Method for screening markers based on data model constructed by GWAWS data set and application of method in diagnosis of diabetic hypomnesia type mild cognitive impairment

Abstract

The invention belongs to the technical field of biomedicine, discloses a method for screening markers based on a data model constructed by a GWAWS data set and application of the method in diagnosis of diabetic hypomnesia type mild cognitive impairment, and discloses a serum exosome miR-125a-5p molecular marker for the first time. Experiments find that the expression change in diabetic patients with hypomnetic mild cognitive impairment is significant, and the expression change is significantly related to occurrence of hypomnetic mild cognitive impairment and poor scene memory performance; the invention further discloses a kit containing the molecular marker, the sensitivity of the kit in diagnosis of hypomnesia type mild cognitive impairment of diabetic patients reaches 0.63, and the specificity reaches 0.67; therefore, the serum exosome miR-125a-5p molecular marker provided by the invention can be used as a marker for risk estimation and detection of hypomemory mild cognitive impairment of diabetic patients, and is beneficial to screening, prevention and treatment of cognitive impairment of diabetic patients.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and discloses a method for screening markers based on a data model constructed based on a GWAWS data set and its application in the diagnosis of diabetic amnesia-type mild cognitive dysfunction, and specifically discloses the use of serum exosome miR-125a-5p in the preparation of a preparation for diagnosing diabetic patients with amnesia-type mild cognitive dysfunction. Background Art

[0002] With the aging of the population, diabetes and its complications seriously threaten the health of the population. Among them, diabetes-related cognitive dysfunction has received increasing attention. According to the development of the disease, cognitive dysfunction can be divided into preclinical, mild cognitive dysfunction and dementia. Mild cognitive dysfunction, especially amnestic mild cognitive dysfunction, has a high risk of transformation to dementia and is an important gateway for early intervention. At present, there is a lack of effective drugs to delay or reverse dementia, so identifying the risk population of amnestic mild cognitive dysfunction is a hot topic and difficulty in research. Different assessment methods have different diagnostic efficacy in the course of cognitive dysfunction. Routine clinical detection methods, such as neuropsychological assessment, are not sensitive to mild changes in cognitive dysfunction. Brain imaging and cerebrospinal fluid testing to find neurodegenerative markers are helpful for early diagnosis of the disease. However, due to the high cost of the examination and the invasive operation, it has not been widely carried out in clinical diabetic patients. More importantly, the diagnostic efficacy of neurodegenerative markers for diabetes-related mild cognitive dysfunction is poor (Aβ: AUC = 0.631). This suggests that diabetes-related cognitive dysfunction may have a mechanism other than classic dementia. Therefore, we need to find specific markers for cognitive dysfunction in this population based on the disease characteristics of cognitive dysfunction in the diabetic population.

[0003] Serum exosomes and their contents have great potential for disease prediction. Serum exosomes and their contents also play an important role in the diagnosis of diabetic complications. Exosomes derived from diabetic adipose tissue can carry microRNAs into the brain, causing synaptic loss and cognitive impairment in mice. The miRNAs carried by exosomes play an important role in this relationship. Targeting miRNAs in adipose tissue or hippocampus reduces synaptic loss in mice and improves cognitive impairment. Adipose tissue is an important source of serum exosomal miRNAs. However, serum exosomal miRNAs are rarely used as molecular markers in the diagnosis of diabetic cognitive dysfunction, and the existing diagnostic markers for diabetic cognitive dysfunction do not have the characteristics of changes in the expression of serum exosomal miRNAs in the disease. Summary of the invention

[0004] To solve the above problems, the present invention discloses a method for screening markers based on a data model constructed based on the GWAWS dataset and its application in the diagnosis of diabetic amnesia-type mild cognitive dysfunction, and specifically discloses the use of serum exosome miR-125a-5p in the preparation of a preparation for diagnosing diabetic patients with amnesia-type mild cognitive dysfunction.

[0005] The present invention includes the following technical solutions:

[0006] The present invention discloses a method for screening markers based on a data model constructed based on a GWAWS data set, comprising the following steps: using Mendelian randomization analysis to screen the relationship between upregulated miRNAs in diabetic adipose tissue exosomes and cognitive function based on the GWAWS data set, matching SNP data through the TwoSampleMR toolkit, using the IVW method to summarize the effect estimation, combining sensitivity analysis and eliminating SNPs one by one to verify stability, and finally drawing a funnel plot and MR-Egger regression to evaluate pleiotropic interference, so as to screen out markers of mild cognitive impairment of diabetic patients with memory impairment.

[0007] The invention also discloses the application of the method in preparing a preparation for diagnosing mild cognitive impairment of memory loss type in diabetic patients.

[0008] The present invention also discloses for the first time the use of serum exosome miR-125a-5p in preparing a preparation for diagnosing mild cognitive impairment of memory impairment type in diabetic patients.

[0009] Furthermore, the present invention found that when the serum exosome miR-125a-5p is upregulated in the serum exosomes of diabetic patients, it leads to cognitive impairment.

[0010] Furthermore, in the above use, the diabetic patient is a type 2 diabetes patient.

[0011] Furthermore, the present invention discloses the use of serum exosome miR-125a-5p in preparing a kit for diagnosing mild cognitive impairment of memory impairment type in diabetic patients.

[0012] The invention discloses a kit for diagnosing diabetic patients with memory impairment type mild cognitive dysfunction, wherein the kit contains serum exosome miR-125a-5p molecular marker standard products.

[0013] Furthermore, the above-mentioned kit also includes a reverse transcription system and a real-time fluorescence quantitative PCR system.

[0014] Furthermore, in the above-mentioned kit, the reverse transcription system includes reverse transcription primers and PCR probes, which are products of Thermo Fisher Scientific with catalog number 4427975.

[0015] Furthermore, in the above kit, the reverse transcription system includes: 2ul of total RNA, 2ul of RT Buffer (5×), 0.5ul of dNTP (10mM), 0.5ul of reverse transcription primer (1μM), 0.5ul of MMLV reverse transcriptase (200U / μl), and 4.5ul of enzyme-free water;

[0016] The reverse transcription program was set as follows: 16°C for 30 min, 42°C for 30 min, and 85°C for 10 min.

[0017] Furthermore, in the above kit, the real-time fluorescence quantitative PCR system includes 0.5ul of reverse transcription product, 2ul of 10× Real-time PCR Buffer, dNTP (Mg 2+ plus) 1.6ul, TaqMan probe 0.3ul, rTaq enzyme 0.1ul, enzyme-free water 15.5ul;

[0018] The amplification conditions were set as follows: 95°C for 3 min, 50 cycles; 95°C for 12 s, 58°C for 40 s.

[0019] Furthermore, in the above-mentioned kit, the sensitivity of the kit in diagnosing mild cognitive impairment of memory impairment type in diabetic patients reaches 0.63, and the specificity reaches 0.67.

[0020] The present invention has the following beneficial effects:

[0021] The present invention discloses a method for screening markers based on a data model constructed based on the GWAWS data set, which can effectively identify the potential causal relationship between diabetes-related miRNAs and cognitive impairment, and can verify the robustness of the results; the present invention also discloses for the first time the use of serum exosome miR-125a-5p in the preparation of a preparation for diagnosing diabetic patients with amnesia-type mild cognitive dysfunction, and screens out a marker exosome miR-125a-5p that can be used to diagnose diabetic patients with amnesia-type mild cognitive dysfunction by detecting the miRNA expression spectrum of adipose tissue exosome samples and by using the Mendelian randomization study method; its expression level in the serum of diabetic patients with amnesia-type mild cognitive dysfunction is significantly higher than that of diabetic patients with normal cognitive function and non-amnesia-type mild cognitive dysfunction, and is significantly correlated with the occurrence of amnesia-type mild cognitive dysfunction and impaired episodic memory. It has important value in preparing diagnostic products for auxiliary diagnosis of diabetic patients with amnesia-type cognitive dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1This is a graph showing the results of the miRNA detection method for differentially expressing exosomes in adipose tissue of type 2 diabetic patients and non-diabetic subjects screened by the present invention;

[0023] Figure 2 The results of Mendelian randomization analysis of the causal relationship between upregulated miRNAs in adipose tissue exosomes of patients with type 2 diabetes and Alzheimer's disease;

[0024] Figure 3 The results of Mendelian randomization analysis of the causal relationship between upregulated miRNAs in adipose tissue exosomes and cognitive impairment in patients with type 2 diabetes;

[0025] Figure 4 This is a comparison chart of serum exosome miR-125a-5p detection results in patients with type 2 diabetes combined with memory impairment, non-memory impairment and normal cognitive function;

[0026] Figure 5 This is the result graph of the correlation between serum exosomal miR-125a-5p and cognitive dysfunction of memory impairment in patients with type 2 diabetes;

[0027] Figure 6 This is the result diagram of the correlation between serum exosomal miR-125a-5p and cognitive function performance in patients with type 2 diabetes;

[0028] Figure 7 ROC curve of serum exosomal miR-125a-5p in diagnosing cognitive dysfunction with memory impairment in type 2 diabetes. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The reagents or instruments used in the examples of the present invention without indicating the manufacturer are all conventional reagent products that can be obtained through commercial purchase.

[0031] Example 1

[0032] High-throughput sequencing and Mendelian randomization studies were combined to screen exosomal miRNA markers associated with cognitive impairment in patients with type 2 diabetes.

[0033] (1) Isolation and extraction of exosomes from adipose tissue

[0034] Collect the visceral fat tissue of the research subjects. Carefully separate and remove other tissues wrapped around the visceral fat, place them in a sterile culture dish and mark them. Replace new sterile scissors again to cut the visceral fat tissue as much as possible, add an appropriate amount of serum-free culture medium to it, and gently shake the culture dish to evenly disperse the cut visceral fat tissue in DMEM. Place it in a 37℃ incubator and place it under 5% CO2 for 24h. Collect the supernatant of the visceral fat tissue culture medium. Centrifuge the supernatant at 4℃ and 3,000g for 20min to remove the tissue and cell fragments precipitated at the bottom of the centrifuge tube; extract the supernatant after centrifugation, centrifuge the supernatant at 4℃ and 10,000g for 30min; take the supernatant and add it to an ultracentrifuge tube, add PBS to balance, and centrifuge it at 4℃ and 100,000g for 80min; immediately use PBS to resuspend the adipose tissue exosomes precipitated at the bottom of the centrifuge tube after centrifugation, and freeze it in liquid nitrogen.

[0035] (2) Total RNA extraction

[0036] Take an appropriate amount of the sample to be tested and add it to a test tube. Add 1ml Trizol (Ambion) lysis buffer and then add 200ul chloroform. Mix thoroughly by inverting and let stand for 5min. Adjust the centrifuge to 4℃ and centrifuge at 12,000rpm for 15min. Take the upper liquid, add 500ul isopropanol, mix thoroughly and let stand for 2 hours at room temperature. Centrifuge at 4℃ and 12,000rpm for 10min. Aspirate the supernatant, add 1mL 75% DEPC ethanol, centrifuge at 4℃ and 12,000rpm for 10min. Aspirate the supernatant and dry at room temperature. After the alcohol is completely evaporated, add 20ul DEPC water to completely dissolve the precipitate. Use Nanodrop 2000 instrument to detect the purity and concentration of the extracted RNA.

[0037] (3) Small RNA sequencing

[0038] Small RNA sequencing analysis was performed using 5 ng RNA. Sequencing was performed by Novogene (Beijing, China). Differentially expressed miRNAs between the type 2 diabetes group and the non-diabetes group were analyzed using the DESeq2 package (|log2FC|>0, P<0.05).

[0039] Figure 1 : is a graph showing the results of differentially expressed miRNAs in adipose tissue exosomes of patients with type 2 diabetes in the present invention. Figure 1 As shown, 13 miRNAs were upregulated in adipose tissue exosomes of patients with type 2 diabetes compared with participants with normal glucose tolerance.

[0040] (4) Screening miRNAs with significant causal relationship with cognitive dysfunction through Mendelian randomization analysis

[0041] The miRNAs screened out and upregulated in adipose tissue exosomes of diabetic patients were subjected to Mendelian randomization analysis to identify risk factors for cognitive impairment. Mendelian randomization study is an analytical method that uses genetic variation as an instrumental variable for specific risk factors to establish a statistical model and then explore the causal relationship between exposure factors and outcomes. First, the instrumental variables for candidate miRNAs and cognitive function-related outcomes were constructed based on the public GWAWS dataset. Table 1 shows the source of genetic variation data for Mendelian randomization study. This study extracted the effect allele (EffectAllele), alternative allele (Other Allele), effect allele frequency (Effect Allele Frequency), beta coefficient, standard error (Standard Error, SE) of the beta coefficient, and p-value associated with the exposure factor for each SNP. The linkage disequilibrium test was based on the European 1000 Genomes Project reference template and was implemented by the R package TwoSampleMR (https: / / github.com / MRCIEU / TwoSampleMR). Matching the exposure and outcome GWAS data corresponding to the SNPs was also achieved using the TwoSampleMR package. For any SNP, its effect on the exposure factor and its effect on the outcome must match the same allele. Otherwise, the SNP will be eliminated from the analysis process. For the screened SNPs, the corresponding data of each item were extracted from the outcome data set. The two-sample MR method was mainly used to evaluate the causal association between miRNAs levels and cognitive function, cerebral cortical surface area, and dementia risk. First, the random effects model inverse variance weighted (IVW) method was used to summarize and merge the causal effect estimates of each SNP to obtain the odds ratio (OR) of each exposure factor for the outcome, which was used as the main statistical result. Secondly, the maximum likelihood estimation method (Maximum Likelihood) and weighted median (WM) method were used as sensitivity analysis to test the stability of the main results. Then, each SNP was eliminated one by one in order to repeat the main results, and the stability of the results was further tested to determine whether it was significantly affected by one or several SNPs. Finally, a funnel plot was drawn to intuitively and qualitatively determine the size of the data heterogeneity, and the Mendelian randomization Egger regression (MR-Egger method) intercept test was combined to quantitatively determine whether there was pleiotropic interference.

[0042] Table 1. Sources of genetic variation data for Mendelian randomization studies

[0043]

[0044] Figure 2 This is a Mendelian randomization analysis result of upregulated miRNAs in adipose tissue exosomes of patients with type 2 diabetes and the risk of Alzheimer's disease. Among them, only the increased level of miR-125a-5p has a significant causal relationship with the increased risk of Alzheimer's disease.

[0045] Figure 3 This is a graph showing the results of a Mendelian randomization analysis of upregulated miRNAs in adipose tissue exosomes of patients with type 2 diabetes and multiple cognitive function assessments. Among them, only increased levels of miR-125a-5p were significantly causally associated with worse episodic memory scores.

[0046] Combining the sequencing results and Mendelian randomization analysis, it was found that among these miRNAs significantly upregulated in adipose tissue exosomes of patients with type 2 diabetes, only increased levels of miR-125a-5p had a significant causal relationship with increased risk of Alzheimer's disease and worse episodic memory performance.

[0047] Example 2

[0048] RT-PCR was used to verify the differential expression of serum exosomal miR-125a-5p and to evaluate its use in the preparation of a preparation for diagnosing mild cognitive impairment of the memory impairment type in diabetic patients.

[0049] (1) Collect serum samples and isolate serum exosomes

[0050] A total of 89 patients with type 2 diabetes with normal cognitive function, 52 patients with type 2 diabetes combined with mild cognitive impairment of amnesia, and 66 patients with type 2 diabetes combined with mild cognitive impairment without amnesia were included. Inclusion criteria: age ≥ 55 years; blood samples were collected. Exclusion criteria: (1) history of stroke, brain trauma, or surgery; (2) other neurological diseases (such as delirium); (3) psychiatric diseases (such as anxiety or depression); (4) uncontrolled hypothyroidism; (5) unable to cooperate with cognitive function assessment; (6) nasal and maxillofacial diseases that affect olfaction; (7) contraindications to MRI examination.

[0051] Collect fasting venous blood in the morning, centrifuge at 3000rpm for 15min, and keep the upper serum in a -80℃ refrigerator. Slowly thaw the frozen serum sample to be tested on ice and take 1ml, centrifuge at 4℃, 3,000g for 20min, remove the tissue and cell debris precipitated at the bottom of the centrifuge tube; extract the supernatant after centrifugation, centrifuge at 4℃, 10,000g for 30min; take the supernatant and add it to an ultracentrifuge tube, add pre-frozen PBS to balance, and centrifuge at 4℃, 100,000g for 80min; immediately resuspend the serum exosomes precipitated at the bottom of the centrifuge tube with PBS after centrifugation, and freeze in liquid nitrogen.

[0052] The exosome RNA was extracted using the method of Example 1. The extracted RNA and miR-125a-5p standard were reverse transcribed to obtain cDNA. The reverse transcription system included 2ul of total RNA, 2ul of RT Buffer (5×), 0.5ul of dNTP (10mM), 0.5ul of reverse transcription primer (1μM), 0.5ul of MMLV reverse transcriptase (200U / μl), and 4.5ul of enzyme-free water. After adding to the centrifuge tube and mixing, the amplification was performed according to the following amplification program: 16°C for 30min, 42°C for 30min, and 85°C for 10min. After the reaction was terminated, the reverse transcription product was immediately taken out and quickly cooled in ice water.

[0053] The cDNA was then subjected to qPCR fluorescence quantitative detection. The real-time fluorescence quantitative PCR system includes 0.5ul of reverse transcription product, 2ul of 10× Real-time PCR Buffer, dNTP (Mg 2+ Plus) 1.6ul, TaqMan probe 0.3ul, rTaq enzyme 0.1ul, and enzyme-free water 15.5ul were added to the centrifuge tube, the solution was mixed and bubbles were removed, and the tube was placed on the real-time fluorescence quantitative PCR system. Amplification was performed according to the following amplification conditions: 95℃ for 3min, 50 cycles; 95℃ for 12s, 58℃ for 40s. After the reaction was terminated, the absolute expression of miRNAs was calculated.

[0054] The reverse transcription primer and TaqMan probe targeting miR-125a-5p were purchased from Thermo Fisher Scientific with a catalog number of 4427975.

[0055] The levels of serum exosomal miR-125a-5p in the three groups were compared using analysis of variance. Correlation analysis was performed to evaluate the relationship between serum exosomal miR-125a-5p and cognitive function performance in patients with memory impairment and cognitive dysfunction in type 2 diabetes. Finally, ROC analysis was used to evaluate the diagnostic ability of serum exosomal miRNA for mild cognitive dysfunction in patients with memory impairment and cognitive dysfunction in type 2 diabetes.

[0056] Figure 4 This is a comparison of the results of serum exosomal miR-125a-5p detection in patients with type 2 diabetes combined with amnesia-type cognitive impairment, non-amnesia-type cognitive impairment, and normal cognitive function. The serum exosomal miR-125a-5p level in patients with type 2 diabetes combined with mild cognitive impairment of amnesia was significantly higher than that in patients with normal cognitive function and patients with type 2 diabetes with non-amnesia-type cognitive impairment (P<0.05).

[0057] Figure 5 The figure shows the correlation between serum exosomal miR-125a-5p and memory impairment-type cognitive dysfunction in patients with type 2 diabetes. Taking patients with type 2 diabetes with normal cognitive function as a reference, elevated serum exosomal miR-125a-5p levels are a risk factor for memory impairment-type mild cognitive dysfunction, but not for non-memory impairment-type mild cognitive dysfunction.

[0058] Figure 6 The results of the correlation between serum exosomal miR-125a-5p and cognitive function in patients with type 2 diabetes. In patients with type 2 diabetes, increased levels of serum exosomal miR-125a-5p were associated with lower immediate memory scores and worse language function performance. Impaired immediate memory is the main manifestation of memory impairment-type cognitive dysfunction, and this result also confirms the relationship between serum exosomal miR-125a-5p and memory impairment-type mild cognitive dysfunction.

[0059] Figure 7 The ROC curve of serum exosomal miR-125a-5p in diagnosing memory impairment-type cognitive dysfunction in type 2 diabetes. In patients with type 2 diabetes, serum exosomal miR-125a-5p has a good diagnostic efficacy for memory impairment-type mild cognitive dysfunction, with an area under the curve of 0.73, a sensitivity of 0.63, and a specificity of 0.67.

[0060] It can be seen from the above examples that the present invention screened out a marker exosome miR-125a-5p that can be used to diagnose diabetic amnesia-type mild cognitive dysfunction by detecting the miRNA expression profile of adipose tissue exosome samples and by using the Mendelian randomization study method; its expression level in the serum of diabetic patients with amnesia-type mild cognitive dysfunction was significantly higher than that of diabetic patients with normal cognitive function and non-amnesia-type mild cognitive dysfunction, and was significantly correlated with the occurrence of amnesia-type mild cognitive dysfunction and impaired episodic memory. It has important value in preparing diagnostic products for auxiliary diagnosis of diabetic amnesia-type cognitive dysfunction.

[0061] The above embodiments show and describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A method for screening markers based on a data model constructed based on a GWAWS dataset, characterized in that: The following steps are involved: Mendelian randomization analysis was used to screen the relationship between upregulated miRNAs in diabetic adipose tissue exosomes and cognitive function based on the GWAWS dataset. SNP data were matched using the TwoSampleMR toolkit, and the IVW method was used to summarize the effect estimate. Sensitivity analysis and SNP elimination were combined to verify the stability. Finally, funnel plots and MR-Egger regression were used to evaluate pleiotropic interference, and markers of mild cognitive impairment of memory impairment type in diabetic patients were screened out.

2. Use of the method according to claim 1 in preparing a preparation for diagnosing mild cognitive impairment of amnesia type in diabetic patients.

3. Use of serum exosomal miR-125a-5p in the preparation of a preparation for diagnosing mild cognitive impairment with memory impairment in diabetic patients.

4. The use according to claim 1, wherein the serum exosomal miR-125a-5p, when expressed up-regulated in the serum exosomes of diabetic patients, leads to cognitive impairment.

5. The use according to claim 3 or 4, characterized in that the diabetic patient is a type 2 diabetic patient.

6. Use of serum exosomal miR-125a-5p in the preparation of a kit for diagnosing mild cognitive impairment with memory impairment in diabetic patients.

7. A kit for diagnosing mild cognitive impairment of memory loss in diabetic patients, characterized in that: The kit contains serum exosome miR-125a-5p molecular marker standard.

8. The kit according to claim 7, characterized in that It also includes a reverse transcription system and a real-time fluorescence quantitative PCR system.

9. The kit according to claim 8, characterized in that The reverse transcription system includes reverse transcription primers and PCR probes, which are products of Thermo Fisher Scientific with catalog number 4427975; The reverse transcription system includes: 2ul of total RNA, 2ul of RT Buffer (5×), 0.5ul of dNTP (10mM), 0.5ul of reverse transcription primer (1μM), 0.5ul of MMLV reverse transcriptase (200U / μl), and 4.5ul of enzyme-free water; The reverse transcription program was set as follows: 16°C for 30 min, 42°C for 30 min, and 85°C for 10 min; The real-time fluorescence quantitative PCR system includes 0.5ul of reverse transcription product, 2ul of 10×Real-time PCR Buffer, dNTP (Mg 2+ plus) 1.6ul, TaqMan probe 0.3ul, rTaq enzyme 0.1ul, enzyme-free water 15.5ul; The amplification conditions were set as follows: 95°C for 3 min, 50 cycles; 95°C for 12 s, 58°C for 40 s.

10. The kit according to claim 9, characterized in that The sensitivity of the kit in diagnosing mild cognitive impairment of memory impairment type in diabetic patients reaches 0.63, and the specificity reaches 0.67.

Images