The Basic And Clinical Study Of Body Fluid MicroRNAs As Disease Biomarkers

MicroRNA(miRNA)are a class of small non-coding single-stranded RNA molecules with the lenth about 22 nucleotides,and can regulate its target gene expression at the post-transcriptional level by completely or partially complementary binding to mRNA 3' end untranslated region sequence,which resulting in target mRNA degradation or translation inhibit.Our group previous studies found human body fluids including serum,plasma,urine,pleural effusion,and milk have numerous stable miRNAs,and further studies showed that serum miRNAs can be used as novel tumor markers of esophageal squamous cell carcinoma,gastric cancer,liver cancer,pancreatic cancer,pleural effusion miRNA can be used as a marker for distinguish between benign and malignant tumors,and milk miRNAs can be used as a new testing standards for the raw milk in milk products.As potential novel disease biomarkers,body fluid miRNAs are quite different from traditional biomarkers,because they are quite stable,easy to detect,and can be accurate quantified.Its development will improve the defect of traditional protein-based biomarkers,and provide a new way to prevent and treat disease.However,as a newborn area,there are still a lot of problems remain to be elucidated.Based on our preliminary studies,in this PhD thesis,we carried out a series of basic and clinical research to study the potential of body fluid miRNAs using as disease diagnostic biomarkers for some higher incidence diseases,and the research was divided in four parts.In the first section,we investigate the seminal plasma miRNA profile in infertile men and identify miRNAs that are altered in infertility and then evaluated their diagnostic value.Seminal plasma samples were obtained from 289 infertile men and 168 age-matched fertile controls.The stability of the miRNAs was first assessed by time-course and freeze-thaw cycle analyses.The Solexa sequencing technology was used for an initial screen of the miRNAs in samples pooled from 45 patients with nonobstructive azoospermia,58 patients with asthenozoospermia,and 100 fertile controls.A stem-loop quantitative reverse-transcription PCR(qRT-PCR)assay was conducted in the training and verification sets to confirm the concentrations of the altered miRNAs in 73 patients with nonobstructive azoospermia,79 patients with asthenozoospermia,34 patients with oligospermia,and 68 fertile controls.The existing form and potential target gene of dysregulated seminal plasma miRNA were also studied.Our data showed that the miRNAs in seminal plasma were stable.The Solexa sequencing demonstrated 19 markedly altered miRNAs in the patient groups compared to the control group.qRT-PCR analysis identified 7 miRNAs(miR-34c-5p,miR-122,miR-146b-5p,miR-181a,miR-374b,miR-509-5p,and miR-513a-5p)being markedly decreased in azoospermia but increased in asthenozoospermia.The area under the ROC curve for these miRNAs ranged from 0.733 to 0.921,markedly higher than for routine biochemical parameters(0.510 to 0.622).Moreover,the concentrations of some selected miRNAs were also increased in the semen sperm of the asthenozoospermia patients.We then studied the exsiting form and potential mechanism roles of seminal plasma miRNA in male infertility.The results showed that MV enrich in seminal plasma,seven miRNA present in different form in seminal plasma,for miR-34c-5p,122,181a,509-5p,513a-5p,which are mainly existed in MV-free form,miR-374b mainly exist in MV form,and miR-146b-5p has cloth relative content both in MV and in MV-free.Further bioinformatics research and Western blotting results showed that unregulated miR-181a both in the seminal plasma and sperm of asthenozoospermia patients may be involved in regulation of Akap4 and lead to low sperm motility.In the second section,we determined the serum miRNA expression profile in lung cancer patients in order to develop a novel diagnostic lung cancer biomarker.An initial screen of miRNA expression was performed in pooled serum samples from 31 patients and 31 controls by TaqMan low density array(TLDA).A TaqMan probe-based stem-loop quantitative reverse transcription polymerase chain reaction assay was employed to confirm the expression of selected miRNAs in serum samples from 19 patients and 19 controls.We used risk score analysis to evaluate the diagnostic value of the serum miRNA profiling system.To assess the serum miRNA-based biomarker accuracy in predicting lung cancer,we performed double-blind testing in 108 lung cancer cases and 104 controls.We also examined the serum dys-regulate miRNAs in five pairs lung cancer and adjacent normal tissues.Bioinformatics' methods and western blotting were also used to investigate in potential targets and function of the selected miRNA.The TLDA results showed that 86 miRNAs are different expressed in patents and controls.Serum miRNA expression profile in lung cancer patients was quite different from controls.qRT-PCR identified a profile of five serum miRNAs including miR-483-5p,193a-3p,214,25 and miR-7 to be biomarkers for lung cancer.More importantly,this panel of five miRNAs can clearly distinguished lung cancer from controls by further confirmed in another double-blind testing include 108 lung cancer and 106 controls,95.4%of 108 lung cancer cases and 88.5%of 106 control samples were classified correctly,and using the optimal cut-off value,the sensitivity and specificity were 88.6%and 94.3%.Furthermore,the five miRNAs' expression also showed changes between lung cancer and adjacent normal tissue,three miRNA including miR-7,25,193a-3p was significantly upregulated in lung cancer tissues than normal tissue.Bioinformatics analysis showed that tumor suppressor gene RB1 is a potential target genes of miR-7.Wsternblot experimental results showed that lung cancer RB1 protein expression was significantly decreased.Luciferase experiments indicate that miR-7 can be combined with RBI 3'-UTR regulating the expression of RBI.In A549 lung cancer cell lines,over-expression of miR-7 can suppress the RBI protein expression,but has no effect on mRNAs' expression.And further in vitro functional assay results showed that over expression of miR-7 can promote A549 lung cancer cells proliferation.In the third section,we focus on our study of the serum miR-200a as a novel RCC diagnostic marker.By using qRT-PCR,we first detect the expression of miR-200a in 19 pairs RCC tissue and adjacent normal tissue.we then examine the expression level of miR-200a in 107 RCC patients and 107 age-gender matched control sera in two stages.MiR-200a concentration was further measured in urine samples of 27 RCC patients and 27 controls;Moreover,bioinformatics' methods and western blotting were used to investigate in potential targets and function of miR-200a in RCC.qRT-PCR data showed that miR-200a is significantly reduced in RCC tissue,and it is also significant down regulated in the serum samples of patients with RCC,further ROC curve analysis showed that miR-200a is a potential biomarker of RCC.Moreover,the absolute concentration of miR-200a were also significantly reduced in RCC patients' urine samples.Bioinformatics analysis showed that the proto-oncogene sperm associated antigen 9(SPAG9)is a potential target gene of miR-200a.Western blot experiment results showed that SPAG9 protein expression level was significantly higher in the RCC tissue than in adjacent normal tissue,which significantly negatively correlated with miR-200a.Luciferase experimental results show that miR-200a can be combined with SPAG9 3'-UTR and regulation SPAG9 expression.In the fourth section,we determined the serum miRNA expression profile in type 1 diabetes(T1D)patients in order to develop a novel diagnostic biomarker for predict ongoing beta-cell destruction and regeneration in children with newly diagnosed Type 1 Diabetes(T1D).An initial screen of miRNA expression was performed in pooled serum samples from 275 12-month after newly onset T1D patients,129 1-month after newly onset T1D patients and 151 healthy controls by Solexa sequencing.qRT-PCR assay was employed to confirm the expression of selected miRNAs in serum samples which used in Solexa sequencing stage.Solexa sequencing data were also assayed in 42 adult T1D patients and 42 controls.The correlation analysis was conduct between the expression level of sera miRNAs and expression levels to beta-cell function and glycaemic control.From Solexa sequencing data,we identified 47 upregulated human miRNAs in T1D patients and of these,24 miRNA were choosen for further confirmation.11 miRNA including miR-152,miR-30a-5p,miR-181a,miR-24,miR-148a,miR-210,miR-27a,miR-29a,miR-26a,miR-27b,miR-25 and miR-200a were showed a same upregulated change with the solexa sequencing data,several of these miRNAs were linked to apoptosis and beta cell networks.Furthermore,we identified miR-25 as negatively associated with residual beta-cell function(p=0.0045),and positively associated with glycaemic control(HbAlc)(p=0.0026),miR-29a also showed negatively associated with residual beta-cell function(p=0.06).The qRT-PCR assay on the 26 miRNA from the Solexa screening phase in 42 adult T1D patients and 42 control showed that 5 serum miRNAs(miR-21,25,29 b,222,200 a)were significant up-regulated and 9 miRNA(miR-26a,210,27a,27b,30a,103,148a,152,181a)were significantly down-regulated in adult T1D patients.Further association analysis showed miR-25 and-21 positive correlated with patients' blood glucose concentration(p<0.05).In summary,we studied the miRNA expression profile in numerous body fluids including seminal plasma,serum,urine and evalute their clinical value for diseases diagnostic,we also investigate the potential role of the selected miRNA in disease and found that:(1)seven seminal plasma miRNA including miR-34c-5p,122,146b-5p,181a,374b,509-5p,513a-5p can be used as a potential diagnostic marker for male infertility,and its diagnostic sensitivity and specificity significantly higher than that the seminal plasma biochemical markers.Seminal plasma contains MV,and miR-34c-5p,122,181a,509-5p,513a-5p mainly existed in MV-free form,miR-146b-5p,374b mainly exist in the form of MV.Up-regulated miR-181a both in the seminal plasma and sperm of asthenozoospermia patients may be involved in regulation of Akap4 and lead to low sperm motility.(2)Serum miR-483-5p,193a-3p,25,214 and 7 can be used as a diagnostic marker of lung cancer.The elevated serum miRNAs in lung patients also showed up-regulated in lung cancer tissues.MiR-7 can regulate the expression of the tumor suppressor gene RBI and involved in the development of lung cancer.(3)Down-regulated miR-200a in RCC tissues and patients' serum can be used as a novel serological marker of RCC and it is also down-regulated in RCC urine.Down-regulated miR-200a in RCC patients can regulate the expression of proto-oncogene SPAG9 and participate in the occurrence and development of RCC.(4)Dysregulated serum miRNA can be used as a novel diagnostic biomarker for predict ongoing beta-cell destruction and regeneration in T1D.