ARK5在肝细胞肝癌对多柔比星耐药性中的作用机制的研究

发布时间：2018-10-12 16:36

【摘要】：实验目的腺苷酸活化蛋白激酶相关激酶5(ARK5)在包括肝细胞肝癌(HCC)在内的多种肿瘤内均高表达。肿瘤细胞内ARK5高表达的病人的预后较差。ARK5可以促进肿瘤的增殖,侵袭,迁移,复发,但是其与肿瘤耐药性的关系尚不清楚。本研究的目的就是观察ARK5在肝癌对多柔比星耐药性中的作用及其内在机制。材料与方法本研究通过多柔比星处理Huh7, Hep3B, SNU449和SNU387四种肝癌细胞系,利用细胞活力检测试剂盒(CCK-8)检测肝癌细胞系的细胞活力,蛋白免疫印迹技术(Western-blot)、免疫荧光技术检测细胞内ARK5、上皮间质细胞转化相关蛋白的表达情况,细胞增殖检测试剂盒(EdU试验)检测肝癌细胞系的增殖情况。ARK5 siRNA干扰肝癌细胞系内ARK5的表达,观察Huh7, Hep3B, SNU387, SNU449四种细胞系的细胞活力、增殖情况以及细胞内上皮间质化相关蛋白的表达情况。为探讨ARKS影响肝癌耐药的机制,我们首先利用多柔比星处理肝癌细胞系,再用ARK5 siRNA干扰肝癌细胞系内ARK5的表达,Wetern-blot检测EMT相关蛋白的表达；利用TWIST SiRNA干扰肝癌细胞系内TWIST的表达,再分别给予多柔比星和多柔比星KARK5 siRNA处理肝癌细胞系,并检测Huh7, Hep3B, SNU387, SNU449四种细胞系对多柔比星的敏感性。为了明确ARK5在低氧诱导的MT的作用,ARK5 siRNA处理的低氧条件培养的肝癌细胞系Huh7、Hep3B,并检测其细胞活力、上皮间质转化相关蛋白。结果实验结果发现：(1)ARK5的表达与肝癌细胞系对多柔比星的敏感性呈负相关；(2)ARK5敲除后可显著增加肝癌细胞系对多柔比星的敏感性；(3)ARK5可调控EMT相关蛋白的表达；(4)干扰多柔比星处理后的肝癌细胞系中ARK5的表达,可以逆转肝癌细胞系的EMT；(5)ARK5通过TWSIT通路调控肝癌细胞系对多柔比星的耐药性；(6)干扰低氧状态下培养的肝癌细胞系内ARK5的表达,可以逆转低氧诱导的EMT。结论：ARK5的高表达可明显降低肝癌细胞系对多柔比星的敏感性,抑制肝癌细胞系内ARK5的表达不仅可以显著增加肝癌细胞对多柔比星的敏感性,还能逆转多柔比星和低氧诱导的肝癌细胞对多柔比星的耐药性。ARK5影响肝癌对多柔比星耐药性机制为ARK5的低表达抑制TWIST通路,引起E-cadherin的表达上调和Vimentin的表达下调,从而抑制肝癌细胞系的上皮间质转化。
[Abstract]:Objective adenylate activated protein kinase associated kinase 5 (ARK5) was overexpressed in various tumors including hepatocellular carcinoma (HCC) (HCC). The prognosis of patients with high expression of ARK5 in tumor cells is poor. ARK5 can promote the proliferation, invasion, migration and recurrence of tumor, but the relationship between ARK5 and tumor resistance is not clear. The aim of this study was to investigate the role and mechanism of ARK5 in the resistance of hepatocellular carcinoma to doxorubicin. Materials and methods in this study, Huh7, Hep3B, SNU449 and SNU387 hepatoma cell lines were treated with doxorubicin, and cell viability was detected by cell viability test kit (CCK-8). Western blotting (Western-blot) and immunofluorescence technique were used to detect the expression of transforming proteins in the epithelial stromal cells of ARK5,. Cell proliferation test kit (EdU test) was used to detect the proliferation of HCC cell lines. ARK5 siRNA interfered with the expression of ARK5 in HCC cell lines and observed the cell viability of four Huh7, Hep3B, SNU387, SNU449 cell lines. Proliferation and expression of epithelial interstitial associated proteins in cells. In order to investigate the mechanism of ARKS affecting drug resistance of HCC, we first treated HCC cell line with doxorubicin, then used ARK5 siRNA to interfere the expression of ARK5 in HCC cell line, detected the expression of EMT related protein by Wetern-blot, and used TWIST SiRNA to interfere the expression of TWIST in HCC cell line. The hepatoma cell lines were treated with doxorubicin and doxorubicin KARK5 siRNA, respectively, and the sensitivity of four Huh7, Hep3B, SNU387, SNU449 cell lines to doxorubicin was detected. In order to elucidate the role of ARK5 in hypoxia induced MT, ARK5 siRNA treated hepatoma cell line Huh7,Hep3B, was cultured under hypoxia condition, and its cell viability and epithelial interstitial transformation associated protein were detected. Results the results showed that: (1) the expression of ARK5 was negatively correlated with the sensitivity of hepatoma cell line to doxorubicin, (2) ARK5 knockout could significantly increase the sensitivity of hepatoma cell line to doxorubicin, (3) ARK5 could regulate the expression of EMT related protein. (4) interfering with the expression of ARK5 in hepatoma cell line treated with doxorubicin could reverse the resistance of EMT; (5) ARK5 to doxorubicin through TWSIT pathway. (6) interfering with the expression of ARK5 in hepatoma cell line cultured under hypoxia can reverse the hypoxia induced EMT.. Conclusion: the high expression of ARK5 can significantly decrease the sensitivity of hepatoma cell line to doxorubicin, and inhibit the expression of ARK5 in hepatoma cell line can not only increase the sensitivity of hepatoma cell line to doxorubicin, but also increase the sensitivity of hepatoma cell line to doxorubicin. In addition, doxorubicin and hypoxia induced drug resistance of hepatoma cells to doxorubicin were reversed. The mechanism of ARK5 affecting drug resistance of HCC to doxorubicin was that the low expression of ARK5 inhibited the TWIST pathway, which resulted in the up-regulation of E-cadherin expression and the down-regulation of Vimentin expression. Thus inhibiting the epithelial mesenchymal transformation of hepatoma cell lines.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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