miRNAs have recently emerged as a novel class of gene expression regulators. Studies had shown that miRNAs were stabilized in the serum, of which the expression level was related to tumor types and development stages[18,19]. Thus, the circulating miRNAs may be a novel kind of potential biomarker for early diagnosis and clinical evaluation of EC patients. However, the mature miRNA is a kind of short RNA of 21-25nt, which easily interfered by homologous sequences and homologous miRNA sequences during the detection process. Also, many miRNAs are less abundant in circulation and difficult to be detected accurately[17]. At present, PCR detection methods for miRNA mainly include SYBR GREEN method and probe method. The SYBR GREEN method has a lower cost, but due to methodological restrictions, the specificity is poor. As for the most commonly used TaqMan probe, it has a long sequence and high experimental cost, which is difficult to design and accept[20]. These limitations make current PCR detections of miRNAs more difficult to promote clinically. The sensitivity and amplification efficiency of miRNA detection are very significant, and the stability and repeatability of the detection method are of great impact on the results, without which, even if the detection method is economically feasible, it is difficult to become a routine examination method in clinical.
Currently, AllGlo probe, the latest generation of quantitative fluorescent probe, has been applied in detecting H7N7, HPV and acute respiratory infection-associated virus. AllGlo probe has higher specificity and sensitivity than common methods as well as satisfactory cost effectiveness[30,31,32]. Since the AllGlo probe is shorter than other probes, the fluorophore will increase the TM value of the probe by 8–10 °C during the PCR reaction, making it more suitable for the detection of small fragment miRNAs. In the detection process by AllGlo probe, as long as there is a base mismatch in the amplification reaction, it will not produce a fluorescent signal, thereby greatly reducing the non-specific fluorescent signals and effectively resolving the interference of the miRNA precursors and homologous sequences. The advantages of this assay system overcome the problems of miRNAs detection and will promote miRNAs as novel tumor biomarkers in clinical diagnosis. In addition, the PCR method can also be applied to gene detection.
This study was the first to design an AllGlo-probe-based absolute quantitative RT-qPCR assay to identify and quantify miRNA, thus solving the above problems. Our method not only overcome the restrictions of miRNAs detection in plasma, but also was quantitative and convenient. In this study, the detection method had good precision with intra-assay CV < 1.5%, daytime CV < 2.0% and total CV < 2.5%, which were all in the acceptance range, indicating that the detection method was stable, reliable and reproducible, which would significantly reduce random error between each test. The method had a great detection accuracy in the linear range with the bias of repeated measurements less than 0.4log10, indicating that the test results were accurate and reliable. Moreover, the test results were not affected by the concentrations of miRNAs in the specimens during the detection process, and there was no contamination or cross-influence between the adjacent wells, proving that the method could detected miRNAs in each reaction well accurately. The overall performance evaluation results of this method proved that the detection method was stable, accurate and sensitive, and had significant application value in scientific research and clinical diagnosis. Compared with the SYBR Green qPCR, the AllGlo qPCR method had a higher sensitivity and a wider linear range (103-1010copies/µL), meaning that we could easily detect the miRNAs which were expressed in low abundance in the circulation. The levels of miRNAs in some body fluids such as urine, cerebrospinal fluid and exosome are much lower than those in serum or plasma, however studies had shown that they have great significance in the process of cancer development or some other diseases[33,34,35].
The established system was initially used to explore the diagnostic efficiency of plasma miRNAs through expanded sample size, which also provided evidence of the reliability of the method. We found that the expression level of miR-34a-5p increased, whereas the expression levels of miR-148a-3p and miR-181a-5p decreased in the plasma of EC. The results indicated that the plasma levels of miR-34a-5p, miR-148a-3p and miR-181a-5p could serve as biomarkers for EC diagnosis. Our results also showed that combination of the 3 kinds of miRNAs could be used as a more comprehensive indicator of tumor detection compared to Cyfra211 and CEA. In particular, we observed significant differences in the expression of miR-181a-5p in EC patients at different stages of development and normal controls. The expression level of miR-181a-5p was lower in early EC patients than that in normal controls with the sensitivity 85.07%, indicating that miR-181a-5p could be used as a biomarker for early diagnosis of EC. In order to obtain the optimal diagnostic performance, we combined clinical indicators (CEA, Cyfra21-1) with these 3 kinds of miRNAs to construct a diagnostic mathematical model. After the indicators were combined and analyzed by logistic regression, the mathematical formula was constructed according to the different weights in the diagnosis process, of which the diagnostic AUC was up to 0.9196, and the sensitivity and specificity were up to 85.45% and 84.71%, respectively. Similar to the Roman index, this mathematical formula has a strong practicality, with which we can evaluate the risk of EC based on the examination results of miR-34a-5p, miR-148a-3p and Cyfra21-1, reducing the false negative rate, thus improving the diagnosis efficiency of EC.
In summary, the novel absolute quantitative RT-qPCR method based on AllGlo probes designed to detect miRNAs possesses the advantage of high stability, accuracy and sensitivity. It has great application value in scientific research and clinical diagnosis. Meanwhile, by using this developed method, we identified that miR-34a-5p, miR-148a-3p and miR-181a-5p may serve as novel plasma biomarkers for EC diagnosis and prognosis. However, this study is still in its infancy, requiring more different types of samples and different kinds of miRNAs to ultimately optimize the detection system and method. We will continue to expand the sample size of EC, especially the patient's postoperative samples and samples during postoperative treatment. Simultaneously, information on the treatment, prognosis, and survival of EC patients will be collected, in order to further study, the specific role of these miRNAs in the prognosis of EC. Additionally, we will take follow-up studies to determine whether the plasma levels of these 3 kinds of miRNAs can predict recurrence / metastasis of EC.