939 was used to inhibit wnt/β-catenin signaling pathway activity in A-375-SHMT2 cells,the change of cell proliferation was observed.Results The results of GEPIA online database analysis showed that SHMT2 was highly expressed in human cutaneous melanoma tissues(P<0.05),and that SHMT2 expression was positively correlated withβ-catenin,c-Myc and Cyclin D1(P<0.05).The A-375-SHMT2 cell line with stable SHMT2 expression and the control cell line A-375-GFP were successfully obtained.The proliferation ability of A-375-SHMT2 cells was significantly higher than that of A-375-GFP cells(P<0.05),and the proportion of A-375-SHMT2 cells in the G0/G1 phase was lower than that of A-375-GFP cells(P<0.05),while the proportion of S and G2/M phase cells was higher(P<0.05).Western blot and TOP/FOP assay showed that SHMT2 enhanced wnt/β-catenin signaling pathway activity and upregulated the expressions ofβ-catenin,c-Myc and Cyclin D1(all P<0.05).The promoted cell growth and the upregulated expressions of Cyclin D1 and c-Myc induced by SHMT2 in A-375-SHMT2 cells were reversed by XAV-939.Conclusion SHMT2 can promote the proliferation of human cutaneous melanoma cells in vitro by activating wnt/β-catenin signaling pathway activity.