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Title: [Doublecortin-like kinase 1 activates Hippo pathway to promote migration, invasion and proliferation of pancreatic cancer cells].
Author: Yan R, Liang ZW, Liu HS, Ge Y, An GY.
Journal: Zhonghua Zhong Liu Za Zhi; 2023 Jul 23; 45(7):594-604. PubMed ID: 37462016.
Abstract:
Objective: To explore the mechanism of Doublecortin-like kinase 1 (DCLK1) in promoting cell migration, invasion and proliferation in pancreatic cancer. Methods: The correlation between DCLK1 and Hippo pathway was analyzed using TCGA and GTEx databases and confirmed by fluorescence staining of pancreatic cancer tissue microarrays. At the cellular level, immunofluorescence staining of cell crawls and western blot assays were performed to clarify whether DCLK1 regulates yes associated protein1 (YAP1), a downstream effector of the Hippo pathway. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to analyze the expressions of YAP1 binding transcription factor TEA-DNA binding proteins (TEAD) and downstream malignant behavior-promoting molecules CYR61, EDN1, AREG, and CTGF. Transwell test of the DCLK1-overexpressing cells treated with the Hippo pathway inhibitor Verteporfin was used to examine whether the malignant behavior-promoting ability was blocked. Analysis of changes in the proliferation index of experimental cells used real-time label-free cells. Results: TCGA combined with GTEx data analysis showed that the expressions of DCLK1 and YAP1 molecules in pancreatic cancer tissues were significantly higher than those in adjacent tissues (P<0.05). Moreover, DCLK1was positively correlated with the expressions of many effectors in the Hippo pathway, including LATS1 (r=0.53, P<0.001), LATS2 (r=0.34, P<0.001), MOB1B (r=0.40, P<0.001). In addition, the tissue microarray of pancreatic cancer patients was stained with multicolor fluorescence, indicated that the high expression of DCLK1 in pancreatic cancer patients was accompanied by the up-regulated expression of YAP1. The expression of DCLK1 in pancreatic cancer cell lines was analyzed by the CCLE database. The results showed that the expression of DCLK1 in AsPC-1 and PANC-1 cells was low. Thus, we overexpressed DCLK1 in AsPC-1 and PANC-1 cell lines and found that DCLK1 overexpression in pancreatic cancer cell lines promoted YAP1 expression and accessible to the nucleus. In addition, DCLK1 up-regulated the expression of YAP1 binding transcription factor TEAD and increased the mRNA expression levels of downstream malignant behavior-promoting molecules. Finally, Verteporfin, an inhibitor of the Hippo pathway, could antagonize the cell's malignant behavior-promoting ability mediated by high expression of DCLK1. We found that the number of migrated cells with DCLK1 overexpressing AsPC-1 group was 68.33±7.09, which was significantly higher than 22.00±4.58 of DCLK1 overexpressing cells treated with Verteporfin (P<0.05). Similarly, the migration number of PANC-1 cells overexpressing DCLK1 was 65.66±8.73, which was significantly higher than 37.00±6.00 of the control group and 32.33±9.61 of Hippo pathway inhibitor-treated group (P<0.05). Meanwhile, the number of invasive cells in the DCLK1-overexpressed group was significantly higher than that in the DCLK1 wild-type group cells, while the Verteporfin-treated DCLK1-overexpressed cells showed a significant decrease. In addition, we monitored the cell proliferation index using the real-time cellular analysis (RTCA) assay, and the proliferation index of DCLK1-overexpressed AsPC-1 cells was 0.66±0.04, which was significantly higher than 0.38±0.01 of DCLK1 wild-type AsPC-1 cells (P<0.05) as well as 0.05±0.03 of DCLK1-overexpressed AsPC1 cells treated with Verteporfin (P<0.05). PANC-1 cells showed the same pattern, with a proliferation index of 0.77±0.04 for DCLK1-overexpressed PANC-1 cells, significantly higher than DCLK1-overexpressed PANC1 cells after Verteporfin treatment (0.14±0.05, P<0.05). Conclusion: The expression of DCLK1 is remarkably associated with the Hippo pathway, it promotes the migration, invasion, and proliferation of pancreatic cancer cells by activating the Hippo pathway. 目的： 探讨双肾上腺素皮质样激酶1(DCLK1)在胰腺癌中促进细胞迁移、侵袭与增殖的分子机制。 方法： 基于癌症基因组图谱(TCGA)数据库与基因型-组织表达(GTEx)数据库，分析DCLK1与Hippo通路的相关性并采用胰腺癌组织芯片荧光染色进一步证实。细胞水平上，通过细胞爬片免疫荧光染色以及Western blot实验验证DCLK1对Hippo通路下游效应分子yes相关蛋白1(YAP1)是否具有调控作用，实时荧光定量聚合酶链反应分析YAP1结合转录因子TEA-DNA结合蛋白(TEAD)以及下游促恶性行为分子CYR61、EDN1、AREG、CTGF的表达。利用Hippo通路抑制剂Verteporfin处理细胞，采用Transwell实验检测DCLK1过表达细胞的迁移、侵袭能力是否受到抑制，采用实时无标记细胞分析实验检测细胞增殖指数的变化。 结果： TCGA和GTEx数据库数据分析显示，胰腺癌组织中DCLK1和YAP1的表达水平高于癌旁正常组织(均P<0.05)。相关性热图提示，DCLK1与YAP1所在的Hippo通路有关，其中LATS1(r＝0.53)、LATS2(r＝0.34)、MOB1B(r＝0.40)等均与DCLK1表达呈正相关(均P<0.05)。胰腺癌患者组织芯片经多色荧光染色显示，胰腺癌DCLK1高表达患者伴随YAP1表达上调。胰腺癌AsPC-1与PANC-1细胞中DCLK1呈低表达，过表达DCLK1可促进YAP1进入细胞核。过表达DCLK1上调YAP1结合转录因子TEAD的表达并增加下游促迁移、侵袭、增殖分子mRNA的表达。使用Hippo通路抑制剂Verteporfin则可降低DCLK1高表达介导的细胞迁移、侵袭、增殖能力增加。AsPC-1 DCLK1过表达细胞的迁移数为(68.33±7.09)个，Verteporfin处理后的细胞迁移数为(22.00±4.58)个，差异有统计学意义(P<0.05)。PANC-1对照组细胞的迁移数为(37.00±6.00)个，DCLK1过表达细胞的迁移数为(65.66±8.73)个，Verteporfin处理后两组细胞迁移数为(19.66±3.05)个和(32.33±9.61)个，差异均有统计学意义(均P<0.05)。DCLK1过表达胰腺癌细胞侵袭数高于DCLK1对照组细胞以及Verteporfin处理后的DCLK1过表达细胞(均P<0.05)。实时无标记细胞分析实验证实DCLK1过表达的AsPC-1细胞增殖指数为0.66±0.04，高于AsPC-1对照细胞的增殖指数(0.38±0.01，P<0.05)，高于Verteporfin处理后的DCLK1过表达AsPC1细胞(0.05±0.03，P<0.05)。而PANC-1 DCLK1过表达细胞的增殖指数为0.77±0.04，高于Verteporfin处理后的DCLK1过表达PANC1细胞(0.14±0.05，P<0.05)。 结论： DCLK1与Hippo通路显著相关且通过激活Hippo通路促进胰腺癌细胞迁移、侵袭与增殖。.

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