The Expression Profiling Of MiRNAs In Tissue And Serum From Lung Cancer Patients And Study On Cytological Function Of MiR-486-5p

Lung cancer is the leading cause of cancer death in the world.In China,the incidence and mortality of lung cancer rank first in cancers.MiRNAs are small,single strand,endogenous non-coding RNAs of about 18-25 nucleotides.Many studies have shown that miRNAs are frequently dsregulated in cancers,including lung cancer.The characterization of miRNAs may provide new insights for the study of pathogenesis,diagnosis and treatment in lung cancer.Compared to traditional methods,the high throughput sequencing has a tremendous advantage in the throughput and effeciency of miRNA detection.This study profiled the miRNAs in paired tissue and serum samples of lung cancer based on high throughput sequencing,and selected miR-486-5p to study its cytological function.(1)miRNA profiling of paied tissue samples in lung cancer: Simultaneously profiled the expression of miRNAs in paired tumor tissue,adjacent tissue and normal tissue of lung cancer based on high throughput sequencing.Various differentially expressed miRNAs were found in squamous cell carcinoma(SCC)and adenocarcinoma(AC),respectively.The levels of some miRNAs gradually changed from normal tissue to adjacent tissue to tumor tissue.Among them,12 gradually changed miRNAs appeared in SCC,3 miRNAs in AC.These gradually changed miRNAs were validated by quantitative RT-PCR,and further analysis found that the target genes of differential miRNAs were enriched in the pathway of Non-small-cell lung cancer pathway.These results indicated that the gradually changed miRNAs might have important roles in the tumorigenesis and development of lung cancer.(2)miRNA profiling of serum samples in lung cancer: Further profiled the miRNAs in paied serum samples of lung cancer patients based on high throughput sequencing.Compared with the differential miRNAs in tissue samples,5 miRNAs were found to have similar expression differences in tissue and serum samples of lung cancer patients.These miRNAs could be used as potential target for further study.The qPCR results showed the levels of three miRNAs were validated,miR-181b-5p and miR-21-5p were significantly up-regulated,miR-486-5p was significantly downregulated in lung cancer.The targets of these 3 miRNAs were also validated in tissue samples,the pathway analysis found that the targets were enriched in the carcinogenesis and development process of lung cancer.The study provided potential target miRNAs to the early diagnosis of lung cancer.(3)Cytological function study of miR-486-5p: Combined with previous research progress,this study selected the down-regulated miR-486-5p to quantify its expression levels in more samples by qPCR.The differential expression was confirmed in tissue,serum and cell samples of lung cancer.And TCGA data also shown that the levels of miR-486-5p were significantly lower in lung squamous cell carcinoma and adenocarcinoma compared to the normal controls.The analysis of clinical data found that down-regulated miR-486-5p was associated with advanced tumor stage and large tumor size.The target prediction and luciferase reporter assay validated that PIK3R1 was a direct target of miR-486-5p.The expression level of miR-486-5p was inversely correlated with that of PIK3R1,and over-expressed miR-486-5p significantly inhibited the expression of PIK3R1 by qPCR and western blot assay.Moreover,over-expressed miR-486-5p effectively inhibited A549 cell proliferation and invasion by targeting PIK3R1 in lentivector transfection assay,and successfully induced cell apoptosis.It is infered that miR-486-5p might act as a tumor-suppressor contributing to the progression of lung cancer by targeting PIK3R1.MiR-486-5p would be a potential target for the treatment of lung cancer.This study profiled the expression of miRNAs in various samples of lung cancer by high throughput sequencing,found some miRNAs gradually changed from normal tissue to tumor tissue,and several miRNAs had the similar differences in serum samples.The cytological function validation showed that miR-486-5p might be a potential diagnostic and therapeutic target for lung cancer.