JAK2信号通路介导人恶性黑素瘤A375细胞的自噬和凋亡机制研究

[摘要]目的：探討JAK2/STAT3信号通路介导人恶性黑素瘤A375细胞的自噬和凋亡活性，为黑素瘤的发生机制和潜在干预靶点提供理论依据。方法：人恶性黑素瘤A375细胞经复苏和传代后随机分为对照组和JAK2抑制剂干预组，比较两组细胞培养12h、24h、48h和72h的增殖率（采用MTT定量法）及凋亡率（采用流式细胞术），培养72h的细胞JAK2、p-JAK2、STAT3、LC3B和p-STAT3蛋白相对表达量（采用Western blot法），检测IL-6和TNF-α水平（采用ELISA法），检测Caspase-3和Bax/Bcl-2 mRNA相对表达量[采用反转录PCR（RT-PCR）法]。结果：两组培养12h、24h、48h和72h的细胞增殖率比较，干预组各时间点均明显小于对照组，而凋亡率明显大于对照组，差异有统计学意义（P<0.05）。干预组培养72h的细胞p-JAK2和p-STAT3蛋白相对表达量、IL-6和TNF-α水平明显低于对照组，但LC3B蛋白、Caspase-3和Bax/Bcl-2 mRNA相对表达量均明显高于对照组，差异有统计学意义（P<0.05）。结论：JAK2/STAT3信号通路异常激活可能是人黑素瘤A375细胞恶性增殖的重要通路之一，靶向干预JAK2/STAT3信号通路可以促进细胞自噬和凋亡活性上调，有望成为临床干预的重要靶点。

[关键词]JAK2/STAT3信号通路;黑素瘤;自噬;凋亡;增殖

[中图分类号]R739.5    [文献标志码]A    [文章编号]1008-6455（2020）04-0098-04

Abstract： Objective  To study the autophagy and apoptotic activity of human malignant melanoma A375 cells via by JAK2/STAT3 signaling pathway，and provide theoretical basis for pathogenesis of melanoma and potential intervention target. Methods  After recovery and generation，human malignant melanoma A375 cells were randomly divided into the control group and the JAK2 inhibitor intervention group. MTT and flow cytometry were used to detect proliferation and apoptosis rates of cells after cultured for 12， 24， 48 and 72 hours， Western blot was used to detect relative expressions of JAK2， p-JAK2， STAT3， LC3B and p-STAT3 proteins， ELISA was used to detect levels of IL-6 and TNF-α and reverse transcription PCR （RT-PCR） was used to detect relative expressions of Caspase-3 and Bax/Bcl-2 mRNAs. Results  The cell proliferation rate of the intervention group was significantly lower than those of the control group at 12h， 24h， 48h and 72h， while the apoptosis rate was significantly higher than those of the control group， the differences were statistically significant（P<0.05）. Whats more， relative expressions of p-JAK2 and p-STAT3 proteins， IL-6 and TNF-α levels after 72h in the intervention group were both significantly lower than those of the control group， while expressions of LC3B protein， relative expressions of Caspase-3 and Bax/Bcl-2 mRNAs in the intervention group were all significantly higher than those of the control group（P<0.05）. Conclusion  Abnormal activation of JAK2/STAT3 signaling pathway may be one of important pathways for malignant proliferation in human melanoma A375 cells. Targeted intervention of JAK2/STAT3 signaling pathway can promote up-regulations of autophagy and apoptotic activities of cells， which is expected to become an important target for clinical intervention.

Key words： JAK2/STAT3 signaling pathway; melanoma; autophagy; apoptosis; proliferation

黑素瘤是最常见的皮肤和黏膜恶性肿瘤之一，近年来发病率明显增多，已引起临床的高度重视，早期以手术切除为主，中晚期以化疗和靶向干预为主[1]。黑素瘤的发生与环境变化和基因突变有关，研究发现[2]，细胞恶性增殖、自噬和凋亡活性下降是黑色素瘤发生、侵袭和转移的重要机制。细胞自噬和凋亡是细胞存活、分化和内环境稳态维持的重要方式，受体内外多个基因和信号通路的共同调控，具有较强的程序控制性[3]。JAK2/STAT3信号通路是真核生物体内参与调控细胞增殖、分化、损伤修复、炎症反应、氧化应激、细胞因子表达等重要细胞内途径之一[4]。IL-6和TNF-α是JAK2/STAT3信号通路的重要诱导剂，在多种恶性肿瘤如结直肠癌、黑素瘤、胃癌的发生和发展中发挥重要作用。JAK2/STAT3信号通路的活化既需要炎症因子IL-6和TNF-α的诱导，同时又可加剧炎症因子的释放，形成炎症瀑布样级联反应，参与恶性肿瘤的凋亡和自噬过程。……