基于分子信标技术对活细胞内miR-155检测用于肺癌诊断
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摘要
目的探讨激光共聚焦检测miR-155 MB对肺癌活细胞中miR-155的识别成像功能。方法设计锁核酸修饰的miR-155分子信标(MB),同时设计不和任何序列结合的随机序列分子信标(RS MB)作为阴性对照;采用液相分子杂交试验验证其灵敏性和特异性,并进一步在肺肿瘤组织水平上验证。以纳米壳聚糖(CS)作为载体转染miR-155 MB,检测miR-155 MB对肺癌细胞中miR-155识别成像功能;同时采用miR-155抑制剂antagomir抑制miR-155的表达后检测miR-155 MB对肺癌细胞中miR-155的识别成像功能。结果液相分子杂交实验中miR-155+miR-155 MB组荧光强度为miR-155 MB组的58倍,RS+RS MB组荧光强度为RS MB组的43倍(P<0.05)。在肺鳞癌、腺癌组织中,激光共聚焦可检测到较强的荧光信号,阴性对照组未检测到明显荧光信号。纳米壳聚糖转染miR-155 MB后,可在肺癌活细胞中检测到较强的荧光信号,antagomir抑制miR-155的表达后,发现荧光信号明显降低(P<0.05)。结论利用分子信标技术可以实现通过识别肺癌活细胞中的miR-155并成像肺癌细胞,从而为发现肺癌细胞并用于肺癌诊断提供了新的理论基础。
Objective Designed the miR-155 molecular beacon( MB) which was modified by lock nucleic acid based on MB technology,detected and imaged the miR-155 in lung cancer cells by the laser confocal microscopy.Methods Designed the miR-155 MB which was modified by lock nucleic acid and the random sequence molecular beacon( RS MB) which was not complementary with any other sequences as the negative control.The specificity and sensitivity of the designed molecular beacon was detected by the hybridization assay and was verified in the lung cancer tissues.Chitosan( CS) nanoparticles were adopted to deliver the miR-155 MB.The laser confocal microscopy was used to detect and image the miR-155 or the miR-155 inhibited by the antagomir in lung cancer cells at the same time.Results Hybridization assay showed that the fluorescence intensity in the miR-155+miR-155 MB group was 58 times higher than that in the only miR-155 MB group,and the fluorescence intensity in the RS+RS MB group was 431 times higher than that in the only RS MB group( P< 0.05).The fluorescence signal could be detected in the lung cancer tissues and could not be detected in the RS MB group.The fluorescence signal could be detected in the lung cancer cells after CS transfect the miR-155 MB.The fluorescence signal was significantly decreased after the miR- 155 expression was inhibited by the antagomir( P<0.05).Conclusion The miR-155 MB could detect the miR-155 in the lung cancer cells and imaging them based on molecular beacon technology and provided new theoretical basis for diagnosis of lung cancer.
Objective Designed the miR-155 molecular beacon( MB) which was modified by lock nucleic acid based on MB technology,detected and imaged the miR-155 in lung cancer cells by the laser confocal microscopy.Methods Designed the miR-155 MB which was modified by lock nucleic acid and the random sequence molecular beacon( RS MB) which was not complementary with any other sequences as the negative control.The specificity and sensitivity of the designed molecular beacon was detected by the hybridization assay and was verified in the lung cancer tissues.Chitosan( CS) nanoparticles were adopted to deliver the miR-155 MB.The laser confocal microscopy was used to detect and image the miR-155 or the miR-155 inhibited by the antagomir in lung cancer cells at the same time.Results Hybridization assay showed that the fluorescence intensity in the miR-155+miR-155 MB group was 58 times higher than that in the only miR-155 MB group,and the fluorescence intensity in the RS+RS MB group was 431 times higher than that in the only RS MB group( P< 0.05).The fluorescence signal could be detected in the lung cancer tissues and could not be detected in the RS MB group.The fluorescence signal could be detected in the lung cancer cells after CS transfect the miR-155 MB.The fluorescence signal was significantly decreased after the miR- 155 expression was inhibited by the antagomir( P<0.05).Conclusion The miR-155 MB could detect the miR-155 in the lung cancer cells and imaging them based on molecular beacon technology and provided new theoretical basis for diagnosis of lung cancer.
引文
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14 Azuma K,Izumi R,Osaki T,et al.Chitin,chitosan,and its derivatives for wound healing:old and new materialsr[J].J Funct Biomater,2015,6(1):104-142.
2 江飞龙,韩静,朱海振,等.人肺腺癌厄洛替尼耐药细胞PC9/ER的建立及奥曲肽改善耐药机制研究[J/CD].中华肺部疾病杂志:电子版,2015,8(2):149-153.
3 Lee J,Moon SU,Lee YS,et al.Quantum dot-based molecular beacon to monitor intracellular microRNAs[J].Sensors(Basel),2015,15(6):12872-12883.
4 Li J,Tan S,Kooger R,et al.MicroRNAs as novel biological targets for detection and regulations[J].Chem.Soc.Rev,2014,43(2):506-517.
5 Xie K,Ma H,Liang C,et al.A functional variant in miR-155regulation region contributes to lung cancer risk and survival[J].Oncotarget,2015,6(40):42781-42792.
6 Peng XH,Cao ZH,Xia JT,et al.Real-time detection of gene expression in cancer cells using molecular beacon imaging:new strategies for cancer research[J].Cancer Res,2005,65(5):1909-1917.
7 Zhu HZ,An JH,Yao Q,et al.Chitosan combined with molecular beacon for mir-155 detection and imaging in lung cancer[J].Molecules,2014,19(9):14710-14722.
8 Chen J,Wu J,Hong Y.The morpholino molecular beacon for specific RNA visualization in vivo[J].Chem Commun(Camb),2016,52(15):3191-3194.
9 Dong H,Ma J,Wang J,et al.A Biofunctional Molecular Beacon for Detecting Single Base Mutations in Cancer Cells[J].Mol Ther Nucleic Acids,2016,5:e302.
10 Giannetti A,Barucci A,Cosi F,et al.Optical fiber nanotips coated with molecular beacons for DNA detection[J].Sensors(Basel),2015,15(5):9666-9680.
11 Li Y,Zhang J,Zhao J,et al.A simple molecular beacon with duplex-specific nuclease amplification for detection of microRNA[J].Analyst,2016,141(3):1071-1076.
12 Morinaga S,Nakamura Y,Atsumi Y,et al.Locked nucleic acid in situ hybridization analysis of microRNA-21 predicts clinical outcome in patients after resection for pancreatic cancer treated with adjuvant gemcitabine monotherapy[J].Anticancer Res,2016,36(3):1083-1088.
13 陈华萍,王关嵩.纳米材料与疾病的诊断和治疗[J/CD].中华肺部疾病杂志:电子版,2015,8(3):374-376.
14 Azuma K,Izumi R,Osaki T,et al.Chitin,chitosan,and its derivatives for wound healing:old and new materialsr[J].J Funct Biomater,2015,6(1):104-142.