miR-135b-5p在口腔鳞状细胞癌中的表达及相关生物信息学分析
石河子大学医学院第一附属医院口腔科,新疆维吾尔自治区 石河子(832000)
Expression and relevant bioinformatics analysis of miR-135b-5p in oral squamous cell carcinoma
Department of Stomatology, the First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi 832000, China
通讯作者: 黎昌学,主任医师,硕士,Email:lichangxue100@163.com, Tel: 86-993-2810069
责任编辑: 周春华
收稿日期: 2021-02-28 修回日期: 2021-04-10
基金资助: |
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Corresponding authors: LI Changxue, Email:lichangxue100@163.com, Tel: 86-993-2810069
Received: 2021-02-28 Revised: 2021-04-10
Fund supported: |
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作者简介 About authors
赵格,住院医师,硕士,Email:zhaoge_1994_y@163.com
目的 探索miR-135b-5p在口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)及癌旁组织中的表达及其临床意义,预测miR-135b-5p靶基因并进行相关生物信息学分析。方法 通过肿瘤与癌症基因图谱(the Cancer Genome Atlas,TCGA)、基因表达数据库(Gene Expression Omnibus,GEO)数据库分析miR-135b-5p在OSCC组织和癌旁组织中的表达并分析其临床意义;临床收集新鲜组织标本,采用实时荧光定量PCR验证不同组织中miR-135b-5p的表达情况。采用生物信息学方法预测miR-135b-5p的靶基因并进行通路富集分析。构建蛋白互作网络筛选关键靶基因。结果 OSCC组织中miR-135b-5p表达量较癌旁组织上调,差异有统计学意义(P < 0.001);miR-135b-5p表达量对OSCC组织具有良好的诊断效能(AUC=0.960,P < 0.001);OSCC组织中miR-135b-5p表达水平与组织病理分级相关(P=0.011);生信分析结果显示,miR-135b-5p的靶基因富集在与肿瘤相关的钙离子、cGMP-PKG、cAMP信号通路中;筛选得到10个关键靶基因:DLG2、ANK3、ERBB4、SCN2B、NBEA、GABRB2、ATP2B2、SNTA1、CACNA1D、SPTBN4。结论 miR-135b-5p可作为一种促癌基因参与OSCC的发生发展,并具有成为OSCC诊断标志物及治疗靶点的潜在应用价值。
关键词:
Objective To observe the clinical significance of miR-135b-5p in oral squamous cell carcinoma (OSCC) tissues and to conduct a bioinformatics analysis of its predicted target genes. Methods The expression levels of miR-135b-5p in OSCC tissues and adjacent normal tissues were compared using data from TCGA and GEO databases, and the correlations of miR-135b-5p expression level with clinicopathologic characteristics were analyzed. Fresh tissues were collected in the clinic, and the expression of miR-135b-5p was verified by quantitative real-time PCR. The target genes with enriched pathways were analyzed by using bioinformatics methods. A protein-protein interaction network was constructed to screen hub genes. Results The expression levels of miR-135b-5p were significantly upregulated in OSCC tissues compared to adjacent normal tissues (P < 0.001) and had a good diagnostic capability (AUC=0.960, P < 0.001). The expression level of miR-135b-5p was positively correlated with histopathological grading (P=0.011). Enrichment analyses revealed that the target genes of miR-135b-5p were significantly associated with tumor-related signaling pathways, such as the calcium signaling pathway, the cGMP-PKG signaling pathway and the cAMP signaling pathway. Ten core target genes were obtained by screening: DLG2, ANK3, ERBB4, SCN2B, NBEA, GABRB2, ATP2B2, SNTA1, CACNA1D, and SPTBN4. Conclusion miR-135b-5p may act as an oncogene miRNA in OSCC and has the potential value of acting as a diagnostic biomarker and therapeutic target for OSCC.
Keywords:
本文引用格式
赵格, 黎昌学, 郭超, 朱慧.
ZHAO Ge, LI Changxue, GUO Chao, ZHU Hui.
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口腔癌为全球第九大恶性肿瘤,约90%以上的口腔癌是口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC),虽然近年来治疗OSCC的手术和放化疗技术有了一定进步,但5年生存率仍仅为50%左右[1]。随着分子生物学的发展,越来越多的研究显示微小RNA(microRNA,miRNA)在肿瘤细胞的生长、分化、增殖和凋亡等过程中发挥了重要的作用[2]。miR-135b-5p在胰腺癌[3]、胃癌[4]、结直肠癌[5]等恶性肿瘤中异常高表达,但miR-135b-5p在OSCC中的表达情况尚不明确。本研究通过对公开发表的转录组测序数据、基因芯片数据进行分析,辅以新鲜组织样本实验验证,探讨miR-135b-5p在OSCC患者中的表达水平及其与临床特征的关系。同时采用生物信息学方法预测miR-135b-5p的靶基因并进行通路富集分析。
1 资料和方法
1.1 研究资料
肿瘤与癌症基因组图谱(the Cancer Genome Atlas,TCGA)中下载与OSCC相关的miR-135b-5p表达数据(247例癌组织,17例癌旁组织)及其完整的临床资料(222例);与OSCC相关的mRNA表达信息(240例癌组织,17例癌旁组织)。基因表达数据库(Gene Expression Omnibus,GEO)下载OSCC相关基因表达谱数据GSE45238(40例癌与癌旁配对组织)。收集石河子大学医学院第一附属医院2017年1月至2020年6月手术切除的30例口腔鳞癌组织和癌旁组织(距癌组织边缘 ≥ 2 cm)。所有OSCC患者手术前均未接受放疗或化疗。所有手术取材标本离体后立即放置在液氮中,后储存在-80 ℃冰箱中。本研究由石河子大学医学院第一附属医院伦理委员会批准(文件编号:AF/SC-08/01.0),本研究收集的标本已取得患者的书面知情同意。
1.2 研究方法
1.2.1 公共数据库中miR-135b-5p表达分析 TCGA、GEO官网下载OSCC组织及癌旁组织中miR-135b-5p原始表达数据,标准化处理后行差异分析。将miR-135b-5p表达信息与临床信息合并,用于后续临床相关性分析。使用R语言edgeR包筛选出TCGA下载的mRNA表达谱中的差异表达的mRNA。
1.2.2 新鲜组织中miR-135b-5p表达分析 依据Biospin miRNA Extraction Kit试剂盒说明完成组织中miRNA提取,测量OD260/280值在1.8~2.0之间。使用miRNA ALL-IN-One cDNA Synthesis Kit将总miRNA反转录成cDNA。以U6为内参(表1),使用EvaGreen miRNA qPCR Master Mix-Low ROX试剂盒进行实时荧光定量聚合酶链式反应(quantitative Real-Time PCR,qRT-PCR)。扩增反应条件如下:预变性95 ℃ 10 min,1个循环;变性95 ℃ 10 s,退火58 ℃ 30 s,延伸72 ℃ 30 s,40个循环。所有反应均设置3个复孔以减少加样等造成的误差影响实验结果。扩增反应结束后分析溶解曲线,判断扩增产物是否存在非特异性扩增。分析扩增曲线,采用2-△△Ct计算miR-135b-5p在OSCC组织中的相对表达量。
表1 miR-135b-5p的实时荧光定量PCR扩增引物序列
Table 1
Gene | Primer | Primer Sequence |
---|---|---|
U6 | Forward | CTCGCTTCGGCAGCACA |
U6 | Reverse | AACGCTTCACGAATTTGCGT |
miR-135b-5p | Forward | TATGGCTTTTCATTCCTATGTGA |
miR-135b-5p | Reverse | TGGTGTCGTGGAGTCG |
1.2.3 miR-135b-5p靶基因预测及功能富集分析 从miRTarBase、targetScan和miRDB三个数据库预测miR-135b-5p的靶基因,选取至少2个数据库中预测到的靶基因与TCGA数据库下载分析的差异mRNA取交集。通过R语言对这些交叉基因进行京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)通路富集分析。
1.2.4 miR-135b-5p关键靶基因筛选 通过STRING数据库绘制蛋白互作网络(protein-protein interaction network,PPI)用于揭示靶基因之间的关系,置信度参数为0.400。使用Cytoscape3.6.1及其插件cytoHubba筛选出前10个关键靶基因。
1.3 统计学方法
使用R语言3.6.3及其附属包进行统计分析并绘图。两组间差异采用Wilcoxon检验。卡方检验分析miR-135b-5p表达情况与OSCC患者临床病理特征关系。通过受试者工作特征曲线(receiver operator characteristic curve,ROC)分析评价miR-135b-5p表达对于OSCC患者诊断的准确性。P < 0.05为差异具有统计学意义。
2 结果
2.1 miR-135b-5p在OSCC组织及癌旁组织中的表达差异
TCGA、GEO官网下载OSCC组织原始表达数据,以及本研究中新鲜组织中miR-135b-5p的qRT-PCR结果的分析显示,OSCC组织中miR-135b-5p表达量高于癌旁组织,差异具有统计学意义(P < 0.001)。见图1。
图1
图1
miR-135b-5p在口腔鳞状细胞癌组织中高表达
Figure 1
miR-135b-5p is frequently increased in oral squamous cell carcinoma cancer tissues
Data were obtained from (a) TCGA database, (b) GEO database and (c) qRT-PCR result of fresh tissues. miR-135b-5p was highly expressed in oral squamous cell carcinoma compared with paracancerous normal tissues
2.2 miR-135b-5p表达水平与临床病理关系
表2 miR-135b-5p表达与临床病理相关性
Clinicopathological factor | miR-135b-5p expression | Case | χ2 | P | |
---|---|---|---|---|---|
High | Low | ||||
Age/year | |||||
≤ 60 | 55 | 56 | 111 | 0.018 | 0.893 |
> 60 | 56 | 55 | 111 | ||
Gender | |||||
Male | 74 | 73 | 147 | 0.020 | 0.887 |
Female | 37 | 38 | 75 | ||
Clinical stages | |||||
Ⅰ+Ⅱ | 28 | 25 | 53 | 0.223 | 0.637 |
Ⅲ+Ⅳ | 83 | 86 | 169 | ||
Histopathological grading | |||||
G1+G2 | 82 | 97 | 179 | 6.490 | 0.011 |
G3 | 29 | 14 | 43 | ||
Lymph node status | |||||
N0 | 51 | 49 | 100 | 0.073 | 0.787 |
N1+N2+N3 | 60 | 62 | 122 |
图2
图2
miR-135b-5p诊断口腔鳞状细胞癌的受试者工作曲线
Figure 2
Receiver operating characteristic curve of miR-135b-5p in the diagnosis of oral squamous cell carcinoma
The area under the receiver operating characteristic curve was 0.960. When the cut-off value was 0.89, the sensitivity was 100%, and the specificity was 83.33%. Thus, this marker has high diagnostic efficacy for oral squamous cell carcinoma, AUC: area under curve
2.3 miR-135b-5p靶基因预测及功能分析
图3
图3
miR-135b-5p的预测靶基因数目韦恩图
Figure 3
Venn diagram of predicted target genes of miR-135b-5p
The target genes of miR-135b-5p were predicted by the miRDB, miRTAR Base and Target Scan databases, and 503 target genes were obtained by intersecting each pair of databases
表3 miR-135b-5p靶基因KEGG信号通路显著性富集分析结果
Table 3
ID | Description | P | Target gene ID |
---|---|---|---|
hsa05032 | Morphine addiction | < 0.001 | PDE1C/GNG7/PDE3B/GABRB2/PDE7B |
hsa04020 | Calcium signaling pathway | < 0.001 | ATP2B3/PDE1C/ERBB4/CACNA1D/ATP2B2 |
hsa04726 | Serotonergic synapse | < 0.001 | GNG7/SLC6A4/GABRB2/CACNA1D |
hsa04723 | Retrograde endocannabinoid signaling | < 0.001 | GNG7/RIMS1/GABRB2/CACNA1D |
hsa04924 | Renin secretion | 0.001 | PDE1C/PDE3B/CACNA1D |
hsa04022 | cGMP-PKG signaling pathway | 0.001 | ATP2B3/PDE3B/CACNA1D/ATP2B2 |
hsa04727 | GABAergic synapse | 0.002 | GNG7/GABRB2/CACNA1D |
hsa04925 | Aldosterone synthesis and secretion | 0.003 | ATP2B3/CACNA1D/ATP2B2 |
hsa04024 | cAMP signaling pathway | 0.004 | ATP2B3/PDE3B/CACNA1D/ATP2B2 |
hsa04724 | Glutamatergic synapse | 0.005 | GRIK3/GNG7/CACNA1D |
KEGG: Kyoto Encyclopedia of Genes and Genomes; PDE1C: phosphodiesterase 1C; GNG7: guaninenucleotide-binding protein γ7; PDE3B: phosphodiesterase 3B; GABRB2: γ-aminobutyric acid neurotransmitter receptor β2 subunit gene; PDE7B: phosphodiesterase 7B; ATP2B3: plasma membrane calcium ATPase 3; ERBB4: erb-B2receptor tyrosine kinase 4; CACNA1D: calcium voltage-gated channel subunit alpha1 D; ATP2B2: plasma membrane calcium ATPase 2; SLC6A4: solute carrier family 6 member 4; RIMS1: rab-interacting molecules 1
2.4 miR-135b-5p关键靶基因筛选
上述得到的60个预测靶基因中,共有35个被过滤到靶基因PPI网络中,筛选得到10个关键靶基因(图4),分别为大同源物2(discs-large homolog 2,DLG2)、锚蛋白重复序列3(ankyrin repeat 3,ANK3)、Erb-B2受体酪氨酸激酶4 (Erb-B2receptor tyrosine kinase 4,ERBB4)、电压门控性钠通道二型beta亚单位(sodium channel voltage-gated type II beta,SCN2B)、蛋白激酶锚定蛋白(neurobeachin,NBEA)、γ-氨基丁酸β2亚基(γ-aminobutyric acid neurotransmitter receptor β2 subunit gene,GABRB2)、质膜钙ATP酶异构体2(plasma membrane calcium ATPase 2,ATP2B2)、α-互养蛋白(α-syntrophin,SNTA1)、钙电压门控通道亚单位α1D(calcium voltage-gated channel subunit alpha1 D,CACNA1D)、血影蛋白β非红细胞4(spectrin beta non-erythrocytic 4,SPTBN4)。
图4
图4
蛋白互作网络的关键靶基因
Figure 4
Hub genes of protein-protein interaction network
Ten hub genes were screened by Cytoscape software: the darker the color, the more important the function in the interaction. DLG2: discs-large homolog 2; ANK3: ankyrin repeat 3; ERBB4: erb-B2receptor tyrosine kinase 4; SCN2B: sodium channel voltage-gated type II beta; NBEA: neurobeachin; GABRB2: γ-aminobutyric acid neurotransmitter receptor β2 subunit gene; ATP2B2: plasma membrane calcium ATPase 2; SNTA1: α-syntrophin; CACNA1D: calcium voltage-gated channel subunit alpha1 D; SPTBN4: spectrin beta non-erythrocytic 4; UTRN: background Utrophin; DTNA: dystrobrevin-α; RIMS1: rab-interacting molecules 1; SLC6A4: solute carrier family 6 member 4; ATP2B3: plasma membrane calcium ATPase 3; GNG7: guaninenucleotide-binding protein γ-7; KCNB1: voltage-gated potassium channel sub-family B member 1; GRIK3: glutamate ionotropic receptor Kainate type subunit 3; PGR: epithelial progesterone receptor; SHISA6: shisa family member 6; PRLR: prolact inreceptor; DCLK1: doublecortin-like kinase1; CTTNBP2: cortactin binding protein 2
3 讨论
OSCC的发生发展是一个多阶段、多步骤、多基因、多通路调控的复杂过程[6]。miRNA表达异常在肿瘤的发生、发展中发挥着重要作用,深入研究miRNA与肿瘤的相关性将有望为肿瘤的诊断及靶向治疗提供新的思路。miR-135b位于1号染色体上,有miR-135b-5p和miR-135b-3p两种剪切成熟体[7]。miR-135b作为一种促癌因子,在多种肿瘤中均有着重要的作用,控制着癌细胞的生物学行为并影响耐药,如:高表达的miR-135b-5p可通过靶向NR3C2促进胰腺癌细胞的迁移、侵袭和上皮间质转化[3],可抑制胃癌细胞凋亡并诱导顺铂耐药[4];抑制miR-135b的表达能够抑制结大肠癌细胞的增殖,提高患者对奥沙利铂的敏感性[8],降低鼻咽癌细胞的增殖、迁移和侵袭能力[9]。
目前miR-135b-5p在OSCC中的表达情况仍存在争议。有研究表明,微阵列实验结果显示miR-135b在OSCC中呈低表达;而另有研究证明miR-135b-5p在OSCC中呈高表达[10]。为了进一步明确miR-135b-5p在OSCC中的表达情况,本研究首先基于TCGA、GEO数据库大样本优势分析了OSCC组织中miR-135b-5p的表达情况,后辅以新鲜组织进行体外实验验证。实验结果均表明miR-135b-5p在OSCC组织中高表达,差异有统计学意义;且miR-135b-5p表达量对于OSCC具有良好的诊断效能。此外OSCC组织中miR-135b-5p表达与病理分级相关,低分化程度患者组织中miR-135b-5p表达量高于高中分化患者,差异具有统计学意义。Kademani等[11]发现每升高一个组织学等级,患者生存率降低44%,组织学等级是预测生存率的独立因素。研究表明低分化的细胞由于缺乏凝聚力,具有单细胞侵袭模式,随着细胞分化程度的降低,淋巴结转移的机会会增加[12]。因此笔者推测,作为诊断标志物的miR-135b-5p的高表达可能预示着不良预后。
miRNAs是转录后水平调控基因表达的关键分子。KEGG富集分析结果显示,miR-135b-5p的预测靶基因集中富集在与肿瘤密切相关的3个通路中:钙离子、cGMP-PKG、cAMP信号通路。这些通路都是与肿瘤增殖、分化、凋亡、侵袭、转移等生物学行为密切相关的信号通路[13,14,15]。这与前述的关于miR-135b-5p影响多种肿瘤细胞不良生物学行为和耐药的实验研究结论相一致[3,4]。构建蛋白互作网络,筛选出10个重要的中枢蛋白:DLG2、ANK3、ERBB4、SCN2B、NBEA、GABRB2、ATP2B2、SNTA1、CACNA1D、SPTBN4。其中CACNA1D和ATP2B2均参与了上述3个癌症相关通路。乳腺癌中miR-135b低表达,ATP2B2高表达,ATP2B2减少了肿瘤细胞内钙离子进而抑制了细胞凋亡[16]。这与笔者预测的ATP2B2参与调控钙离子通路的结果一致。CACNA1D在肾癌和肺癌中均低表达[17],前列腺癌中低表达的CANCA1D提示肿瘤的恶性程度更高,侵袭性更强[18],有望成为癌症治疗的新靶点。本实验通过生物信息学分析,得到的miR-135b-5p的关键靶基因及癌症相关的靶向调控的信号通路,为后续机制研究提供方向。
【Author contributions】 Zhao G collected, processed and analyzed the data and wrote the article. Li CX designed the study. Guo C revised the article. Zhu H directed the tissue samples collection. All authors read and approved the final manuscript as submitted.
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Regulation of Ca(2+) signaling for drug-resistant breast cancer therapy with mesoporous silica nanocapsule encapsulated doxorubicin/siRNA cocktail
[J].Multidrug resistance (MDR) is the key cause that accounts for the failure of clinical cancer chemotherapy. To address the problem, herein, we presented an alternative strategy to conquer drug-resistant breast cancer through the combinatorial delivery of Ca channel siRNA with cytotoxic drugs. Mesoporous silica nanocapsules (MSNCs) with mesoporous and hollow structure were fabricated for co-delivery of T-type Ca channel siRNA and doxorubicin (DOX) with high drug loading efficiency. The DOX/siRNA co-loaded MSNCs showed a synergistic therapeutic effect on drug-resistant breast cancer cells MCF-7/ADR, while had only an additive effect on the drug-sensitive MCF-7 counterpart. It was found that the combination of T-type Ca channel siRNA and DOX had a similar effect on MCF-7 and MCF-7/ADR in the knockdown of overexpressed T-type Ca channels and decrease in cytosolic Ca concentration ([Ca]), but it specifically induced G/G phase cell-cycle arrest and intracellular drug accumulation enhancement in MCF-7/ADR. The in vitro and in vivo results demonstrated that the MSNCs with good biocompatibility had a high efficiency for conquering the drug-resistant breast cancer with the DOX/calcium channel siRNA cocktail co-delivery. It provides a biological target for drug/gene delivery enhanced cancer therapy with nanoformulations.
Phase separation of a PKA regulatory subunit controls cAMP compartmentation and oncogenic signaling
[J].
Propranolol selectively inhibits cervical cancer cell growth by suppressing the cGMP/PKG pathway
[J].
PMCA2 regulates apoptosis during mammary gland involution and predicts outcome in breast cancer
[J].
Voltage-gated calcium channels: novel targets for cancer therapy
[J].
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