摘要
背景与目的
肺癌是最常见的恶性肿瘤,其总的5年生存率不足15%,严重威胁人类健康。有效的早期诊断方法是提高肺癌患者生存率的关键,基于肿瘤干细胞(cancer stem cell,CSC)理论的新策略有望为肺癌早诊带来突破。CD133是经典的CSCs标记物,成功用于包括脑胶质瘤、结肠癌、肾癌、肝癌以及胰腺癌的CSCs的鉴定与分离。最近,Eramo A等证实CD133是肺癌干细胞(lung cancer stem cells,LCSCs)新的标记物,为研究以LCSCs为靶标的肺癌早期诊断策略提供了契机。此外,已有不少的研究表明:特异的微小RNA(miRNA)在维持肺上皮干细胞(lung epithelium stem cells,LSCs)生物学特性、促进肺癌发生发展过程中起重要的作用,是潜在的肺癌早期诊断的分子标签。基于此,我们设想:基于LCSCs和miRNAs双靶标的检测手段可能是更理想的肺癌早期诊断策略。因此,本实验拟经Real-time PCR观察miRNAs在CD133(+)LCSCs中的表达水平差异,以期获得CD133(+)LCSCs相关的miRNAs。
敏感的完整靶细胞内特异性分子实时成像观察是实现肺癌早期诊断的有效手段。分子信标(molecular beacon,MB)是根据荧光共振能量转移(fluorescence resonance energy transfer,FRET)原理设计的一种高灵敏性、高特异性检测手段。较之于Real-time PCR、荧光原位杂交(fluorescence in situ hybridization,FISH )等传统的miRNAs检测方法,分子信标荧光技术可实现完整细胞内特异性靶分子的实时成像观察,是了解活细胞内生物大分子相互作用的新的手段。目前,基于分子信标技术的特异性miRNAs实时成像检测的文献报道较少。
类似于反义寡核苷酸(antisense oligonucleotides,ASO),分子信标活细胞内应用面临生物稳定性差、转染效率低等诸多方面的困难。有研究提示用对DNA有保护作用的载体并阻止分子信标分子的核内流是提高分子信标稳定性的有效的策略。在众多的核酸载体中,壳聚糖纳米颗粒(chitosan nanoparticle,CS-NP)具有DNA保护作用、缓释效应等特性,可能是较为理想的分子信标载体。然而,壳聚糖纳米能否用作分子信标技术的载体尚未见报道。
为此,本研究在分离、鉴定CD133(+)LCSCs并分析其miRNAs表达的基础上,以CD133(+)LCSCs高表达的has-miR-155为研究对象,设计、合成靶向结合成熟miR-155及其前体的特异性分子信标(miR-155 MB)。制备miR-155分子信标壳聚糖纳米复合物实时成像观察SPC-A1肺癌细胞内miR-155的表达,为建立实时成像CD133(+)LCSCs特异性miRNAs表达的技术平台奠定基础。
实验方法
用CD133单抗观察不同类型非小细胞肺癌(non-small cell lung cancer,NSCLC)组织中的CD133(+)LCSCs。用免疫磁珠分选方法自NSCLC组织富集CD133(+)LCSCs。结合文献,以肺癌相关miRNA(hsa-miR-155、hsa-miR-205a、hsa-miR-191、hsa-miR-21、hsa-miR-210、hsa-miR-17-5p和hsa-miR-106a)为检测对象,经Real-time PCR初步筛选CD133(+)LCSCs相关miRNA。
根据成熟has-miR-155序列设计、合成特异性miR-155分子信标。同时,设计、合成不能与任何内源性序列互补结合的随机序列MB(random sequence molecular beacon,RS MB)作阴性对照。液相杂交实验观察miR-155分子信标和随机序列分子信标的特异性和热力学稳定性。用miR-155分子信标孵育丙酮固定的人NSCLC组织切片和H446、SPC-A1肺癌细胞观察miR-155在肺癌内的表达并经Real-time PCR方法验证。同时,用Vero细胞和PC-3前列腺癌细胞作对照。
吸附法制备分子信标壳聚糖纳米复合物。DNase I消化实验、细胞蛋白提取物孵育实验观察分子信标壳聚糖纳米复合物稳定性。激光共聚焦观察壳聚糖纳米介导miR-155分子信标转染SPC-A1肺癌细胞及其检测miR-155的表达能力。同时,用随机序列分子信标和Vero细胞作为阴性对照,评估活细胞内荧光信号的特异性。
结果
1、免疫荧光和流式分析发现12例不同类型的人NSCLC组织内存在比例约为0.11～1.2%的CD133(+)LCSCs,而正常肺组织未见CD133(+)细胞。免疫磁珠分选、富集CD133(+)LCSCs并经Real-time PCR检测发现hsa-miR-17-5p、hsa-miR-155在富集的AC133(+)LCSCs中高表达,较之于AC133(-)肺癌细胞,表达上调分别为1.44、1.13倍。
2、液相杂交实验显示分子信标有很好的特异性和热动力学稳定性。miR-155分子信标孵育固定的NSCLC组织切片和H446、SPC-A1肺癌细胞后发现肺癌细胞胞浆内可见特异的红色荧光,而背景很低,阴性对照组未见到明显荧光信号。Real-time PCR结果证明荧光信号来源于miR-155分子信标与靶分子特异性结合。
3、经吸附法制备分子信标壳聚糖纳米复合物。琼脂糖凝胶电泳和荧光屏蔽实验显示当WCS-NP/WODN≥5:1时复合物结合较为完全,粒径和Zeta电位分析显示分子信标壳聚糖纳米复合物适合于细胞转染。DNase I保护实验表明分子信标壳聚糖纳米复合物有抗核酸酶降解作用。胞浆蛋白孵育2h后裸分子信标荧光强度增加约2～3倍,而核蛋白孵育裸分子信标后荧光强度增加约9倍,表明胞核蛋白对裸分子信标构象稳定性影响明显。用胞核蛋白孵育分子信标壳聚糖纳米复合物2h后发现分子信标荧光信号无明显增强,提示壳聚糖纳米能有效阻止分子信标与蛋白的非特异性结合。继续孵育24h后荧光信号增强约5~6倍,显示分子信标壳聚糖纳米复合物的缓释效应。进一步用壳聚糖纳米作为载体介导miR-155分子信标转染SPC-A1肺癌细胞24h后发现:大约40~50%的SPC-A1细胞内可见较强的红色荧光,以胞浆内较为明显,显示转染效率较高,且SPC-A1细胞浆内的荧光信号主要来源于miR-155分子信标与其靶分子的特异性结合。核内可见弱的荧光信号表明壳聚糖纳米可阻止分子信标迅速、大量在核内聚集,用壳聚糖纳米作为载体是减少分子信标核内非特异性信号的有效策略。
4、实时成像SPC-A1肺癌细胞移植瘤的实验研究正在进行中。
结论
1、不同类型人NSCLC组织内存在极低比例的CD133(+)LCSCs。免疫磁珠分选富集CD133(+)LCSCs并经Real-time PCR初步检测发现hsa-miR-17-5p、hsa-miR-155在富集的CD133(+)LCSCs中有较显著的过表达。
2、成功设计、合成特异性miR-155分子信标并应用于固定的肺癌细胞内miR-155基因表达的检测。
3、成功用壳聚糖纳米包裹分子信标并实时成像SPC-A1肺癌细内miR-155基因的表达,为建立实时成像CD133(+)LCSCs内特异性miRNAs表达的技术平台奠定了基础。
Background and Objective
Lung cancer is the most common cancer in the world with the total 5-year survival rate of less than 15% and threaten seriously on human health. Operative approaches for early diagnosis of lung cancers could improve significantly total 5-year survival.New strategies based on cancer stem cell(CSC)theory were promising. CD133 was widely considered as classical CSCs marker and had been successfully used to isolate and identify CSCs in various solid cancers, including glioma, colon cancer, renal cancer, liver cancer and pancreatic cancer. Recently, Eramo A et al proved CD133 as new lung cancer stem cells(LCSCs)marker, which provided opportunities for design of early diagnosis strategies of lung cancer targeting to lung cancer stem cells. In addition, A body of researches suggested miRNAs playing an important role in maintenance of biological characteristics of lung epithelium stem cells (LSCs) and in occurrence and development of lung cancer were potent molecular signatures for early diagnosis of lung cancer. Accordingly, we presumed that new strategies for early diagnosis of lung cancer using dual targets including lung cancer stem cells and miRNAs were more potent.
Therefore,we investigated CD133(+)LCSCs related-miRNAs differentially expressing in CD133(+)LCSCs through Real-time PCR in this study.
Sensitive Real-time imaging of specific markers in intact cells were operative approaches for early diagnosis of lung cancers.Molecular beacon(MB)fluorescence designed according to fluorescence resonance energy transfer(FRET)was a sensitive and specific approach showing many advantages in Real-time imaging detection of expression of specific targets and studying interactions between biological molecules in intact living cells compared to conventional approaches such as Real-time PCR and fluorescence in situ hybridization(FISH). But few was reported about Real-time imaging detection of expression of miRNAs using molecular beacons so far.
There were many difficulties applying molecular beacons in living cells similar to antisense oligonucleotides, including biostability and transfection efficiency. Some studies indicated that it was feasible to improve biostability of molecular beacons using protective carriers and prevent sequenstration in nucleus. Chitosan nanoparticle with superordinary properties of DNA protection and decaying release was promising molecular beacon carrier among various candidates. However, little was known if chitosan nanoparticle could be used as molecular beacons carrier so far.
For thess proposes, has-miR-155 differentially over-expressing in isolating CD133(+)LCSCs was selected to designed and synthetized specific molecular beacon(miR-155 MB). miR-155 molecular beacon nanoparticle complexes were further prepared for Real-time imaging detection of expression of miR-155 gene in living SPC-A1 lung cancer cells, which settled foundation for Real-time imaging detection of expression of specific miRNAs in CD133(+)LCSCs.
Methods
1.CD133(+)LCSCs were identified in NSCLC tissues with different pathological types using CD133 monoclonal antibody, and were further enriched from NSCLC tissue specimens using immunomagnetic bead sorting. Expression of several lung cancer- related-miRNAs (hsa-miR-155, hsa-miR-205a, hsa-miR-191, hsa-miR-21, hsa-miR-210,hsa-miR-17-5p and hsa-miR-106a)According to reported results were further investigated in enriched CD133(+)LCSCs using Real-time PCR approach.
2.Specific miR-155 molecular beacon targeting mature miR-155 and its precursors was designed and synthetized according to sequence of mature has-miR-155,and random sequence molecular beacon(RS MB)not perfectly complementary to any endogenous sequences was also synthetized as negative control. Specificity and thermodynamic of molecular beacons were evaluated by solution hybridization assay. Expression of miR-155 gene in fixed H446,SPC-A1 lung cancer cells and frozen NSCLC tissue sections was investigated using miR-155 molecular beacon and further verified through Real-time PCR approach. PC-3 prostatic carcinoma cells and Vero cells were used as control.
3.Molecular beacon nanoparticle complexes were prepared using incubation procedure. Biostability of Molecular beacon nanoparticle complexes were investigated by DNase I protection assay and incubation assay with cell lysates. Transfection and Real-time imaging detection using molecular beacon nanoparticle complex was further performed in living SPC-A1 lung cancer cells and observed through laser confocal microscope. Random sequence molecular beacon and Vero cells were also used as negative controls to evaluate specificity of fluorescence signal generated in cells.
Results
1.Immunofluorescence analysis performed on NSCLC patient-derived tumor sections indicated the existence of extremely low percentage of CD133(+)LCSCs ranging from 0.11% to 1.2% in all 12 of NSCLC specimens analyzed and absence of CD133(+)cells within control lung tissues derived from normal tissue samples surrounding the tumor of the same patient. CD133(+)LCSCs were further enriched CD133(+)LCSCs from NSCLC tissue specimens using immunomagnetic bead sorting. Mature hsa-miR-17-5p and hsa-miR-155 differentially over-expressed in enriched CD133(+)LCSCs with about 1.44 and 1.13 fold increase respectively compare to CD133(-) cells by Real-time PCR.
2. MiR-155 molecular beacon and RS molecular beacon showed perfect specificity and thermodynamic stability by solution hybridization assay. Strong red fluorescent signal was observed mainly in the cytoplasm of fixed H446, SPC-A1 lung cancer cells and NSCLC tissues with extremely low background similar to passive control PC-3 prostatic carcinoma cells after incubating with miR-155 molecular beacon. In contrast, no significant fluorescent signal was generated in cells of negative groups including H446 and SPC-A1 lung cancer cells and NSCLC tissue sections incubating with negative control random sequence molecular beacon and Vero cells incubating with miR-155 molecular beacon. Specific binding of miR-155 molecular beacon with its targets in lung cancer cells was further verified by Real-time PCR.
3. Molecular beacon nanoparticle complexes were prepared using incubation procedure. Complete condense of molecular beacons with chitosan nanoparticle was determined at the WCS-NP/WODN ratio of above five by agarose gel electrophoresis and fluorescence retard array and physiochemical properties analysis indicate the complex was suitable for further transfection.
DNase I protection assay indicated that molecular beacon nanoparticle complexes were protected from enzyme degradation. About 2- to 3-fold fluorescent enhancement was detected in naked molecular beacons solution compared to molecular beacons background after incubating with cytosolic fractions of cell lysates for 2 hours. In contrast, about 9-fold fluorescent enhancement was detected in naked molecular beacons solution compared to molecular beacons background after incubating with nuclear fractions of cell lysate at the same conditions,indicating nuclear protein influence significantly on conformation of molecular beacons produsing strong non-specific fluorescence. Molecular beacon chitosan nanoparticle complexes were further incubated with nuclear fractions of cell lysates for 2 hours at the same conditions and no significant fluorescent enhancement happened, implying chitosan nanoparticle prevented molecular beacon from binding with proteins. However, about 5- to 6-fold fluorescent enhancement was detected in molecular beacon chitosan nanoparticle complexes solution after incubating until 24 hours, indicating decayed release potential of chitosan nanoparticle. Strong fluorescence was observed in about 40~50% of mainly cytoplasm of transfected SPC-A1 lung cancer cells after further transfecting with miR-155 molecular beacon chitosan nanoparticle at the WCS-NP/WODN ratio of six, showing a high transfection efficiency and specificity. Weaker fluorescent signal in the nucleus of SPC-A1 cells compare in cytoplasm implied that using chitosan nanoparticle as carrier of molecular beacon was a feasible approach to control nuclear sequestration and decrease nuclear non-specific fluorescence.
4. Researches on Real-time imaging detection of expression of miR-155 gene in xenograft tumors are in process.
Conclusions
1.Extremely low percentage of CD133(+)LCSCs ranging from 0.11% to 1.2% existed in NSCLC tissues. CD133(+)LCSCs were enriched from NSCLC tissues using immunomagnetic bead sorting. Mature hsa-miR-17-5p and hsa-miR-155 differentially over-expressed with about 1.44 and 1.13 fold increase respectively compare to CD133(-) cells by Real-time PCR.
2. Specific miR-155 molecular beacon was successfully designed and synthetized, and was further used to detect expression of miR-155 gene in fixed lung cancer cells.
3. Specific miR-155 molecular beacon incorporated into with chitosan nanoparticle was successfully deliver into living SPC-A1 lung cancer cells for Real-time imaging detection of expression of miR-155 gene, which set foundation for Real-time imaging researches of several specific miRNAs in living CD133(+)LCSCs based on molecular beacon fluorescence approach.
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