Application of combination of two miRNAs to preparation of kit for diagnosing early diabetic nephropathy

Abstract

The invention discloses application of combination of two miRNAs to preparation of a kit for diagnosing early diabetic nephropathy. According to the application disclosed by the invention, the miR-3680-3p and the miR-4463 in serum are taken as markers and are jointly used for diagnosing and distinguishing pure DM and early DN, so that an excellent diagnosis effect is achieved. Based on the technical scheme, the DN can be timely found in the early stage when the DN appears in the pure DM by irregularly detecting the pure DM. According to the technical scheme, screening is fast, cost is low, invasion is avoided, and damage to the body of a patient is avoided. Therefore, the miR-3680-3p and the miR-4463 have the prospect of being developed into the serum detection kit for diagnosing the early diabetic nephropathy.

Description

Technical Field

[0001] The invention belongs to the field of disease diagnosis, relates to the diagnosis of early diabetic nephropathy, and specifically relates to the use of two miRNAs combined to prepare a kit for diagnosing early diabetic nephropathy. Background Art

[0002] Diabetic nephropathy (DN) is one of the common microvascular complications in patients with diabetes mellitus (DM), and its incidence in diabetic patients is as high as 30% to 40%. With the increase in the incidence of diabetes worldwide, the incidence of DN is also on the rise, which has brought a heavy burden to the quality of life of patients and the medical system.

[0003] Early detection and early treatment are important means to delay the progression of diabetic nephropathy. The clinical diagnosis of DN is based on renal puncture biopsy as the gold standard. Although it can accurately determine the patient's condition, it is an invasive operation in its application and will cause damage to the patient's body. In addition, most patients have already developed to the middle and late stages when they are tested, which affects the patient's prognosis. In recent years, with the discovery of more and more studies, clinical use of components contained in blood, urine, etc. as diagnostic markers can be used for early screening and diagnosis of DN. This method is not only fast and low-cost, but also non-invasive and will not cause damage to the patient's body. However, the markers that can be used are still too few to meet clinical needs.

[0004] miRNA (microRNA) is a type of small non-coding RNA molecule consisting of 21 to 23 nucleotides. They mainly bind to each other through the 3′ uncoding region (3′UTR) in the target mRNA sequence, thereby regulating the expression of the target gene, generally affecting the translation of the target gene by inhibition. At present, miRNA has been proven to play a key regulatory role in various life activities of the body and has been used as a diagnostic marker for a variety of diseases.

[0005] The inventors have discovered miRNAs markers that can be used for early diagnosis of DN, and have proposed the present invention. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for preparing a kit for diagnosing early diabetic nephropathy by combining two miRNAs.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] The use of miR-3680-3p and miR-4463 in combination for preparing a serum detection kit for diagnosing early diabetic nephropathy; wherein the diagnosis of early diabetic nephropathy refers to the diagnosis of early diabetic nephropathy in diabetic patients.

[0009] Preferably, the diabetes is type 2 diabetes, and the early diabetic nephropathy is early type 2 diabetic nephropathy.

[0010] More preferably, the serum detection kit contains reagents for detecting the contents of miR-3680-3p and miR-4463 in serum.

[0011] Beneficial effects:

[0012] The present invention finds that using miR-3680-3p and miR-4463 in serum as markers, combined for diagnosis and differentiation of simple DM and early DN, has excellent diagnostic effect. Based on this technical solution, simple DM can be detected irregularly, and DN can be found in time in the early stage of simple DM. This technical solution is not only fast in screening and low in cost, but also non-invasive and will not cause damage to the patient's body. Therefore, miR-3680-3p and miR-4463 have the prospect of being developed into a serum detection kit for diagnosing early diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the ROC curve for distinguishing simple DM from early DN in the training set; where:

[0014] ① is the ROC curve of miR-3680-3p alone for distinguishing simple DM from early DN; ② is the ROC curve of miR-4463 alone for distinguishing simple DM from early DN; ③ is the ROC curve of miR-3680-3p and miR-4463 combined for distinguishing simple DM from early DN. DETAILED DESCRIPTION

[0015] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0016] 1. Experimental Sample

[0017] Simple type 2 diabetes (simple DM, UAER < 30 mg / 24h) and early type 2 diabetic nephropathy (early DN, 30 mg / 24h≤UAER < 300 mg / 24h) collected from September 2021 to September 2024 in the Third Xiangya Hospital of Central South University were selected as the research subjects, including: 128 simple DM samples, 66 males, 62 females, aged 45 to 76 years old, with an average age of (56.5±6.3) years old; 140 early DN samples, 73 males, 67 females, aged 42 to 75 years old, with an average age of (54.6±5.9) years old. There was no significant difference in gender and age between the two groups of samples. All cases were diagnosed by the gold standard (renal puncture biopsy). Inclusion criteria: 1. Meet the relevant diagnostic criteria in the "Guidelines for the Prevention and Treatment of Type 2 Diabetes in China (2020 Edition)" and the "Expert Consensus on Multidisciplinary Diagnosis, Treatment and Management of Diabetic Nephropathy"; 2. Age > 18 years old. Exclusion criteria: 1. Type 1 DM; 2. Combined autoimmune diseases, severe infections or malignant tumors; 3. History of nephrotic syndrome; 4. History of liver disease; 5. History of renal transplantation; 6. History of immunosuppressants or hormone drugs within 2 weeks before admission.

[0018] 2. Experimental Methods

[0019] 1. Serum specimen collection

[0020] All subjects had 5 ml of peripheral venous blood collected in the early morning on an empty stomach and immediately sent to the laboratory for centrifugation at 3600 r / min for 10 min with a centrifugal radius of 12.5 cm. After centrifugation, 2 ml of the upper serum was collected and stored in a -80°C refrigerator for testing.

[0021] 2. Dataset grouping and usage positioning

[0022] A total of 268 samples (128 simple DM samples and 140 early DN samples) were randomly divided into the following three groups. The number of samples and the purpose of each group were as follows:

[0023] Test set: 5 simple DM samples + 5 early DN samples, used to compare the differences in miRNAs expression levels;

[0024] Training set: 80 simple DM samples + 80 early DN samples, used to evaluate the diagnostic efficacy of miRNAs for simple DM and early DN, and to construct ROC curves to determine sensitivity and specificity;

[0025] Validation set: 43 simple DM samples + 55 early DN samples, used to verify the diagnostic efficacy of miRNAs for simple DM and early DN, and calculate the diagnostic accuracy.

[0026] 3. Serum miRNAs detection

[0027] Total RNA was extracted from serum using a total RNA extraction kit. Then, it was reverse transcribed into cDNA using a reverse transcription kit. qPCR amplification was performed using the miRcute enhanced miRNA fluorescence quantitative detection kit (SYBR Green) with cDNA as a template. The total reaction system was 20 μl. The miRNAs primers were designed and synthesized by Shanghai Shenggong Biotechnology Co., Ltd., with β-actin as the internal reference gene. The reaction conditions were: 95°C for 10 min, 95°C for 15 s, 60°C for 60 s, and 45 cycles. 2 －ΔΔCt The relative expression levels of miRNAs were calculated by the method. miR-3680-3p forward: 5'-GGTTTTGCATGACCCTGGG-3'; miR-3680-3p reverse: 5'-CAGTGCGTGTCGTGGAGT-3'; miR-4463 forward: 5'-GGCCCACCCCAGT CTC-3'; miR-4463 reverse: 5'-GCAGGAGACTGGGGTG-3'; β-actin forward: 5'-CTTACGCAACTGA GTCC-3'; β-actin reverse: 5'-ATCTGACTAGTGACTATCAT-3'.

[0028] 4. Statistical analysis

[0029] In the training set, the ROC curve of miRNAs alone for distinguishing simple DM samples from early DN samples was established, and the area under the curve (AUC) and 95% confidence interval (95% CI), sensitivity, and specificity were calculated. For the joint diagnosis of multiple miRNAs, a Logistic regression equation was established to generate a set of new variables logit[p]. The ROC curve analysis of the new variables was performed to calculate the area under the curve (AUC) and 95% confidence interval, sensitivity, and specificity. In the validation set, the best cut-off value obtained by the ROC curve in the training set was used as the threshold to calculate the accuracy of miRNAs joint diagnosis to distinguish simple DM samples from early DN samples. SPSS22.0 software was used for statistical analysis. The relative expression of miRNAs in each sample was input into the software, and the AUC, 95% CI, sensitivity, specificity, regression equation, and cut-off value were automatically generated through the ROC curve analysis provided by the software.

[0030] 3. Experimental Results

[0031] 1. Difference in expression levels of target miRNAs in serum between simple DM and early DN (test set)

[0032] The relative expression levels of miR-3680-3p and miR-4463 in simple DM and early DN serum are shown in Table 1. The results in Table 1 show that compared with simple DM, the expression levels of miR-3680-3p and miR-4463 in early DN serum were significantly upregulated, and the difference was statistically significant (P < 0.05).

[0033] Table 1 Expression levels of miRNAs in serum

[0034] Simple DM Early DN Relative expression level of miR-3680-3p 0.75±0.16 1.28±0.25 Relative expression level of miR-4463 0.89±0.23 1.55±0.29

[0035] 2. Efficacy of target miRNAs in serum for distinguishing simple DM from early DN and construction of ROC curve (training set)

[0036] The relative expression levels of miR-3680-3p and miR-4463 in the serum of all samples in the training set were input into SPSS software, and the ROC curve and related data of miR-3680-3p and / or miR-4463 for diagnosing and distinguishing simple DM from early DN were automatically obtained with simple DM and early DN as the groups. Figure 1 The relevant data are shown in Table 2.

[0037] Table 2 ROC curve data of target miRNAs alone or in combination for distinguishing simple DM from early DN

[0038] AUC 95% CI Sensitivity Specificity Youden Index P miR-3680-3p 0.615 0.652～0.733 83.35 64.69 0.495 <0.001 miR-4463 0.673 0.687～0.814 86.37 80.02 0.646 <0.001 Combination of the two 0.906 0.798～0.992 95.67 92.65 0.889 <0.001

[0039] Those skilled in the art know that the area under the ROC curve AUC indicates the accuracy of the diagnostic test. The area under the ROC curve AUC has been widely recognized as an inherent accuracy indicator for evaluating the authenticity of diagnostic tests. When AUC is 0.5, it means that there is no diagnostic significance; when AUC is 0.5-0.7, it means that the diagnostic accuracy is low; when AUC is 0.7-0.9, it means that the diagnostic accuracy is medium; when AUC>0.9, it means that the diagnosis has a high accuracy. As can be seen from the results in Table 2, although the expression levels of miR-3680-3p and miR-4463 in the serum of early DN are upregulated compared with simple DM, the accuracy of miR-3680-3p and miR-4463 used alone to diagnose and distinguish simple DM from early DN is low, which is also in line with the common phenomenon in this field, that is, it does not mean that a higher diagnostic distinction efficiency can be obtained if there is a difference in content. However, the accuracy of the combined diagnosis of miR-3680-3p and miR-4463 in distinguishing simple DM from early DN was significantly improved to above 0.9, and the sensitivity and specificity were increased to more than 90%, indicating that the combined diagnosis of miR-3680-3p and miR-4463 for distinguishing simple DM from early DN had complementary effects in improving the accuracy, sensitivity and specificity, and the combined use had obvious diagnostic value. According to SPSS software, the binary logistic regression equation for the combined diagnosis of miR-3680-3p (independent variable X1) and miR-4463 (independent variable X2) for distinguishing simple DM from early DN was logit[p]=-3.22+1.73X1+1.91X2, and the cut-off value was 0.869.

[0040] 3. Final verification of diagnostic accuracy (validation set)

[0041] In the validation set, the relative expression levels of miR-3680-3p and miR-4463 in the serum samples of simple DM and early DN were substituted into the above binary logistic regression equation, and the regression value of each sample was calculated and compared with the cut-off value of 0.869. Samples above the cut-off value were predicted to be early DN, otherwise they were predicted to be simple DM. The accuracy of the combined diagnosis of miR-3680-3p and miR-4463 in distinguishing simple DM from early DN was 96.9% (95 / 98), which was the number of correctly predicted samples divided by the total number of samples.

[0042] The above experimental results show that the combined use of serum miR-3680-3p and miR-4463 as markers to diagnose and distinguish simple DM from early DN has an excellent diagnostic effect. Based on this technical solution, simple DM can be detected irregularly to detect DN in the early stage of simple DM. This technical solution is not only fast and low-cost, but also non-invasive and will not cause damage to the patient's body. Therefore, miR-3680-3p and miR-4463 have the prospect of being developed into a serum detection kit for diagnosing early diabetic nephropathy.

[0043] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. The use of miR-3680-3p and miR-4463 in combination for preparing a serum detection kit for diagnosing early diabetic nephropathy; wherein, The diagnosis of early diabetic nephropathy refers to the diagnosis of early diabetic nephropathy in diabetic patients.

2. The use according to claim 1, characterized in that: The diabetes is type 2 diabetes, and the early diabetic nephropathy is early type 2 diabetic nephropathy.

3. The use according to claim 1 or 2, characterized in that: The serum detection kit contains reagents for detecting the contents of miR-3680-3p and miR-4463 in serum.

Images