舌鳞癌化疗耐药相关线粒体微小RNA的筛选和鉴定

doi:10.12016/j.issn.2096-1456.2019.07.002

摘要/Abstract

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:

R739.8

林欣祤,陈伟雄,雷新元,欧展鹏,范松,李劲松. 舌鳞癌化疗耐药相关线粒体微小RNA的筛选和鉴定[J]. 口腔疾病防治, 2019, 27(7): 417-422.

Xinyu LIN,Weixiong CHEN,Xinyuan LEI,Zhanpeng OU,song FAN,Jinsong LI. Screening and identification of mitochondrial miRNAs related to chemotherapy resistance in tongue squamous cell carcinoma[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2019, 27(7): 417-422.

图/表 3

表1 qRT-PCR引物序列

Table 1 Nucleotide sequences of the qRT-PCR primers

引物	序列（5′→3′）
β-actin-F	5′-CTACCTCATGAAGATCCTCACCGA-3′
β-actin-R	5′-TTCTCCTTAATGTCACGCACGATT-3′
GAPDH-F	5′-GGACCTGACCTGCCGTCTAG-3′
GAPDH-R	5′-GTAGCCCAGGATGCCCTTGA-3′
CYTB-F	5′-CAAACTAGGAGGCGTCCTTG-3′
CYTB-R	5′-CTGGTTGTCCTCCGATTCAG-3′
ND5-F	5′-CTATCACCACTCTGTTCGCAG-3′
ND5-R	5′-GTGGTTGGTTGATGCCGATTG-3′

图1 在舌鳞癌耐药细胞和其亲本细胞中筛选差异表达的mitomiRs a：Western blotting显示线粒体的纯度。β-actin和GAPDH作为胞浆内参, CYTB、ND5和TOM20作为线粒体内参;b：qRT-PCR显示线粒体的纯度。β-actin和GAPDH作为胞浆内参,CYTB和ND5作为线粒体内参;c：qRT-PCR显示线粒体的纯度,β-actin和GAPDH作为核内参,CYTB和ND5作为线粒体内参;d：CAL-27和CAL-27-re细胞线粒体（Mito）、细胞质（Cyto）和总胞浆（Total）中miRNA表达聚类图。在所有6个组分中共检测到263个miRNAs。红色表示线粒体中miRNAs显著上调;蓝色表示线粒体中miRNAs表达显著下调;e：miRNA的探针分布,CAL-27-re细胞的线粒体（Mito）/细胞质（Cyto）（左上图）,CAL-27-re/CAL-27总体miRNA（右上图）。结果显示,11个mitomiRs在CAL-27-re表达上调（≥ 1.5倍）,5个mitomiRs表达下调（左下图）（≥ 1.5倍）;f：维恩图显示了3个组分mitomiRs的交集,11个mitomiRs在CAL-27-re中高表达（≥ 1.5倍）;g：3个组分mitomiRs表达量系统聚类分析,11个mitomiRs在CAL-27-re细胞中表达上调。mitomiRs：线粒体微小RNA

Figure 1 Screening of the differentially expressed mitochondrial microRNAs in the tongue squamous cell carcinoma cisplatin-resistant and parental cell lines

图2 舌鳞癌细胞及临床样本中mitomiRs的表达a：qRT-PCR验证显示11个mitomiRs主要富集于线粒体,12SrRNA作为内参;b：qRT-PCR结果显示,与CAL-27细胞相比,CAL-27-re细胞中11种mitomiRs上调。U6作为内参;c：MTT法检测顺铂耐药以及敏感患者的肿瘤原代细胞的顺铂敏感性;d：在临床样本中对11个mitomiRs进行检测,#表示P < 0.05,**表示P < 0.001,具有统计学意义,NS表示不具有统计学意义。 mitomiRs：线粒体微小RNA

Figure 2 Quantification of mitomiR expression in the mitochondria of tongue squamous cell carcinoma cells and tumors

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