| 郑方静,赖红华,赖晓兰.微小RNA-216a调控JAK2/STAT3通路对鼻咽癌细胞增殖、侵袭、自噬及血管生成的影响[J].肿瘤学杂志,2021,27(11):905-914. |
| 微小RNA-216a调控JAK2/STAT3通路对鼻咽癌细胞增殖、侵袭、自噬及血管生成的影响 |
| Effects of microRNA-216a on Proliferation， Invasion， Autophagy and Microangiogenesis in Nasopharyngeal Carcinoma Cells Through Regulation of JAK2/STAT3 Signaling Pathway |
| 投稿时间：2021-08-18 |
| DOI： |
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| 中文关键词: 微小RNA-216a 酪氨酸蛋白激酶2/信号转导和转录激活因子3信号通路 鼻咽癌 增殖 侵袭 自噬 血管生成 |
| 英文关键词:microRNA-216a tyrosine protein kinase 2/signal transducer and activator of transcription 3 signaling pathway nasopharyngeal carcinoma proliferation invasion autophagy angiogenesis |
| 基金项目:福建省科技厅自然科学基金面上项目（2018J01222）；福建省卫生计生青年科研课题资助计划（2018-1-99） |
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| 中文摘要: |
| 摘 要：［目的］ 探究微小RNA（microRNA，miR）-216a靶向调控酪氨酸蛋白激酶2/信号转导和转录激活因子3（Janus protein tyrosine protein kinase 2/ signal transducer and activator of transcription 3，JAK2/STAT3）信号通路对鼻咽癌细胞增殖、侵袭、自噬及血管生成的影响。［方法］ Target Scan Human预测miR-216a与JAK2的结合位点，双荧光素酶实验验证miR-216a与JAK2是否结合；免疫组化染色检测正常鼻咽黏膜组织、鼻咽癌组织标本p-JAK2、p-STAT3的表达水平，并分析p-JAK2、p-STAT3表达水平与患者临床病理参数的关系。采用LipofectamineTM3000将miR-216a 模拟物双链小RNA、阴性对照双链小RNA转染至CNE-2细胞构建过表达miR-216a细胞系，细胞分为对照组、miR-NC组、miR-216a组。将抑制剂双链小RNA，抑制剂对照双链小RNA转染至CNE-2细胞构建低表达miR-216a细胞系，细胞分为对照组、抑制miR-NC组、抑制miR-216a组。采用qRT-PCR检测细胞JAK2、STAT3 mRNA表达水平；MTT法、Transwell小室、皮下种植瘤模型、小管形成实验、吖啶橙染色分别检测细胞增殖、侵袭、移植瘤生长、血管形成和自噬泡形成的能力；Western blot检测JAK2、p-JAK2、STAT3、p-STAT3、微管相关蛋白轻链3-Ⅱ（microtubule-associated protein light chain 3-Ⅱ，CL3-Ⅱ）、酵母自噬相关基因6的哺乳动物同源体（autophagy related protein ATG6，Beclin1）、血管内皮生长因子（vascular endothelial growth factor，VEGF）的表达水平。［结果］ 双荧光素酶实验验证了miR-216a对JAK2的靶向调控作用。鼻咽癌组织p-JAK2、p-STAT3阳性表达率（75.0%，68.3%）明显高于正常鼻咽黏膜组织（10%，13.3%）（P<0.05）。p-JAK2、p-STAT3表达越高，浸润深度越严重，越容易发生淋巴结和远处转移（P均<0.05）。细胞实验显示：与miR-NC组相比，miR-216a组细胞24、48、72、96 h吸光度值（0.32±0.03 vs 0.15±0.04、0.58±0.07 vs 0.24±0.06、0.86±0.10 vs 0.41±0.06、1.48±0.11 vs 0.75±0.07）、细胞侵袭数目（101.00±11.00 vs 22.67±5.77）、血管形成数目（127.53±10.61 vs 74.36±8.48）明显降低，移植瘤体积［（1 545.21±70.71）mm3 vs （485.24±91.92）mm3）］减小，JAK2、p-JAK2、p-STAT3、VEGF蛋白表达量（1.85±0.07 vs 0.93±0.11、1.03±0.07 vs 0.32±0.05、0.92±0.09 vs 0.44±0.08、1.17±0.05 vs 0.55±0.07）明显减少（P均<0.05）；细胞自噬能力、Beclin1、CL3-Ⅱ蛋白表达量（0.30±0.08 vs 1.03±0.11、0.38±0.04 vs 1.00±0.08）均显著性升高（P<0.05）。与抑制miR-NC组相比，抑制miR-216a组JAK2、p-JAK2、p-STAT3蛋白表达量（1.35±0.07 vs 1.93±0.11、0.55±0.07 vs 1.28±0.11、0.45±0.07 vs 1.23±0.11）明显升高（P均<0.05）。 ［结论］ JAK2、STAT3在鼻咽癌组织及细胞中表达量升高且发挥促癌作用，其作用机制可能是miR-216a通过靶向调控JAK2/STAT3信号通路，提高了细胞自噬水平，抑制了增殖、迁移和血管形成能力，对鼻咽癌患者的治疗和预后具有指导意义。 |
| 英文摘要: |
| Abstract： ［Objective］ To investigate the effects of microRNA-216a（miR-216a） on the proliferation， invasion， autophagy and angiogenesis of nasopharyngeal carcinoma cells and its relation with Janus protein tyrosine protein kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3) signal pathway. ［Methods］ TargetScanHuman was used to predict the binding site of miR-216a and JAK2， dual luciferase assay was used to verify whether miR-216a binds to JAK2， immunohistochemical staining was used to detect the expression levels of p-JAK2 and p-STAT3 in normal nasopharyngeal mucosal tissues and nasopharyngeal carcinoma tissue specimens， and the relationship between the expression levels of p-JAK2， p-STAT3 and the clinical pathological parameters of patients was analyzed. The miR-216a agomir and miR-NC agomir were transfected into CNE-2 cells with lipofectamineTM3000； and the antimiR-216a agomir and antimiR-NC agomir were transfected into CNE-2 cells. MTT method， transwell test， subcutaneous tumor model， tubule formation assay and acridine orange staining were used to detect the ability of cell proliferation， invasion， tumor growth， angiogenesis and autophagic vesicle formation， respectively. Western blot was used to detect the expression level of p-JAK2， p-STAT3， microtubule-associated protein light chain 3-Ⅱ(CL3-Ⅱ)， yeast autophagy-related gene 6 mammals homolog(autophagy related protein ATG6， Beclin1)， vascular endothelial growth factor(VEGF). ［Results］ The dual luciferase assay verified the targeted regulation of miR-216a on JAK2. The positive expression rate of p-JAK2 and p-STAT3 in nasopharyngeal carcinoma tissues(75.0%， 68.3%) was significantly higher than that of normal nasopharyngeal mucosal tissues(10.0%， 13.3%)(P<0.05). And the expression of p-JAK2 and p-STAT3 was positively correlated with the depth of invasion， lymph node and distant metastasis(P all <0.05). Compared with the miR-NC group， the absorbance value at 24 ， 48 ， 72， 96 h(0.32±0.03 vs 0.15±0.04， 0.58±0.07 vs 0.24±0.06， 0.86±0.10 vs 0.41 ±0.06， 1.48±0.11 vs 0.75±0.07)， number of cell invasion(101.00±11.00 vs 22.67±5.77)， number of blood vessel formation(127.53±10.61 vs 74.36±8.48) and transplanted tumor volume［(1 545.21±70.71) mm3 vs (485.24±91.92) mm3）］ were significantly reduced in the miR-216a group. The expression of JAK2， p-JAK2， p-STAT3 and VEGF protein(1.85±0.07 vs 0.93±0.11， 1.03±0.07 vs 0.32±0.05， 0.92±0.09 vs 0.44±0.08， 1.17±0.05 vs 0.55±0.07) was significantly reduced(P all <0.05). The expression of Beclin1， CL3-Ⅱ protein(0.30±0.08 vs 1.03±0.11， 0.38±0.04 vs 1.00±0.08) were significantly increased(P<0.05). Compared with the antimiR-NC group， the expression of JAK2， p-JAK2 and p-STAT3 protein in the antimiR-216a group(1.35±0.07 vs 1.93±0.11， 0.55±0.07 vs 1.28±0.11， 0.45±0.07 vs 1.23± 0.11) was significantly increased(P all <0.05). ［Conclusion］ The expression of JAK2 and STAT3 is up-regulated in nasopharyngeal carcinoma tissue and cells， which may play a cancer-promoting effect. The mechanism may be that miR-216a increases the level of autophagy and inhibits the ability of proliferation， migration and angiogenesis via regulating JAK2/STAT3 signaling pathway. |
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