口腔疾病防治 ›› 2019, Vol. 27 ›› Issue (7): 417-422.DOI: 10.12016/j.issn.2096-1456.2019.07.002

• 基础研究 • 上一篇    下一篇

舌鳞癌化疗耐药相关线粒体微小RNA的筛选和鉴定

林欣祤,陈伟雄,雷新元,欧展鹏,范松,李劲松()   

  1. 中山大学孙逸仙纪念医院口腔颌面外科,广东 广州(510120)
  • 收稿日期:2019-03-03 修回日期:2019-04-23 出版日期:2019-07-20 发布日期:2019-07-24
  • 通讯作者: 李劲松
  • 作者简介:林欣祤,医师,硕士研究生在读,Email: 860340417@qq.com
  • 基金资助:
    国家自然科学基金项目(81872194,81672676,81772890)

Screening and identification of mitochondrial miRNAs related to chemotherapy resistance in tongue squamous cell carcinoma

Xinyu LIN,Weixiong CHEN,Xinyuan LEI,Zhanpeng OU,song FAN,Jinsong LI()   

  1. Department of Oral and Maxillofacial Surgery, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, Guangzhou 510120, China
  • Received:2019-03-03 Revised:2019-04-23 Online:2019-07-20 Published:2019-07-24
  • Contact: Jinsong LI

摘要:

目的 探讨舌鳞癌细胞线粒体微小RNA(mitochondrial microRNAs,mitomiRs)的表达,筛选出与化疗耐药相关的mitomiRs。方法 提取舌鳞癌细胞系CAL-27及其顺铂耐药细胞株CAL-27-re的线粒体、细胞质、总细胞RNA,采用高通量miRNA芯片筛选出耐药和亲本细胞中差异表达的mitomiRs,应用qRT-PCR验证在CAL-27和CAL-27-re中表达上调的mitomiRs,并在化疗耐药及敏感的舌鳞癌患者样本中验证表达上调的mitomiRs。结果 芯片结果显示,在CAL-27和CAL-27-re的线粒体、细胞质和总细胞RNA共6个组分中共检测到263个miRNA,其中共有57个mitomiRs和134个细胞质微小RNA(cytoplasmic microRNAs,cytomiRs);与总miRNA相比,CAL-27-re细胞中有35个mitomiRs表达上调,CAL-27细胞中有31个mitomiRs表达上调(≥ 1.5倍)。进一步比较分析亲本和耐药细胞中差异表达的mitomiRs,在CAL-27-re细胞中有miR-2392、miR-4462、miR-1290、miR-4449、miR-1268a、miR-1246、miR-371a-5p、miR-3934-5p、miR-4271、miR-513p、miR-664b-3p共11个mitomiRs表达上调;而5个表达下调的mitomiRs则分别是miR-188-5p、miR-1973、miR-3653、miR-4499、miR-5787;其他41个mitomiRs在两种细胞株中的表达水平几乎相同。qRT-PCR与miRNAs 芯片结果相符合,并且在CAL-27和CAL-27-re中得到验证的11个表达上调的mitomiRs中,有miR-1268a、miR-2392、miR-4462、miR-1290、miR-4449共5个mitomiRs在化疗耐药舌鳞癌临床样本中也同样表达上调。结论 顺铂化疗耐药和敏感的舌鳞癌细胞中存在差异表达的mitomiRs,特异高表达的mitomiRs:miR-1268a、miR-2392、miR-4462、miR-1290、miR-4449可能在舌鳞癌化疗耐药中具有重要作用。

关键词: 线粒体miRNAs, 化疗耐药, 线粒体转录, 舌鳞癌, 高通量, miRNA芯片, miRNA表达谱

Abstract:

Objective To investigate the differential expression of mitochondrial microRNAs (mitomiRs) in tongue squamous cell carcinoma (TSCC) and to screen out mitomiRs related to chemotherapy resistance. Methods Mitochondrial, cytoplasmic, and total cellular RNAs were extracted from the squamous cell carcinoma cell line CAL-27 and the cisplatin-resistant cell line CAL-27-re. High-throughput miRNA microarrays were used to screen for differentially expressed mitomiRs between the drug-resistant and parental cells. The upregulated mitomiRs in the CAL-27 and CAL-27-re cells and in samples from chemoresistant and chemosensitive tongue squamous cell carcinoma patients were verified by qRT-PCR. Results The microarray detected 263 miRNAs in 6 components of the mitochondrial, cytoplasmic and total cellular RNAs from the CAL-27 and CAL-27-re cells, including 57 mitomiRs and 134 cytoplasmic microRNAs (cytomiRs). Compared with the total miRNAs, 35 mitomiRs were upregulated in the CAL-27-re cells, and 31 mitomiRs were upregulated in the CAL-27 cells (≥ 1.5-fold). Further comparative analysis of mitomiRs that were differentially expressed between the parental and drug-resistant cells identified 11 upregulated mitomiRs (miR-2392, miR-4462, miR-1290, miR-4449, miR-1268a, miR-1246, and miR-371a-5p, miR-3934-5p, miR-4271, miR-513p, and miR-664b-3p) and 5 downregulated mitomiRs (miR-188-5p, miR-1973, miR -3653, miR-4499, and miR-5787); the expression levels of the other 41 mitomiRs were almost identical in both cell lines. The qRT-PCR results were consistent with the miRNA microarray results. The 11 upregulated mitomiRs that were validated between the CAL-27 and CAL-27-re cells included miR-1268a, miR-2392, miR-4462, and miR-1290. Additionally, 5 mitomiRs, including miR-4449, were upregulated in the clinical chemotherapy-resistant tongue squamous cell carcinoma samples. Conclusion Differentially expressed mitomiRs were found between cisplatin-resistant and cisplatin-sensitive tongue squamous cell carcinoma cells. mitomiRs with high expression levels (miR-2392, miR-4462, miR-1290, miR-4449 and miR-1268a) may play important roles in the drug resistance of tongue squamous cell carcinoma.

Key words: mitochondrial microRNA, chemoresistance, mitochondrial transcription, tongue squamous cell carcinoma, high throughput, miRNA array, miRNA expression spectrum

中图分类号: