循环肿瘤细胞和外泌体miR-21检测在肺磨玻璃样病变诊断中的应用

doi:10.13481/j.1671-587x.20190320

摘要/Abstract

摘要： 目的：评价循环肿瘤细胞（CTC）联合外泌体miR-21检测方法用于肺磨玻璃样病变（GGO）临床诊断的实用性和可行性，为GGO的诊断提供依据。方法：按照GGO诊断标准和排除标准筛选出70例患者，均进行胸腔镜下肺段切除，术后病理诊断为肺癌的患者为病例组（n=24），术后病理诊断为良性病变的患者为对照组（n=46）。术前所有患者通过免疫磁珠负向富集方法从外周血中捕获CTC，采用肿瘤特异性叶酸配体-寡核苷酸偶合物标记捕获的CTC，同时检测同一标本的外泌体，纯化鉴定外泌体，检测患者血清外泌体miR-21表达水平。利用受试者工作特征（ROC）曲线评价CTC和外泌体miR-21对GGO患者的诊断效能。结果：病例组患者CTC阳性率高于对照组（P<0.05）。病例组和对照组不同病理类型患者CTC阳性率比较差异无统计学意义（P>0.05）。纯化样品具备外泌体特征。病例组患者血清外泌体miR-21表达水平高于对照组（P<0.05）。CTC的ROC曲线下面积（AUC）为0.891，外泌体miR-21的AUC为0.711，均具有较好的特异性。二者联合检测的灵敏度和特异度分别为85.64%和86.79%。结论：CTC结合外泌体miR-21检测可以应用于早期肺癌的临床诊断，其灵敏度和特异度较高。

关键词: 叶酸受体, 循环肿瘤细胞, 外泌体, 肺磨玻璃样病变, miR-21

Abstract: Objective:To evaluate the practicability and feasibility of detection of circulating tumor cells (CTC) combined with exosome miR-21 in the diagnosis of ground glass opacity(GGO), and to provide the evidence for the diagnosis of GGO. Methods:Seventy patients were selected according to the diagnostic and exclusion criteria of GGO. All the patients underwent thoracoscopic segmentectomy. The patients diagnosed as lung cancer after operation were regarded as case group (n=24), and the patients diagnosed as benign lesion after operation were regarded as control group (n=46). Before operation, CTC was captured from the peripheral blood by negative enrichment of immunomagnetic beads. CTC was labeled with tumor-specific folate ligand-oligonucleotide conjugate. The exosomes of the same specimen were detected, purified and identified, and the expression levels of serum exosome miR-21 of the patients in two groups were detected. The diagnostic efficacies of CTC and exosome miR-21 in the GGO paitents were evaluated by ROC curves. Results:The positive rate of CTC in the patients in case group was higher than that in control group (P<0.05). There were no significant differences in the positive rates of CTC between the patients with different pathological types in case group and control group (P>0.05). The purified samples showed exosome characteristics. The expression level of serum exosome miR-21 in the patients in case group was higher than that in control group (P<0.05). The area under ROC curve (AUC) of CTC was 0.891, and the AUC of exosome miR-21 was 0.711, which showed good specificity. The sensitivity and specificity of the combined detection were 85.64% and 86.79%,respectively. Conclusion:The detection of CTC combined with exosome miR-21 can be used in the clinical diagnosis of early lung cancer with high sensitivity and specificity.

Key words: folate receptor, circulating tumor cell, exosome, ground glass opacity, miR-21

中图分类号:

R563.9

李刚, 王建生, 秦思达, 孙欣, 任宏, 张靖, 李保成. 循环肿瘤细胞和外泌体miR-21检测在肺磨玻璃样病变诊断中的应用[J]. 吉林大学学报(医学版), 2019, 45(03): 587-594.

LI Gang, WANG Jiansheng, QIN Sida, SUN Xin, REN Hong, ZHANG Jing, LI Baocheng. Application of detection of circulating tumor cells and exosome miR-21 in diagnosis of lung ground glass opacity[J]. Journal of Jilin University(Medicine Edition), 2019, 45(03): 587-594.

参考文献

[1] JAKOBSEN K R,PAULSEN B S,BAEK R,et al. Exosomal proteins as potential diagnostic markers in advanced non-small cell lung carcinoma[J]. J Extra Cell Vesicles, 2015, 2(4):26659.
[2] FAIELLA E, FRAUENFELDER G, SANTUCCI D, et al. Percutaneous low-dose CT-guided lung biopsy with an augmented reality navigation system:validation of the technique on 496 suspected lesions[J]. Clin Imaging, 2018,49(5):101-105.
[3] NADEALIAN Z, NAZARI B, SADRI S, et al. Detection of pulmonary nodules in low-dose computed tomography using localized active contours and shape features[J].J Med Signals Sens, 2017, 7(4):203-212.
[4] PENG Y, WANG Y, HAO X, et al. Utility of multiple increased lung cancer tumor markers in treatment of patients with advanced lung adenocarcinoma[J]. Chin J Lung Cancer, 2017, 20(10):690-694.
[5] XU-WELLIVER M, CARBONE D P. Blood-based biomarkers in lung cancer:prognosis and treatment decisions[J]. Transl Lung Cancer Res, 2017, 6(6):708-712.
[6] MANICONE M, SCAINI M C, RODRIQUENZ M G, et al. Liquid biopsy for monitoring anaplastic lymphoma kinase inhibitors in non-small cell lung cancer:two cases compared[J]. J Thorac Dis, 2017, 9(Suppl 13):S1391-S1396.
[7] TARTARONE A, LEROSE R, RODRIQUENZ M G, et al. Molecular characterization and prognostic significance of circulating tumor cells in patients with non-small cell lung cancer[J]. J Thorac Dis, 2017, 9(Suppl 13):S1359-S1363.
[8] RECLUSA P, TAVERNA S, PUCCI M, et al. Exosomes as diagnostic and predictive biomarkers in lung cancer[J]. J Thorac Dis, 2017, 9(Suppl 13):S1373-S1382.
[9] RⅡHIMAKI M, THOMSEN H, HEMMINKI A, et al. Comparison of survival of patients with metastases from known versus unknown primaries:survival in metastatic cancer[J]. BMC Cancer, 2013, 13(1):36-44.
[10] HARRIS D A, PATEL S H, GUCEK M, et al. Exosomes released from breast cancer carcinomas stimulate cell movement[J]. PLoS One, 2015, 10(3):e0117495.
[11] MOREAU F, SEYFRITZ E, TOTI F, et al. Early effects of liver regeneration on endocrine pancreas:in vivo change in islet morphology and in vitro assessment of systemic effects on betacell function and viability in the rat model of two-thirds hepatectomy[J]. Horm Metab Res, 2014, 46(13):921-926.
[12] YOO C E, KIM G, KIM M, et al. A direct extraction method for microRNAs from exosomes captured by immunoaffinity beads[J]. Anal Biochem, 2012, 431(2):96-98.
[13] 周清华, 范亚光, 王颖, 等. 中国肺部结节分类、诊断与治疗指南(2016年版)[J]. 中国肺癌杂志, 2016, 19(12):793-798.
[14] 刘春全, 崔永. 肺结节评估四大指南比较分析[J]. 中国肺癌杂志, 2017, 20(7):490-498.
[15] XIAO F, JIANG M Y, DU D S, et al. Orexin A regulates cardiovascular responses in stress-induced hypertensive rats[J]. Neuropharmacology, 2013, 67:16-24.
[16] JIANG M Y, WANG Q, KARASAWA T, et al. Sodium-glucose transporter-2(SGLT2; SLC₅A2) enhances cellular uptake of aminoglycosides[J]. PLoS One, 2014, 9(9):e108941.
[17] 连欢欢, 丁志丹, 袁东风, 等. 应用FR靶向PCR法检测CTC在肺癌诊断中的临床价值:初步研究[J].中国肺癌杂志, 2016, 19(12):813-820.
[18] WANG L,WU C Y,QIAO L H,et al. Clinical significance of folate receptor-positive circulating tumor cells detected by ligand-targeted polymerase chain reaction in lung cancer[J]. J Cancer, 2017, 8(1):104-110.
[19] RODRÍGUEZ M, SILVA J, LÓPEZ -ALFONSO A, et al. Different exosome cargo from plasma/bronchoalveolar lavage in non-small-cell lung cancer[J]. Genes Chromosomes Cancer, 2014, 53(9):713-724.
[20] LI J, YANG F, LI X, et al. Characteristics, survival, and risk factors of Chinese young lung cancer patients:the experience from two institutions[J]. Oncotarget, 2017, 8(51):89236-89244.
[21] GHOSSEIN R A, CARUSONE L, BHATTACHERYA S. Review:poly-merase chain reaction detection of micrometastase and circulating tumor cells:application to melanoma, prostate, and thyroid carcinomas[J]. Diagn Mol Pathol, 1999, 8(4):165-175.
[22] HUANG Q Q, WANG Y, CHEN X X, et al. Nanotechnology-Based strategies for early cancer diagnosis using circulating tumor cells as a liquid Biopsy[J]. Nanotheranostics, 2018, 2(1):21-41.
[23] TKACH M, THÉRY C. Communication by extracellular vesicles:where we are and where we need to go[J]. Cell, 2016, 164(6):1226-1232.
[24] LI A C, ZHANG T B, ZHENG M, et al. Exosomal proteins as potential markers of tumor diagnosis[J]. J Hematol Oncol, 2017, 10(1):175-184.
[25] KAI K, DITTMAR R L, SEN S. Secretory microRNAs as biomarkers of cancer[J]. Semin Cell Dev Biol, 2018, 78:22-36.
[26] LI M, WANG Y, SONG Y, et al. MicroRNAs in renal cell carcinoma:A systematic review of clinical implications (Review)[J]. Oncol Rep, 2015, 33(4):1571-1578.
[27] PFEFFER S R, YANG C H, PFEFFER L M. The role of miR- 21 in cancer[J]. Drug Dev Res, 2015, 76(6):270-277.
[28] GAUR A B, HOLBECK S L, COLBURN N H, et al. Downregulation of Pdcd4 by mir-21 facilitates glioblastoma proliferation in vivo[J]. Neurol-oncology, 2011, 13(6):580-590.
[29] XU L, WU Z, CHEN Y, et al. MicroRNA-21(miR-21) regulates cellular proliferation, invasion, migration, and apoptosis by targeting PTEN, RECK and Bcl-2 in lung squamous carcinoma, Gejiu City, China[J]. PLoS One, 2014, 9(8):e103698.
[30] VICINUS B, RUBIE C, STEGMAIER N, et al. miR-21 and its target gene CCL20 are both highly overexpressed in the microenvironment of colorectal tumors:Significance of their regulation[J]. Oncol Rep, 2013, 30(3):1285-1292.
[31] 褚佩英. 肿瘤转移相关标志物:循环肿瘤细胞(CTC)[J]. 临床检验杂志电子版, 2014, 3(3):675-677.
[32] ZHOU B, XU J W, CHENG Y G,et al. Early detection of pancreatic cancer:Where are we now and where are we going?[J]. Int J Cancer, 2017, 141(2):231-241.
[33] 徐亚楠,赵伟,李铭,等.基于支持向量机的肺CT图像三维磨玻璃结节的提取和识别[J].中国医学物理学杂志,2019,36(4):425-430.