| 孙海力,林文巧,李绍霄,陈恩就.MiR-19a在鼻咽癌细胞上皮间质转化中的作用*[J].浙江中西医结合杂志,2022,32(3): |
| MiR-19a在鼻咽癌细胞上皮间质转化中的作用* |
| Role of miR-19a in the epithelial-to-mesenchymal-transition (EMT) of nasopharyngeal cancer |
| 投稿时间：2021-05-18 修订日期：2021-10-16 |
| DOI： |
| 中文关键词: 鼻咽癌 miR-19a KRAS 上皮间质转化 |
| 英文关键词: |
| 基金项目:]:2018年度浙江省医药卫生科技计划项目 |
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| 中文摘要: |
| [摘 要] 目的：研究微小RNA-19a（microRNAs-19a, miR-19a）在鼻咽癌细胞中的表达水平以及对体外鼻咽癌细胞上皮间质转化（epithelial-mesenchymal transition, EMT）的作用。方法：利用实时荧光定量PCR（quantitative real-time PCR, RT-qPCR）法检测miR-19a在鼻咽癌细胞株CNE2、SUNE-1、HNE1、58F及人正常鼻咽上皮细胞株NP69中的转录水平；使用miR-19a模拟物（miR-19a mimics）、miR-19a抑制剂（miR-19a inhibitor）及其阴性对照瞬时转染体外鼻咽癌细胞；光学显微镜下观察细胞形态；Transwell小室实验检测细胞侵袭；Western blot实验测定miR-19a对鼻咽癌细胞EMT行为过程的调控作用；通过生物信息学工具TargetScan研究miR-19a可能靶向调控基因，荧光素酶报告实验测定荧光素酶活性在鼻咽癌细胞中的表达；通过挽救实验验证miR-19a是否直接作用于靶基因来调控鼻咽癌细胞的EMT行为。结果：miR-19a在NP69细胞中的表达水平分别为CNE2、SUNE-1、HNE1和58F细胞的5.88、3.57、11.11和6.67倍，差异均有统计学显著性（P < 0.001）；SUNE-1细胞转染miR-19 mimics促进miR-19表达后，细胞侵袭能力明显下降[(25.6±4.4)比(10.5±2.1), P < 0.01]，HNE1细胞转染miR-19 inhibitor抑制miR-19表达后，细胞侵袭能力明显增强[(16.9±2.5)比(34.2±4.9), P < 0.01]；通过生物学信息预测相关的靶基因和荧光素酶报告实验验证KRAS是miR-19a的直接作用靶点；mimics NC组、miR-19a mimics组和miR-19a mimics+KRAS mimics组侵袭细胞数分别为（28.3±3.4）、（11.6±1.5）和（23.7±2.8），上调KRAS的表达可部分逆转miR-19a对鼻咽癌细胞侵袭能力的抑制作用（P < 0.01）；Western blot实验结果表明，上调miR-19a的表达可抑制N-cadherin、p-FAK和p-SRC蛋白的表达，而E-cadherin的表达上调，差异均有统计学意义（P < 0.05）；细胞经转染KRAS mimics后，miR-19a mimics对E-cadherin、N-cadherin、p-FAK和p-SRC蛋白表达的调控作用明显被削弱。结论：miR-19a可抑制鼻咽癌细胞EMT，该抑癌作用可能是miR-19a 通过靶基因KRAS负性调控FAK/SRC信号通路来实现的。 |
| 英文摘要: |
| [ABSTRACT] AIM: To investigate the expression of miR-19a in four nasopharyngeal cancer cell lines and the function of miR-19a on the process of nasopharyngeal cancer EMT.
METHODS: The relative level of miR-19a in a panel of nasopharyngeal cancer cell lines was assessed by RT-qPCR, namely CNE2, SUNE-1, HNE1 and 58F, along with NP69, which is a normal human nasopharyngeal epithelial cell line. MiR-19a-overexpression SUNE-1 cells and miR-19a-knockdown HNE1 cells were established by transiently transfection with miR-19a mimics and miR-19a inhibitor, respectively. The migratory capacity of nasopharyngeal cancer cells was assessed using transwell migration chambers. Western blot was employed to analyze the effect of miR-19a on the EMT process of nasopharyngeal cancer cells. Bioinformatics software TargetScan was employed to detect the possible target gene of miR-19a, and such predication was verified by luciferase reporter experiment. Following, the rescue experiments were used to confirm whether miR-19a regulates EMT of nasopharyngeal cancer by affecting the target gene directly. RESULTS: The level of miR-19a in NP69 cells was almost 5.88, 3.57, 11.11 and 6.67 times than that in CNE2, SUNE-1, HNE1 and 58F cells, respectively. After transfection with miR-19a mimic, the migratory capacity of SUNE-1 cells decreased significantly [(25.6±4.4) vs (10.5±2.1), P < 0.01]. On the contrary, down-regulation of miR-19a dramatically promoted the migratory capacity of HNE1 cells [(16.9±2.5) vs (34.2±4.9), P < 0.01]. Biology information forecast the target genes and luciferase assay confirmed that KRAS was a direct target gene of miR-19a. The invaded cells in mimics NC, miR-19a mimics, miR-19a mimics+KRAS groups were (28.3±3.4), (11.6±1.5) and (23.7±2.8), respectively. The promotion of KRAS could reverse the inhibitory effect of miR-19a on the invasion of nasopharyngeal cancer cells. Western blot experiment confirm that the levels of N-cadherin, p-FAK and p-SRC were inhibited after upregulation of miR-19a, whereas the expression of E-cadherin was increased. The regulatory effect of miR-19a on the levels of E-cadherin, N-cadherin, p-FAK and p-SRC was attenuated by the promotion of KRAS.
CONCLUSION: MiR-19a inhibits the EMT process of nasopharyngeal cancer cells. The tumor suppressor function, at least partly, is realized by targeting gene KRAS and negatively regulate the FAK/SRC signaling pathway. |
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