Diagnostic Biomarker Screening Of Exosomal MiRNA For Cervical Cancer

Cervical cancer ranks second among all female malignant cancers,which the incidence is 98.9 in every 100,000 women and the morality is 30%.The incidence rate and morality rate still have an increased trend every year.Cervical cancer occurs after a long term of persistent HPV infection,and about 90%cancerous patients resulted from this persistent virus infection.From precancerous lesions to carcinoma,it could last about several years to more than ten years.Precancerous patients and early grade cervical carcinoma patients could be cured by loop electrosurgical excision procedure or transabdominal hysterectomy.Cervical cancer screening tests can find cervical cancer and precancer in the early grades.In current clinical screening,patients are first examined with Papanicolaou(Pap)Test and Thinprep Cytological Test(TCT).Patients with an abnormal Pap or TCT results are then performed with Colposcopy Test.However,the accuracy of these two tests are about 68.86%based on inquiring clinical medical records.Therefore,it’s urgent to develop a more accurate diagnostic method for the early detection of cervical cancer.Exosomes play an important role in this communication process.MiRNA expression is frequently dysregulated in tumour cells and can be reflected by distinct exosomal miRNA profiles isolated from the bodily fluids of cancer patients.The major objective of the present study is to identify circulating exosomal miRNAs as biomarkers for the early detection of cervical cancer with the ultimate goal of improving patient outcome.In our experiments,121 subjects were recruited,including 93 advanced precancerous Ⅱ or cancerous patients,23 healthy women and 5 precancerous grade Ⅰpatients.Exosome-associated miRNAs in plasma samples from these subjects were profiled using small RNA next generation sequencing.Eight differentially expressed miRNAs were identified between CINI-group(including precancerous grade Ⅰ patients and healthy volunteers)and CINII+ group(including advanced precancerous grade Ⅱpatients).They are let-7a-3p,let-7d-3p,miR-30d-5p,miR-144-5p,miR-182-5p,miR-183-5p,miR-215-5p,and miR-4443.This panel of miRNAs was able to distinguish patients in CINII+ group from those in CINI-group.These differentially expressed miRNAs were then validated in 46 new cervical tumors and their matched adjacent tissues using quantitative PCR(qPCR).Three of them(let-7d-3p,let-7a-3p,miR-30d-5p)exhibited a significant consistent trend with the sequencing data,and 2(miRNA-144-5 and miR-4443)with a significant reverse changing trend between tumor and normal tissues.miR-30d-5p and let-7d-3p that expressed highly in plasma samples and showed a consistent trend with the sequencing data were validated successfully in independent samples using droped digital PCR(ddPCR).In ddPCR validation,4 miRNAs within less 5%variable coefficient and high abundance in plasma samples were chosen as internal references to normalize the miRNAs expression.These internal reference controls included miR-128-3p,miR-129-5p,miR-320a,and let-7i-5p.As expected,miR-30d-5p and let-7d-3p showed significant different expressions between CINI-and CINII+ groups in ddPCR results.They show good separation between CINI-and CINII+ groups with AUC values of 0.79 and 0.822 respectively,while the combination of two miRNAs yielded a high AUC value of 0.828.Compared with the next-generation sequencing result of theses two miRNAs with a AUC value of 0.922,the result still had a good classification effect with a slight variation.Moreover,targets of the eight successfully validated miRNAs in tissues were predicted by using DIANA database.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis was then carried out on these target genes.This analysis identified 25 significant pathways(p<0.01)related to more than 5 miRNAs.Many of them are cancer-related pathways,such as cell cycle,p53 signaling pathway,pathways in cancer,proteoglycans in cancer,AMPK signaling pathway and so on.Interestingly,several pathways are specifically associated with female malignancies,including viral carcinogenesis that is at the top of list,bacterial invasion of epithelial cells,HTLV-1 infection,Epstein-Barr virus infection,oocyte meiosis,etc.Among them,many important cancer-related genes were identified,including RAC1,IGF1R,NRAS,TP53,CCND1,CDC27,YWHAG,CDK6 and MAP3K1.Finally,7 miRNAs(miR-128-3p,miR-129-5p,miR-320a,let-7i-5p,miR-30a-5p,miR-30d-5p and let-7d-3p)were evaluated for their expression correspondence between tissues and exosomes.They had different miRNA profiles,and their correlation between tissues and exosomes are modest with a correlation coefficient of about 0.50.This suggests that exosomal miRNAs are selectively secreted from tumor cells.In summary,exosomal miR-30d-5p and let-7d-3p in plasma samples could effectively distinguish patients in CINII+ from CINI-groups.They are valuable diagnostic biomarkers for the early detection of cervical cancer.Large sample sizes are required for further validation of the utility of these miRNAs in the diagnosis.These miRNAs are selectively secreted into exosomes from tumor cells that potentially play an important role in post-transcriptional regulation.Functional investigation of these miRNAs can give novel insight into their mechanism and physiologic relevance in the progression of cervical cancer.