RT-ddPCR同时检测血清EpCAM特异性外泌体中lncRNA RP11-77G23.5和PHEX-AS1在肺癌诊断中的临床价值研究

Tumor-specific exosomes play important roles in the occurrence and development of tumors. The protective effect of their lipid bilayer membranes makes their content long non-coding RNAs (lncRNAs) have unique advantages as tumor markers. The applicant isolated tumor marker, epithelial cell adhesion molecule (EpCAM)-specific exosomes from the serum of patients with lung cancer, and demonstrated that they promoted lung cancer progression in vitro and in vivo. We further screened out two lung cancer growth-associated lncRNAs RP11-77G23.5 and PHEX-AS1. Since reverse transcription-droplet digital polymerase chain reaction (RT-ddPCR) has the advantages of high sensitivity and accuracy, it is especially suitable for the absolute quantification of RNA-typed markers in blood. Accordingly, in this project, we will optimize the primers’ concentrations and the size of amplified fragments to achieve the simultaneous quantification of RP11-77G23.5 and PHEX-AS1 by RT-ddPCR, and test the stabilities of lncRNAs in EpCAM-specific exosomes, and analyze the copy number changes of RP11-77G23.5 and PHEX-AS1 within serum EpCAM-specific exosomes among healthy controls, patients with pneumonia, benign and malignant lung tumors, as well as their correlations with clinical parameters, and finally determine their value in the diagnosis of lung cancer.

肿瘤特异性外泌体在肿瘤发生发展中起重要作用。其脂质双层膜的保护作用使其内容物长链非编码RNA（lncRNA）在作为肿瘤标志物方面独具优势。申请人从肺癌患者血清中分离出肿瘤标志物EpCAM特异性外泌体，并经体外和体内实验证明其促进肺癌进展。我们进一步从EpCAM特异性外泌体中筛选出与肺癌生长相关的lncRNA RP11-77G23.5和PHEX-AS1。由于逆转录-微滴数字PCR（RT-ddPCR）具有灵敏度、精确度高等优点，特别适用于绝对定量血液中RNA类标志物。为此，本项目将优化引物浓度和扩增片段长度，实现RT-ddPCR同时定量检测RP11-77G23.5和PHEX-AS1；检验EpCAM特异性外泌体中lncRNA的稳定性；分析健康人、肺炎、良性肺肿瘤和肺癌4组人血清EpCAM特异性外泌体中RP11-77G23.5和PHEX-AS1拷贝数及其与临床资料的相关性，确定其对肺癌的诊断价值。

肿瘤特异性外泌体在肿瘤发生发展中起重要作用。其脂质双层膜的保护作用使其内容物长链非编码RNA（lncRNA）在作为肿瘤标志物方面独具优势。我们首先采用链霉亲和素磁珠成功从肺癌患者血清中分离出肿瘤标志物EpCAM特异性外泌体，并经体外细胞和体内裸鼠实验证明了其能够促进细胞增殖、迁移、侵袭和肺瘤组织生长。接下来，我们采用lncRNA芯片分析了肺癌患者血清EpCAM特异性外泌体中lncRNA表达谱，并从中筛选出肺癌相关lncRNA RP11-77G23.5和PHEX-AS1。由于逆转录-微滴数字PCR（RT-ddPCR）具有灵敏度、精确度高等优点，特别适用于绝对定量血液中RNA类标志物。因此，我们通过优化引物浓度和扩增片段长度，实现了在RT-ddPCR同一反应体系中同时绝对定量lncRNA RP11-77G23.5和PHEX-AS1。在此基础上，我们采用RT-ddPCR检测了健康人、肺炎、良性肺肿瘤和肺癌4组人血清EpCAM特异性外泌体中lncRNA RP11-77G23.5和PHEX-AS1拷贝数，并且分析了两者在不同样本类型中的拷贝数差异，及其与临床资料（肿瘤大小、病理类型、病理分期、淋巴结转移、远处转移）的相关性，并与传统肺癌标志物进行比较。我们发现相较于健康、肺炎和良性肺肿瘤组，肺癌患者血清EpCAM特异性外泌体中lncRNA RP11-77G23.5和PHEX-AS1拷贝数显著升高，并且能够有效区分良恶性肺肿瘤。其中lncRNA RP11-77G23.5拷贝数在肺腺癌患者中明显高于肺鳞癌患者，能够有效区分鳞癌和腺癌两种不同的病理类型。LncRNA PHEX-AS1拷贝数则在具有更高的病理分期或发生远处转移肺癌患者中升高更为显著，表明其与肺癌恶性进展密切相关。总之，我们建立了在RT-ddPCR同一反应体系中同时绝对定量两种lncRNA的方法，并明确了EpCAM特异性外泌体中lncRNA RP11-77G23.5和PHEX-AS1对肺癌诊断具有更高的灵敏度和特异性，可以作为肺癌早期诊断的新型标志物。