Study On Detection Value Of MicroRNAs As Circulating HPV-DNA Positive Non-small Cell Lung Cancer Biomarkers

Objective:The aim of this study was to screen high-efficient universal primers for HPV-DNA,and to determine the potential risk of circulating HPV-DNA in non-small cell lung cancer(NSCLC)with the selected primers to detect.Further,we intended to explore abnormally expressed microRNAs(miRNAs)in the plasma of circulating HPV-DNA-positive NSCLC patients and analyze the potential value of these miRNAs in identifying circulating HPV-DNA-positive NSCLC.Methods:1.By comparing the effect of 6 pairs of universal primers on the detection of 9types of HPV-DNA,the universal primer with the best efficiency was selected for detecting circulating HPV-DNA in the following study.2.A total of 100 patients with NSCLC were enrolled,and peripheral blood samples were collected for HPV-DNA detection.The genotypes of HPV-DNA were analyzed by HPV-specific nested primers and gene sequencing.3.Based on the clinical and pathological data of patients,the relationship between the presence of HPV-DNA in the blood circulation and the clinicopathological characteristics of NSCLC patients was analyzed.4.Differentially expressed miRNAs in NSCLC were screened based on the TCGA database.Then,expression level of the screened miRNAs in the plasma of circulating HPV-DNA-positive NSCLC patients,circulating HPV-DNA-negative NSCLC patients,and healthy individuals was measured by real time fluorescent quantitative PCR.5.Abnormally expressed miRNAs in plasma of circulating HPV-DNA-positive NSCLC patients were finally identified with statistical analysis.6.The receiver operating characteristic curve(ROC curve)was used to analyze the potential value of different miRNAs in identifying circulating HPV-DNA-positive NSCLC patients.Results:1.The universal primer SPF1 / GP6 ++ had the best detection effect among the 6pairs of universal primers in detecting 9 types of HPV-DNA.2.In the study,the positive rate of HPV-DNA in peripheral blood of patients with NSCLC was 16%.The results also showed that the detected HPV type of the NSCLC patients was HPV16.3.Circulating HPV16-DNA-positive NSCLC patients were significantly different from circulating HPV16-DNA-negative ones in terms of tumour pathological type,degree of differentiation,tumour stage,and lymph node metastasis,but there was no significant difference in tumour diameter between the two groups.Lung adenocarcinoma,poorly differentiated cancer,advanced lung cancer,and lymph node metastasis were more common in circulating HPV16-DNA-positive NSCLC than in circulating HPV16-DNA-negative NSCLC.4.A total of 8 differentially expressed miRNAs were screened out based on the TCGA database.Four of these miRNAs were upregulated including miR-183,miR-9-3,miR-210 and miR-182.Four miRNAs were downregulated,namely,miR-144,miR-451 a,miR-486-1 and miR-486-2.5.The expression level of miR-183,miR-210 and miR-182 in circulating HPV16-DNA-positive patients were significantly higher than those in circulating HPV16-DNA-negative patients.The expression level of miR-144 was significantly lower in circulating HPV16-DNA-positive patients than circulating HPV16-DNA-negative patients.6.When using single miRNA to identify circulating HPV16-DNA-positive NSCLC patients,miR-210 had a higher area under the ROC curve(AUC)than other miRNAs,and its sensitivity and specificity were also higher.In addition,the combination of two miRNAs was more effective than a single miRNA.Among them,miR-210+miR-144 had the highest AUC value and showed the best prediction performance.Conclusions:1.The PCR method of universal primers is simple,efficient and fast,which can be used for HPV-DNA screening.However,the accuracy of universal primers for HPV-DNA screening needs further consideration,and more precise universal primers need to be optimized and designed.2.Circulating HPV-DNA is closely related to the pathological type,degree of differentiation,tumor stage and lymph node metastasis in NSCLC patients,and may act as an early warning factor for poor prognosis in NSCLC patients.3.Plasma miR-183,miR-210,miR-182 and miR-144 can be used as reliable biomarkers to assist the diagnosis of circulating HPV16-DNA-positive NSCLC.The combined detection of two miRNAs can improve the detection effect of circulating HPV16-DNA-positive NSCLC.However,more samples are still needed to increase the accuracy of the detection results.The specific molecular mechanism needs further study.