微信公众号
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.
参考文献
The prognostic value of immune checkpoints in oral squamous cell carcinoma
[J].
Despite the importance of immune checkpoints in immunotherapy, the prognostic value of these molecules remains controversial in oral squamous cell carcinoma (OSCC). We performed a systematic review to investigate the prognostic significance of the immune checkpoints in OSCC.A systematic search was conducted in Ovid Medline, Scopus and Cochrane libraries, and all studies that evaluated the prognostic significance of immune checkpoints in OSCC were systematically retrieved.Twelve immune checkpoints/modulators were studied for their prognostic values in OSCC patients between 1985 and 2017. Seven immune checkpoints (FKBP51, B7-H4, B7-H6, ALHD1, PD-L1, B7-H3 and IDO1) were reported to be associated with poor patients' survival in at least one study, and five (CTLA-4, TLT-2, VISTA, PD-L2 and PD-1) did not have a significant prognostic value. PD-L1 results were controversial as it was reported to be associated with both better and worse patients' survival.Even though immune checkpoint markers had high expectation for OSCC prognostication, our systematic review revealed that the majority of them had been studied only once. The other molecules, which had been studied more than once, had controversial findings, except B7-H3.© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.
口腔鳞状细胞癌患者预后相关基因标志物的生物信息学分析
[J].
Identification of potential genetic markers in prognosis of oral squamous cell carcinoma patients by bioinformatic analysis
[J].
miR-135b-5p promotes migration, invasion and EMT of pancreatic cancer cells by targeting NR3C2
[J].
Helicobacter pylori-induced miR-135b-5p promotes cisplatin resistance in gastric cancer
[J].
miRNAs-Based molecular signature for KRAS mutated and wild type colorectal cancer: an explorative study
[J].
Comprehending the crosstalk between Notch, Wnt and Hedgehog signaling pathways in oral squamous cell carcinoma - clinical implications
[J].
miR-135b-5p靶向KLF4基因调控胃癌SGC-7901细胞生物学行为的研究
[J].
The miR-135b-5p targeting KLF4 gene regulates the biological behavior of gastric cancer SGC-7901 cells
[J].
Knockdown of Mir-135b sensitizes colorectal cancer cells to Oxaliplatin-Induced apoptosis through increase of FOXO1
[J].
miR-135b-5p调控TIMP3的表达对鼻咽癌细胞迁移和侵袭的影响
[J].
miR-135b-5p regulates the expression of TIMP3 and affects the migration and invasion of nasopharyngeal carcinoma cells
[J].
MicroRNA meta-signature of oral cancer: evidence from a meta-analysis
[J].
Prognostic factors in intraoral squamous cell carcinoma: the influence of histologic grade
[J].
Immunity, inflammation, and cancer
[J].Inflammatory responses play decisive roles at different stages of tumor development, including initiation, promotion, malignant conversion, invasion, and metastasis. Inflammation also affects immune surveillance and responses to therapy. Immune cells that infiltrate tumors engage in an extensive and dynamic crosstalk with cancer cells, and some of the molecular events that mediate this dialog have been revealed. This review outlines the principal mechanisms that govern the effects of inflammation and immunity on tumor development and discusses attractive new targets for cancer therapy and prevention.2010 Elsevier Inc. All rights reserved.
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].
