Peifei Li1, Shengcan Chen1, Huilin Chen2, Xiaoyan Mo1, Tianwen Li1, Yongfu Shao1, Bingxiu Xiao3, Junming Guo4. 1. Department of Biochemistry and Molecular Biology, Ningbo University School of Medicine, Ningbo 315211, China; Zhejiang Provincial Key Laboratory of Pathophysiology, Ningbo University School of Medicine, Ningbo 315211, China. 2. Ningbo College of Health Sciences, Ningbo 315010, China. 3. Department of Biochemistry and Molecular Biology, Ningbo University School of Medicine, Ningbo 315211, China; Zhejiang Provincial Key Laboratory of Pathophysiology, Ningbo University School of Medicine, Ningbo 315211, China. Electronic address: xiaobingxiu@nbu.edu.cn. 4. Department of Biochemistry and Molecular Biology, Ningbo University School of Medicine, Ningbo 315211, China; Zhejiang Provincial Key Laboratory of Pathophysiology, Ningbo University School of Medicine, Ningbo 315211, China. Electronic address: guojunming@nbu.edu.cn.
Abstract
BACKGROUND: Circular RNAs (circRNAs), a class of endogenous RNAs, have emerged as an enigmatic class of RNAs. Little is known about their value in the diagnosis of cancers. METHODS: The targeted circRNA of this study was selected using two circRNA databases: CircBase (http://circbase.org/) and circ2Traits (http://gyanxet-beta.com/circdb/). Divergent primers, rather than commonly used convergent primers, for the circRNA were designed. The circRNA levels in 101 paired gastric cancer tissues and adjacent nontumorous tissues from surgical gastric cancer patients and 36 paired plasma samples from preoperative and postoperative gastric cancer patients were analyzed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The specificity of the amplified products was measured by melting curve analysis and DNA sequencing. To observe the stability of circRNA, three randomly selected samples of gastric cancer tissues were stored at room temperature, 4°C and -20°C, and then, their circRNA levels were analyzed. To verify the reproducibility of qRT-PCR, circRNA levels were detected in a set of specimens (n=15) in two independent experiments with an interval of one day. Then, the correlation of their Ct values was determined. The relationships between circRNA expression levels and clinicopathological factors of patients with gastric cancer were further analyzed by one-way analysis of variance. A receiver operating characteristic (ROC) curve was established to evaluate the diagnostic value. RESULTS: Hsa_circ_002059, a typical circular RNA, was first found to be significantly downregulated in gastric cancer tissues compared with paired adjacent nontumorous tissues (p<0.001). Its levels in plasma collected from postoperative gastric cancer patients were found significantly different from those from preoperative gastric cancer patients. The area under the ROC curve was 0.73. Importantly, we further found that lower expression levels were significantly correlated with distal metastasis (P=0.036), TNM stage (P=0.042), gender (P=0.002) and age (P=0.022). The stability of circRNAs and the reproducibility of the qRT-PCR method for detecting circRNA levels were determined. CONCLUSION: These results suggested that circRNAs are highly stable in mammalian cells and that one specific circRNA, hsa_circ_002059, may be a potential novel and stable biomarker for the diagnosis of gastric carcinoma.
BACKGROUND: Circular RNAs (circRNAs), a class of endogenous RNAs, have emerged as an enigmatic class of RNAs. Little is known about their value in the diagnosis of cancers. METHODS: The targeted circRNA of this study was selected using two circRNA databases: CircBase (http://circbase.org/) and circ2Traits (http://gyanxet-beta.com/circdb/). Divergent primers, rather than commonly used convergent primers, for the circRNA were designed. The circRNA levels in 101 paired gastric cancer tissues and adjacent nontumorous tissues from surgical gastric cancerpatients and 36 paired plasma samples from preoperative and postoperative gastric cancerpatients were analyzed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The specificity of the amplified products was measured by melting curve analysis and DNA sequencing. To observe the stability of circRNA, three randomly selected samples of gastric cancer tissues were stored at room temperature, 4°C and -20°C, and then, their circRNA levels were analyzed. To verify the reproducibility of qRT-PCR, circRNA levels were detected in a set of specimens (n=15) in two independent experiments with an interval of one day. Then, the correlation of their Ct values was determined. The relationships between circRNA expression levels and clinicopathological factors of patients with gastric cancer were further analyzed by one-way analysis of variance. A receiver operating characteristic (ROC) curve was established to evaluate the diagnostic value. RESULTS: Hsa_circ_002059, a typical circular RNA, was first found to be significantly downregulated in gastric cancer tissues compared with paired adjacent nontumorous tissues (p<0.001). Its levels in plasma collected from postoperative gastric cancerpatients were found significantly different from those from preoperative gastric cancerpatients. The area under the ROC curve was 0.73. Importantly, we further found that lower expression levels were significantly correlated with distal metastasis (P=0.036), TNM stage (P=0.042), gender (P=0.002) and age (P=0.022). The stability of circRNAs and the reproducibility of the qRT-PCR method for detecting circRNA levels were determined. CONCLUSION: These results suggested that circRNAs are highly stable in mammalian cells and that one specific circRNA, hsa_circ_002059, may be a potential novel and stable biomarker for the diagnosis of gastric carcinoma.