长链非编码RNA MCM3AP-AS1在胆管癌细胞中的表达及其功能

Expression of long non-coding RNA MCM3AP-AS1 in cholangiocarcinoma cells and its function

摘要:

背景与目的 长链非编码RNA MCM3AP-AS1（MCM3AP-AS1）在原发性肝癌、乳腺癌及胶质母细胞瘤等多种肿瘤中发挥癌基因功能，然而MCM3AP-AS1在胆管癌中的表达、功能及作用机制尚知之甚少。因此，本研究观察MCM3AP-AS1在胆管癌细胞中的表达及其对细胞增殖和侵袭的影响，并初步探讨机制。方法 采用qRT-PCR法检测胆管癌细胞株（CCLP、RBE、9810、HuCCT1）及人肝内胆管上皮细胞株（HIBEC）中MCM3AP-AS1的表达。将胆管癌细胞转染MCM3AP-AS1 siRNA后，以转染无义序列的胆管癌细胞为阴性对照，分别用MTT实验和Transwell实验检测细胞增殖与侵袭能力的变化，用Western blot法检测JAK/STAT3信号通路与上皮间质转化（EMT）相关蛋白表达的变化；最后，用JAK/STAT3通路激动剂白血病抑制因子（LIF）行功能拯救实验验证。结果 所有胆管癌细胞系中MCM3AP-AS1表达量均明显高于HIBEC（均P<0.05）。与阴性对照组比较，CCLP细胞转染MCM3AP-AS1 siRNA后增殖能力与侵袭能力均明显减弱（均P<0.05）；JAK1/2与STAT3表达量无明显变化（均P>0.05），但p-JAK1/2与p-STAT3表达量明显降低（均P<0.05），同时，EMT相关蛋白E-cadherin表达升高、vimentin表达降低（均P<0.05）。功能拯救实验结果显示，同时加入LIF后，MCM3AP-AS1沉默对CCLP细胞后的以上作用均被取消，与阴性对照组间差异均无统计学意义（均P>0.05）。结论 胆管癌细胞中MCM3AP-AS1表达上调，MCM3AP-AS1可能通过活化JAK/STAT3通路与EMT过程而促进胆管癌细胞增殖和侵袭。

Abstract:

Background and Aims Long non-coding RNA MCM3AP-AS1 (MCM3AP-AS1) plays an oncogene function in various tumors such as primary liver cancer, breast cancer and glioblastoma. However, the expression, function and mechanism of MCM3AP-AS1 in cholangiocarcinoma are still poorly understood. Therefore, this study was conducted to observe the expression of MCM3AP-AS1 in cholangiocarcinoma cells and its effect on cell proliferation and invasion, and preliminarily investigate the underlying mechanism.Methods The expressions of MCM3AP-AS1 in cholangiocarcinoma cell lines (CCLP, RBE, 9810, HuCCT1) and human intrahepatic bile duct epithelial cell line (HIBEC) were detected by qRT-PCR. In cholangiocarcinoma cells after transfection with MCM3AP-AS1 siRNA, using cholangiocarcinoma cells transfected with scrambled sequences as negative control, the changes in proliferative and invasion abilities were determined by MTT assay and Transwell assay, and the changes in expressions of the proteins associated with the JAK/STAT3 signaling pathway and epithelial-mesenchymal transition (EMT) process were determined by Western blot. Finally, functional rescue experiment was performed using the JAK/STAT3 pathway agonist leukemia inhibitory factor (LIF) for validation.Results The expressions of MCM3AP-AS1 in all cholangiocarcinoma cell lines were significantly higher than that in HIBEC (all P<0.05). In CCLP cells after transfection with MCM3AP-AS1 siRNA compared with negative control, the proliferative and invasion abilities were significantly decreased (both P<0.05); the expression levels of JAK1/2 and STAT3 showed no significant changes (both P>0.05), but the expression levels of p-JAK1/2 and p-STAT3 were significantly decreased (both P<0.05), and meanwhile, the expression of EMT-related protein E-cadherin was increased and vimentin was decreased (both P<0.05). The results of functional rescue experiment showed that the effects of MCM3AP-AS1 silencing on CCLP cells were all abolished by simultaneous addition of LIF, and all parameters had no significant differences with those in negative control group (all P>0.05).Conclusion The expression MCM3AP-AS1 is up-regulated in cholangiocarcinoma cells, and it promotes the cell proliferation and invasion of the cholangiocarcinoma cells probably through activating the JAK/STAT3 signaling pathway and EMT process.