Biological Role of RNF41 in Regulating Proliferation and Metastasis of Cholangiocarcinoma Cells
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摘要:
目的 探讨环指蛋白41（ring finger protein 41，RNF41）在胆管癌发生和发展过程中的作用。 方法 通过生物信息学、Western blot和免疫组织化学分析2010年1月至2016年12月期间在昆明医科大学第二附属医院和昆明市甘美医院接受全手术切除的84例胆管癌患者肿瘤组织和癌旁正常组织中RNF41的mRNA和蛋白质的表达；利用TIMER 2.0数据库中临床资料分析RNF41对胆管癌患者预后生存的影响，分析RNF41与肿瘤临床特点的关系；RNF41 siRNA经转染HCC9810、RBE、HUCCT1细胞后，利用CCK-8、Edu、克隆形成实验和Western blot实验，分析敲低RNF41组和对照组胆管癌细胞的增殖作用的变化；利用Transwell实验和EMT标记物及迁移标记物检测分析敲低RNF41组和对照组胆管癌细胞的侵袭作用的变化，Western blot实验检测敲低RNF41对胆管癌细胞上皮间质的转化作用；12只BALB/c小鼠，随机分为两组，对照组和敲低RNF41组，每组各6只，进行裸鼠成瘤实验、Western blot实验和免疫组织化学染色分析敲低RNF41对动物体内肿瘤生长的影响。 结果 实时定量荧光PCR分析显示胆管癌肿瘤组织中RNF41 mRNA表达水平明显高于相应的癌旁组织（P < 0.01），免疫组织化学染色评价RNF41的蛋白表达水平结果与其一致。TIMER 2.0数据库分析RNF41与胆管癌的分级，分期、淋巴结转移及生存期的关系，结果显示RNF41的表达与CHOL组织学分级和肿瘤淋巴结转移阶段密切相关（P < 0.01）；生存分析显示RNF41的高表达又与CHOL患者的不良预后密切相关。CCK-8、EdU、克隆形成实验显示在胆管癌细胞中与对照组组相比，敲低RNF41组抑制肿瘤细胞的增殖能力；Westernblot结果显示与对照组相比干扰RNF41组肿瘤细胞EMT标志物E-cadherin的水平显著提高，N-cadherin和MMP9的水平降低（P < 0.05）。然而，通过在裸鼠体内进行异体肿瘤移植实验和检测EMT标志蛋白发现，与体外细胞实验分析一致的EMT相关蛋白检测结果（P < 0.01）；此外与对照组相比RNF41敲低组抑制了裸鼠体内胆管癌细胞皮下成瘤的能力（P < 0.05）。 结论 RNF41可促进胆管癌的发生和发展，并与临床病理特征及患者的不良预后密切相关，敲低RNF41对胆管癌细胞的增殖、侵袭、EMT和肿瘤形成都有抑制作用。 Abstract:Objective To explore the role of ring finger protein 41 （RNF41） in the initiation and progression of cholangiocarcinoma. Methods The expression levels of RNF41 mRNA and protein in tumor tissues and adjacent normal tissues from 84 CHOL patients who underwent total surgical resection at the Second Affiliated Hospital of Kunming Medical University and Kunming Ganmei Hospital between January 2010 and December 2016 were analyzed using bioinformatics, Western blot, and immunohistochemistry. The TIMER 2.0 database was used to analyze the impact of RNF41 on the prognosis and survival of CHOL patients and the relationship between RNF41 and tumor clinical characteristics. RNF41 siRNA was transfected into HCC9810, RBE, and HUCCT1 cells. CCK-8, Edu, colony formation, and Western blot assays were conducted to evaluate the changes in proliferation of cholangiocarcinoma cells between the RNF41 knockdown group and the control group. Transwell assays and detection of EMT and migration markers were performed to assess changes in the invasion ability of cholangiocarcinoma cells between the RNF41 knockdown and control groups. Western blot was used to examine the effect of RNF41 knockdown on epithelial-mesenchymal transition in cholangiocarcinoma cells. Twelve BALB/c mice were randomly divided into two groups: a control group and an RNF41 knockdown group, with six mice in each group. Tumor formation assays, Western blot assays, and immunohistochemistry staining were carried out to investigate the effect of RNF41 knockdown on tumor growth in nude mice. Results Real-time quantitative fluorescence PCR analysis revealed that the expression level of RNF41 mRNA in cholangiocarcinoma tissues was significantly higher than that in the corresponding adjacent non-tumor tissues （P < 0.01）, and this trend was corroborated at the protein level by immunohistochemical staining. Using the TIMER 2.0 database, we further analyzed the correlation between RNF41 expression and clinicopathological features, including histological grade, tumor stage, lymph node metastasis, and patient survival. The results indicated that elevated RNF41 expression was significantly associated with advanced histological grade and lymph node metastasis in cholangiocarcinoma （P < 0.01）. Survival analysis demonstrated that high RNF41 expression was closely linked to poor prognosis in patients with cholangiocarcinoma （CHOL）. Functional assays, including CCK-8, EdU incorporation, and colony formation, showed that RNF41 knockdown significantly inhibited the proliferation of cholangiocarcinoma cells compared with the control group. Western blot analysis revealed that, following RNF41 silencing, the expression of the epithelial-mesenchymal transition （EMT） marker E-cadherin was markedly upregulated, whereas the levels of mesenchymal markers N-cadherin and MMP9 were significantly reduced （P < 0.05）. These findings were consistent with the results obtained from in vitro experiments （P < 0.01）. Moreover, in vivo studies showed that RNF41 knockdown suppressed subcutaneous tumor formation in nude mice （P < 0.05）. Conclusion RNF41 plays a critical role in promoting the occurrence and progression of cholangiocarcinoma and is closely associated with adverse clinicopathological features and poor prognosis in patients. The knockdown of RNF41 effectively suppresses the proliferation, invasion, epithelial-mesenchymal transition （EMT）, and tumorigenicity of cholangiocarcinoma cells. -
Key words:
- Cholangiocarcinoma /
- Ring finger protein 41 /
- Proliferation /
- Invasion
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图 1 RNF41在胆管癌中的表达水平及临床意义
A：TIMER数据库中RNF41在不同肿瘤组织与正常组织的mRNA表达水平（*P < 0.05；**P < 0.01；***P < 0.001）；B：人CHOL组织RNF41 mRNA的表达水平（**P < 0.01）；C：RNF41的表达与胆管癌分级、淋巴结转移及分期的关系（**P < 0.01）；D：胆管癌患者肿瘤组织及癌旁正常组织RNF41免疫组织化学染色分析（比例尺 = 20 μm，×40）；E：RNF41表达水平与胆管癌患者总生存率的相关性。
Figure 1. Expression level and clinical significance of RNF41 in cholangiocarcinoma
图 2 敲低RNF41抑制胆管癌细胞的增殖（$\bar x \pm s $，n ≥ 3）
A：CCK-8细胞增殖试剂盒对HCC9810/REB/HUCCT1细胞进行活性检测实验，使用酶标仪在OD 450 nm监测吸光度；B：EdU实验检测HCC9810/REB/HUCCT1细胞转染siRNF41后细胞增殖能力（比例尺 = 20 μm，×40）；C：用HCC9810/REB/HUCCT1细胞进行集落形成实验，检测干扰RNF41后细胞的增殖能力。
Figure 2. Knockdown of RNF41 inhibits the proliferation of cholangiocarcinoma cells （$\bar x \pm s $，n ≥ 3）
图 3 敲低RNF41对CHOL细胞侵袭及EMT的影响（$\bar x \pm s $，n ≥ 3）
A：用 HCC9810、REB及HUCCT1 对照细胞（Con）及 HCC9810、REB及HUCCT1干扰RNF41细胞（siRNF41）进行Western blot检测干扰RNF41对EMT标记物及迁移标记物的影响（*P < 0.05，**P < 0.01，***P < 0.001）；B：Transwell侵袭实验检测细胞侵袭能力（比例尺 = 20 μm，×40）。
Figure 3. Effects of RNF41 knockdown on invasion and EMT in CHOL cells （$\bar x \pm s $，n ≥ 3）
图 4 体内试验检测RNF41与CHOL发生发展之间的关系（$\bar x \pm s $，n ≥ 3）
A：接种shRNF41后5，10，15 d小鼠的体重检测及肿瘤体积分析结果（每组n = 5，**P < 0.01；NS：无统计学差异）；B：来自接种HCC9810-shRNF41或HCC9810 contrl细胞的小鼠的异种移植肿瘤图像（每组n = 6，比例尺 = 1 cm）；C：提取接种HCC9810-shRNF41或HCC9810 contrl细胞的小鼠组织蛋白进行Western blot分析增殖、迁移相关因子的表达水平，结果表示为平均值±SD（**P < 0.01）。
Figure 4. In vivo experiments examining the relationship between RNF41 and CHOL development （$\bar x \pm s $，n ≥ 3）
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