Screening Of Circulating MicroRNA Biomarkers For Gastric Cancer Diagnosis And MicroRNA Detection Based On DNA-Templated Silver Nanoclusters

Gastric cancer(GC)is the fourth most common cancer in the world and the third common cancer and leading cause of cancer-related death in China.To reduce the population of gastric cancer and the related death,the early diagnosis and monitoring of GC is urgently needed.The currently used serum protein biomarkers and their detection methods based on ELISA(enzyme-linked immuno sorbent assay)cannot provide the exact information for the development of GC.Meanwhile,the newly identified circulating microRNA(miRNA)biomarkers,which are closely related to the tumor genesis and development,are greatly helpful for non-invasive early detection and monitoring of gastric cancer.Notably,it is critical to develop the sensitive and reliable detection methods for the screening and application of miRNA biomarkers.However,the traditional methods for in vitro and in vivo or in-situ detection of miRNAs are usually expensive and time and energy-exhausted,failing to be used for point-of-care or on-site detection.To address these problems,our research focused on the screening of miRNA biomarkers for the diagnosis of GC and the development of in vitro and in-situ miRNA detection methods in an easy and rapid way.Firstly,a total of 171 plasma samples of gastric cancer cases,30plasma samples from precancerous lesions,18 paired samples from gastric cancer cases with gastrectomy and 129 normal controls plasma samples were collected and detected by genome-wide miRNA profiling microarrays and quantitative reverse transcription-PCR method.Results showed that based on two normalization methods,miR-16-5p and miR-19b-3p in plasma were found to be capable of distinguishing normal population from GC cases with different TNM stages and differentiation grades,particularly including the early cancer cases(P<0.05).And the two miRNAs were down-regulated in GC cases(FC<0.5).Especially,the down-regulation degree was correlated with the progression of the GC cases from the early stage to the advanced stage(r_s>0.2,P<0.01).And the same weak down-regulation of the two biomarkers as the early GC occurred initially in the precancerous diseases(P<0.05).The corresponding performance of the two miRNAs to detect GC in ROC analysis gradually performed better with disease progressing from the earlier stages or lower grades to the advanced stages(TNM?stage:AUC=0.832 for miR-16-5p;TNM?stage:AUC=0.822 for miR-19b-3p)or high grade of differentiation(Poorly differentiated:AUC=0.801,0.791respectively for miR-16-5p and miR-19b-3p).In conclusion,miR-19b-3p and miR-16-5p could be prospective biomarkers to detect gastric cancer and indicate its progression,and thus own great potential in applications such as early screening and progression evaluation of gastric cancer in the near future.Secondly,hairpin DNA-templated silver nanoclusters(AgNCs/HpDNA)were prepared and integrated into strand-displacement amplification(SDA)as a novel beacon for miRNA detection.The light-up platform was established based on guanine(G)-rich fluorescence enhancement that essentially converted the excitation/emission pair of AgNCs/HpDNAs from a shorter wavelength to a longer wavelength where the fluorescent enhancement was achieved,and was sepctra and double strand DNA-specific.Based on the validation of the method,the single and duplex detection were conducted in the two plasma biomarkers(miR-16-5p and miR-19b-3p)for the diagnosis of gastric cancer.The probe(AgNCs/RED 16(7s)C)utilized for miR-16-5p detection adopted a better conformation with high specificity to recognize single-base mismatches by producing dramatically opposite signals(increase or decrease at 580nm ex/640nm em),while the probe(AgNCs/GRE 19b(5s)C)for miR-19b-3p generated dual signals(increase at 490nm ex/570nm em and decrease at 430nm ex/530nm em)with bright fluorescence in one reaction during the amplification,but unexpectedly was partially digested.This is for the first time to allow the generation of enhanced fluorescent AgNCs and the target recognition integrated into a single process,which offers great opportunity for specific miRNA detection in an easy and rapid way.Thirdly,using the capture probe in the form of hairpin DNA and the signal probe of DNA-templated silver nanoclusters(AgNCs/DNAs),a fluorescence in-situ hybridization method was established for miRNA detection in cells.Based on the G-rich fluorescence enhancement effect,the method was found to own a relatively weak red fluorescence enhancement upon the hybridization of AgNCs/DNA with the capture probe having a G-rich overhang.When used to detect miRNAs including miR-16-5p,miR-19b-3p and miR-101-3p in MGC803 cells and its miR-101-3p overexpressed cells,miRNAs were only located in the nucleus of cells.The developed method enables researchers to obtain the subcellular distribution information of miRNAs.