CST1调节Notch信号通路对非小细胞肺癌细胞增殖、迁移和侵袭的影响

Effects of CST1 on proliferation, migration and invasion of non-small cell lung cancer cells via Notch signaling pathway

目的 探讨半胱氨酸蛋白酶抑制剂(CST1)调控Notch信号通路影响非小细胞肺癌(NSCLC)细胞增殖、迁移和侵袭生物学行为的潜在机制。方法 通过GEPIA分析癌症基因图谱(TCGA)数据库中NSCLC组织的CST1水平。采用实时荧光定量PCR(qPCR)检测NSCLC细胞的CST1水平。向A549细胞转染靶向CST1的小干扰RNA序列si-CST来评估CST1对A549细胞生物学行为的影响,后续在转染si-CST基础上给予Notch通路激活剂(VPA-d4)处理来评估CST1调控Notch信号通路对A549细胞生物学行为的影响,单独给予Notch通路抑制剂(IMR-1)处理来验证Notch信号通路对A549细胞生物学行为的促进作用。CCK-8、划痕实验和Transwell小室实验检测细胞增殖、迁移和侵袭情况。qPCR和Western blotting检测Notch1、发状分裂相关增强子1(Hes1)和基质金属蛋白酶9(MMP-9)的水平。结果 CST1在NSCLC组中的水平高于正常组织(P<0.05),且其在H1299、H460、H1975、SPC-A1、H1650和A549细胞中的水平高于BEAS-2B细胞(P<0.05)。A549细胞转染si-CST或经IMR-1处理后的增殖活力、划痕愈合率和穿膜数量较对照组细胞降低且伴有Notch1和Hes1的表达下调(P<0.05);A549细胞经si-CST和VPA-d4联合处理后的增殖活力、划痕愈合率和穿膜数量较单独转染si-CST的细胞增多且伴有Notch1、Hes1和MMP-9的表达上调(P<0.05)。结论 CST1在NSCLC组织和细胞中上调且可能通过激活Notch信号通路在NSCLC中发挥促癌作用,其有望成为NSCLC治疗的新型生物标志物和潜在治疗靶点。

Objective To explore the potential mechanism by which Cystatin SN(CST1) affects the proliferation, migration and invasion of non-small cell lung cancer(NSCLC) cells via Notch signaling pathway. Methods GEPIA was used to analyze the CST1 level in NSCLC tissues from The Cancer Genome Atlas(TCGA) database. Levels of CST1 in NSCLC cells were determined by real time-quantitative polymerase chain reaction(qPCR). Effects of CST1 on the biological behavior of A549 cells was evaluated by transfecting the small interfering RNA sequence si-CST targeting CST1 into A549 cells. Subsequently, Notch pathway activator(VPA-d4) was given on the basis of transfection with si-CST to evaluate the effect of CST1 regulating Notch signaling pathway on the biological behavior of A549 cells. Notch pathway inhibitor(IMR-1) was given alone to verify the promotion of Notch signaling pathway on the biological behavior of A549 cells. CCK-8, scratch assay and Transwell chamber assay were conducted to evaluate the cell proliferation, migration and invasion abilities. Moreover, qPCR and Western blotting were used to detect the levels of Notch1, hairy and enhancer of split 1(Hes1) and matrix metallopeptidase-9(MMP-9). Results CST1 were up-regulated in NSCLC tissues(P<0.05). Levels of CST1 in NSCLC cells including H1299, H460, H1975, SPC-A1, H1650 and A549 were increased compared with BEAS-2B cell(P<0.05). A549 cells treated with si-CST or IMR-1 had lower proliferative activity, scratch healing rate and number of penetrating membranes than control cells as accompanied by down-regulated Notch1 and Hes1(P<0.05). A549 cells treated with si-CST plus VPA-d4 had higher proliferative activity, scratch healing rate and number of penetrating membrane than cells treated with si-CST alone as accompanied by up-regulated Notch1, Hes1 and MMP-9(P<0.05). Conclusion CST1 is up-regulated in NSCLC tissues and cells and may play a role in promoting cancer in NSCLC by activating Notch signaling pathway. It is expected to become a new biomarker and potential therapeutic target for NSCLC treatment.