| BackgroundParkinson’s disease(PD)is one of the world’s second largest neurodegenerative diseases associated with age.65 years of age in the population incidence is 1 to 2%,85 years of age for 4 to 5%.And as the population ages,the number of PD patients is increasing every year.PD clinical symptoms include resting tremor,muscle rigidity,movement retardation and postural gait disorders and other motor symptoms,as well as olfactory disorders,constipation,neuropsychiatric disorders,cognitive disorders,autonomic dysfunction and other non-motor symptoms.With the development of the disease,most patients will eventually lose their self-care ability,seriously affecting the daily life and work of patients,to the family and society to immeasurable burden.At present,the diagnosis and treatment of PD problems are:(1)pathogenesis is unclear:PD incidence by a variety of factors,now that is mainly based on the aging,genetic and environmental interaction caused.Many studies have shown that the degeneration of neuronal degeneration in substantia nigra of PD may be related to a series of mechanisms such as mitochondrial dysfunction,oxidative stress,lack of neurotrophic factor,excitotoxicity,immunoregulation abnormality and cell apoptosis.Epidemiological studies have shown that about 5 to 10%of patients with Parkinson’s disease have a family history.But only six were confirmed to cause hereditary single gene mutation,indicating that the incidence of Parkinson’s disease and genetic factors are closely related.(2)The lack of objective laboratory diagnostic methods:PD heterogeneity is high,in addition to the current image method alone,The Unified Parkinson’s Disease Rating Scale(UPDRS)and Hoehn-Yahr grading scale is the PD clinical diagnosis and treatment of common methods.According to the British brain brain Parkinson’s disease diagnostic criteria,patients with less dynamic,coupled with static tremor,muscle rigidity,posture gait abnormalities in one of three symptoms,can be diagnosed as PD.However,due to the complexity of the pathogenesis of different patients with clinical symptoms and signs may be different,and the same patient at different times the condition will change,and the results are often subject to the patient and the doctor’s main and objective factors,making The results of the lack of comparable,to the clinical diagnosis of difficulties.Therefore,it is important to screen out the PD-related biomarkers for revealing the pathogenesis and developing an objective diagnostic method so as to achieve the purpose of prevention,control and treatment.MicroRNA(miRNA)is an endogenous single-stranded short-sequence non-protein-encoded RNA with length of 19-23 nucleotides,co-transcribed with the host gene,involved in the expression of approximately 1/3 gene,widely present in eukaryotes Is an important regulatory molecule that regulates the expression of other functional genes and participates in the regulation of a variety of cellular processes,such as generation,differentiation,cell proliferation and apoptosis,and its abnormal regulation leads to the onset of a variety of diseases,including cancer and neurodegenerative diseases.In mature and immature brains,the expression levels of miRNAs are very high in different brain regions and in different cell types,and some miRNAs appear in specific positions such as synapses,dendrites and axons in cells.They can improve the homeostasis,regulate stress response,adjust a lot of parameters related to synaptic plasticity.Compared to proteins,miRNAs are a barrier that is easier to pass through the blood brain,placenta,and small molecules that are present in body fluids.MiRNAs have been detected in cytoplasm,serum,urine,saliva and milk,which are protected by molecules such as exosomes and other particles,proteins,liquids,and the like.As a result of the appearance of apoptotic bodies,the budding and shedding of microbubbles,the active secretion of high density lipoproteins leadsto the release of miRNAs into the cells,followed by cell death,miRNAs appear in the body fluid in all forms of exfoliated miRNAs in the blood and other body fluids were highly stable.The secretion of miRNAs is selective and can be significantly altered by different pathological processes.It is easy to detect the characteristics of prompt its wide prospects as a diagnostic marker.Studies have shown that PD is a brain disease,so brain tissue is the best tissue sample that reflects the nature of the disease.However,due to the particularity of brain tissue,while PD has no clear pathogenic brain area,can only take the body brain tissue as a research object,making the brain tissue related biomarkers difficult to be used in clinical diagnosis.As we all know,cerebrospinal fluid and brain tissue at all times in the molecular exchange,in a timely manner,more accurate and dynamic to reflect some changes in brain tissue.In the study of PD biomarkers,many scholars use cerebrospinal fluid as a biological sample,but because of the use of lumbar puncture method to take cerebrospinal fluid there is the risk of infection and bleeding,it is difficult to be accepted by patients.Because miRNAs are stable in blood plasma or serum,the use of today’s sensitive analytical methods can be reliably detected at low concentrations,so the study of serum miRNAs is a good sample of noninvasive early diagnosis and prognosis.ObjectiveThe miRNA expression profiles of PD patients were screened out and the miRNAs were validated to determine their possible role in the pathogenesis of PD.To provide a basis for PD serum biomarkers of miRNA,evaluate its value in the diagnosis of PD.Method1.Sample collection:According to the British PD brain bank diagnostic criteria,and according to the unified Parkinson’s disease rating scale(UPDRS)and improved Hoehn&Yahr classification and magnetic resonance imaging and neuropsychological tests to give diagnosis of 138 cases of Parkinson patient.There were 112 normal volunteers with age and gender matching as control group.2.Solexa sequencing:The expression of miRNAs in mixed serum composed of 12 patients with PD and 12 healthy controls was detected by Solexa sequencing.MiRNAs were significantly screened in PD group and healthy control group.3.Verification:The real-time quantitative PCR(quantification real time PCR)technique was used to verify the miRNAs of 112 cases and 138 cases of healthy control group.4.Further verification:Real-time quantitative PCR was used to further verify the miRNAs with significant differences in PD patients.The final diagnostic value was evaluated by Spearman rank correlation analysis and ROC curve.Result1.Solexa sequencing results:Solexa sequencing 16 kinds of miRNAs,there are significant differences in PD group and healthy controls.They are miR-141,miR-146a-5p,miR-193a-3p,miR-214,miR-133b,miR-15b,miR-181a,miR-185,miR-195,miR-221,miR-29c,miR-29a,miR-19b,miR-24,miR-34b,miR-148b。2.Validation:using the real-time fluorescent quantitative PCR to 16 kinds of miRNAs verification,we found that four kinds of miRNAs in PD patients with serum expression and healthy human serum were significantly different,they are miR-29c,miR-146a,miR-214 和 miR-221.3.Further validation:These four miRNAs were further validated by real-time fluorescence quantitative PCR.The results showed that the expression of miR-29c,miR-146a,miR-214 and miR-221 in the serum of patients with PD was down-regulated compared with healthy people.(1)miR-221 test results and UPDRS-Ⅲ scale score results,the rank test found that the correlation coefficient of 0.4702,the P value of less than 0.0001;Also found its score with the UPDRS-V score,the correlation coefficient was 0.4788,the P value was less than 0.0001.The miR-221 gene was positively correlated with the UPDRS-Ⅲ and UPDRS-Ⅴ scales and was statistically significant.(2)The correlation coefficients of miR-29c,miR-146a,miR-214 and UPDRS-Ⅲ scale were 0.1218,0.1669 and 0.1931 respectively,and the p value was more than 0.05.At the same time,it was found that the correlation coefficient with the UPDRS-V score was 0.3333,0.1394,0.5476,p value was greater than 0.05.Therefore,the predictive effects of miR-29c,miR-146a and miR-214 on PD are not yet scientific.(3)We also validated the ROC(Receiver Operating Characteristic Curve)of the four miRNAs under the curve area,where the area under the curve of miR-221 was 0.8639±0.00223,and the other three results were as follows:miR-29c under the curve of the area of 0.7565 ± 0.0333,miR-146a under the curve of the area of 0.5752 ± 0.0373,miR-214 under the curve area of 0.8240±0.0288.ConclusionPD patients and healthy people have abundant and stable miRNAs.The expression profiles of serum miRNAs in patients with PD are significantly different from those of healthy people.Our findings show that miR-29c,miR-146a,miR-214 and miR-221 However,the miR-221 gene was positively correlated with the UPDRS-Ⅲ and UPDRS-V scales,and the area under the ROC curve was statistically significant.However,the expression of the miR-221 gene was significantly higher in the serum of PD patients than in healthy subjects.Serum miR-221 may be a potential biomarker for the diagnosis of PD. |
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