Biomarker for diabetic cataract and application

Abstract

The invention relates to the technical field of diabetic cataract detection, in particular to a biomarker for diabetic cataract and application. The research shows that the expression of the lncRNA-HIF1A-AS2 in the anterior lens capsule and aqueous humor of a diabetic cataract patient is obviously up-regulated, the diabetic cataract can be effectively diagnosed based on the expression detection of the lncRNA-HIF1A-AS2 in an aqueous humor sample, and the diagnosis accuracy is 88.0%, the sensitivity is 92.0% and the AUC value is 0.878 through the ROC curve verification. Therefore, the lncRNA-HIF1A-AS2 can be used as a marker for detecting the diabetic cataract, and has an important application prospect.

Description

Technical Field

[0001] This application relates to the technical field of diabetic cataract detection, specifically to biomarkers and applications of diabetic cataract. Background Art

[0002] Diabetic Cataract (DC) is a common ocular complication of diabetes. Its pathogenesis is closely associated with the abnormal activation of the polyol pathway induced by hyperglycemia. In a long-term hyperglycemic environment, the activity of aldose reductase in the lens is enhanced, promoting the conversion of glucose into sorbitol and its accumulation in the lens, which in turn leads to osmotic imbalance, oxidative stress, and non-enzymatic glycosylation of proteins, ultimately causing apoptosis and turbidity of lens fiber cells. Epidemiological studies have shown that the incidence of cataracts in diabetic patients occurs 10 - 15 years earlier than in the non-diabetic population, and the disease progression is significantly accelerated.

[0003] Aqueous humor, as the intraocular fluid, is in a complex system of dynamic circulation, and its composition is closely related to the local physiological and pathological states of the eye. Compared with blood, aqueous humor can more timely and accurately reflect the changes in the intraocular microenvironment. Therefore, the detection and analysis of secreted proteins and biomarkers in aqueous humor have become an important means to reveal the state of ocular diseases.

[0004] In recent years, long non-coding RNAs (lncRNAs) have gradually become a research hotspot in the field of biomedicine. These RNA molecules with a length exceeding 200 nucleotides and no protein-coding function are deeply involved in the occurrence and development of diabetic complications through various complex mechanisms such as epigenetic regulation and chromatin remodeling. However, the current research on lncRNA biomarkers for diabetic cataract is still in its infancy, and no biomarker that can meet the requirements of early diagnosis and has good specificity has been found. This situation has restricted the development of precision detection technologies based on molecular biomarkers to a certain extent and also hindered the formulation and implementation of targeted intervention strategies. Therefore, the development of effective lncRNA biomarkers for diabetic cataract has important clinical significance and research value. Summary of the Invention

[0005] In view of this, this application provides lncRNA-HIF1A-AS2 as a biomarker and application of diabetic cataract. This lncRNA is significantly upregulated in the anterior lens capsule and aqueous humor of diabetic cataract patients and can be used as a diagnostic biomarker.

[0006] Therefore, the embodiments of this application disclose at least the following technical solutions:

[0007] The embodiment discloses the application of a reagent for detecting a biomarker in a sample in the preparation of a product for detecting diabetic cataract, wherein the biomarker is lncRNA-HIF1A-AS2, and the nucleotide sequence of the lncRNA-HIF1A-AS2 is SEQ ID NO: 1.

[0008] Further, the application of the reagent for detecting the biomarker in the sample selects at least one of the following: preparing a kit for detecting diabetic cataract; preparing a kit for assisting in displaying biological information related to diabetic cataract, especially early diabetic cataract; preparing a kit for assisting in diagnosing diabetic cataract; screening candidate drugs for treating diabetic cataract or reducing the risk of early diabetic cataract.

[0009] Further, the reagent uses nucleic acid amplification technology to detect the expression level of lncRNA-HIF1A-AS2.

[0010] Further, the product includes primers, probes, nucleic acid membrane strips or kits for detecting the expression level of lncRNA-HIF1A-AS2 in the sample.

[0011] Further, the product includes the primer pairs shown in SEQ ID NO: 2 and 3.

[0012] Further, the product includes the primer pairs shown in SEQ ID NO: 2 and 3, gDNA Remover Mix and 5×HiFiScript RT Master Mix.

[0013] Further, the sample is collected from aqueous humor.

[0014] The beneficial effects of this application at least include the following:

[0015] This application first reveals that IncRNA-HIF1A-AS2 is specifically highly expressed in the lens anterior capsule and aqueous humor of patients with diabetic cataract (DC). The detection method based on the expression of IncRNA-HIF1A-AS2 in aqueous humor samples can effectively diagnose diabetic cataract. Verified by the ROC curve, its diagnostic AUC value reaches 0.878, the accuracy rate reaches 88.0%, and the sensitivity is 92.0%. Therefore, it provides a quantifiable index for the early screening of diabetic cataract. Description of the Drawings

[0016] Figure 1 It is a box plot for evaluating the quality of RNA chip data of lens anterior capsule samples provided by the embodiment, divided into three groups: NC (normal control), ARC (age-related cataract), and DC (diabetic cataract), and each group contains 3 independent samples (such as NC-1 to NC-3).

[0017] Figure 2 It is the result diagram of high-throughput analysis and screening of lncRNAs related to the occurrence of diabetic cataract provided by the embodiment; wherein:

[0018] Figure 2 A is the hierarchical clustering heat map of lncRNA expression profiles of samples in the DC group and the NC group;

[0019] Figure 2 B is the hierarchical clustering heat map of lncRNA expression profiles of samples in the DC group and the ARC group;

[0020] Figure 2 C and Figure 2 D are the Venn diagrams of the intersections of differentially expressed lncRNAs;

[0021] Figure 2 C is the intersection of up-regulated lncRNAs. The intersection area in the figure represents the lncRNAs that are up-regulated in both comparisons of DC vs NC (red circle) and DC vs ARC (blue circle);

[0022] Figure 2 D is the intersection of down-regulated lncRNAs; the intersection area in the figure represents the lncRNAs that are down-regulated in both comparisons of DC vs NC (green circle) and DC vs ARC (orange circle).

[0023] Figure 3 It is to verify the high-throughput chip screening results by quantitative PCR provided by the embodiment, and detect the relative expression levels of lncRNA-HIF1A-AS2 in the lens anterior capsule samples of the DC, ARC and NC groups.

[0024] Figure 4 It is the statistical chart of the expression levels of lncRNA-HIF1A-AS2 by quantitative PCR analysis in the aqueous humor samples of the DC group and the ARC group provided by the embodiment.

[0025] Figure 5 It is the ROC curve analysis result of the content of lncRNA-HIF1A-AS2 in 50 DC aqueous humor samples and 50 ARC aqueous humor samples provided by the embodiment. Detailed implementation manners

[0026] In order to make the objectives, technical solutions and advantages of the present application clearer and more understandable, the present application will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. The reagents not described in detail and separately in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail and particularly are all conventional experimental methods and can be learned from the prior art.

[0027] In this application, differential expression lncRNAs were screened from the anterior lens capsule samples of patients clinically diagnosed with diabetic cataract, age-related cataract, and the control group. After verification by quantitative PCR and combined with signal pathway analysis, it was found that the expression level of lncRNA-HI1A-AS2 was significantly upregulated in diabetic cataract, and it could effectively diagnose diabetic cataract.

[0028] In a screening process, anterior lens capsule samples were collected from patients clinically diagnosed with diabetic cataract (DC), age-related cataract (ARC), and the control group (NC) from the eye bank; for differential expression lncRNA screening: the Arraystar Human lncRNA Microarray platform was used to analyze the lncRNAs related to the occurrence of diabetic cataract; after verifying the experimental results of sequencing analysis by quantitative PCR and combined with GO and KEGG signal pathway analysis, a target was finally determined for subsequent measurement; the expression differences of the target lncRNA-HI1A-AS2 in the aqueous humor of patients with diabetic cataract and patients with age-related cataract were verified.

[0029] Sequence Information Table

[0030]

[0031]

[0032] The implementation mode of this application will be described below in combination with more specific embodiments, but it does not constitute a limitation to the implementation mode of this application.

[0033] Screening process of lncRNA-HIF1A-AS2

[0034] The embodiment discloses a more specific screening process of lncRNA-HIF1A-AS2. Specifically, it includes:

[0035] 1. Sample preparation:

[0036] Anterior lens capsule samples were collected from patients clinically diagnosed with diabetic cataract (DC), age-related cataract (ARC), and the control group (NC) from the eye bank. RNA was extracted using Trizol (Invitrogen) reagent and stored at -80 °C for later use.

[0037] 2. Screening of differentially expressed lncRNAs:

[0038] After the samples passed the test, the Arraystar Human lncRNA Chip V4.0 was used to detect lncRNAs; after removing rRNA from the total RNA, mRNA (mRNA-ONLY TMEukaryotic mRNA Isolation Kit, Epicentre), and then each sample was amplified and transcribed into fluorescent cRNA using the random primer method. Subsequently, the labeled cRNAs were purified using the RNeasy Mini Kit (Qiagen), and the concentration and activity were detected using NanoDrop ND-1000 to obtain the labeled RNA. Then, it was hybridized with the microarray, and the hybridized microarray was washed, fixed, and scanned (Agilent DNA Microarray Scanner (part number G2505C)). The microarray image was obtained using Agilent Feature Extraction software (v11.0.1.1), and the values were read to obtain the raw data. The raw data was subjected to Quantile normalization and subsequent data processing using GeneSpring GX v12.1 software (Agilent Technologies). After normalization of the raw data, high-quality probes were selected for differential expression analysis of lncRNAs. The distribution of each sample is as Figure 1 shown. Differential expression analysis of lncRNAs was performed on the normalized data (see Figure 2 A and 2B).

[0039] Furthermore, in order to search for up-regulated lncRNAs in DCs, as Figure 2 shown in C, the red circle of "DC VS NC" represents 279 up-regulated lncRNAs that are only expressed in the DC group compared with the NC group, and the blue circle of "DC VS ARC" represents 11 up-regulated lncRNAs that are only expressed in the DC group compared with the ARC group. The overlapping shaded area indicates that 70 identical up-regulated lncRNAs are expressed in the red circle and the blue circle, which is also the number of up-regulated lncRNAs we are looking for.

[0040] Furthermore, in order to search for down-regulated lncRNAs in DCs, as Figure 2 shown in D, the green circle of "DC VS NC" represents 153 down-regulated lncRNAs that are only expressed in the DC group compared with the NC group, and the orange circle of "DC VS ARC" represents 349 down-regulated lncRNAs that are only expressed in the DC group compared with the ARC group. The overlapping shaded area indicates that 489 identical down-regulated lncRNAs are expressed in the green circle and the orange circle, which is also the number of down-regulated lncRNAs we are looking for.

[0041] Verification by quantitative PCR showed that, as Figure 3 shown, there were significant differences in lncRNA-HIF1A-AS2 among the DC group, ARC group, and NC group. Among them, 20 typical cases were selected from each group.

[0042] Expression detection of lncRNA-HIF1A-AS2 in aqueous humor

[0043] In this part, the expression level of lncRNA-HIF1A-AS2 in aqueous humor was detected by RT-PCR. The specific steps are as follows:

[0044] 1) Sample separation

[0045] Aqueous humor samples from 15 patients in each of the experimental group (diabetic cataract) and the control group (age-related cataract) were collected and stored at -80 °C for the detection of lncRNA.

[0046] 2) RNA extraction

[0047] In the aqueous humor sample, add Trizol and let it stand at room temperature for 10 min to fully lyse the sample (Note: If the next step is not carried out, the sample can be stored at -70 °C for a long time). Add 200 μL of chloroform to every 1 mL of Trizol, mix vigorously and let it stand at room temperature for 3 - 5 min to separate naturally. Centrifuge at 12,000 rpm at 4 °C for 15 min. The sample will be divided into three layers: the orange organic phase, the middle layer, and the colorless aqueous phase. RNA is mainly in the aqueous phase. Transfer the aqueous phase to a new tube. Add an equal volume of ice-cold isopropanol to the supernatant and let it stand at room temperature for 15 min. Centrifuge at 12,000 rpm at 4 °C for 10 min and discard the supernatant. The RNA precipitate is at the bottom of the tube. Add 1 mL of 75% ethanol (prepared with RNase-free water) to the RNA precipitate, gently shake the centrifuge tube to suspend the precipitate. Add 1 mL of 75% ethanol to every 1 mL of Trizol. Centrifuge at 8,000 rpm at 4 °C for 5 min and discard the supernatant. Let it dry at room temperature, and then add 50 μL of RNase-free water to the precipitate to fully dissolve the RNA and store it at -70 °C.

[0048] 3) RNA quality detection

[0049] At the absorbance at 260 nm and 280 nm, the concentration of RNA was measured using an ultraviolet spectrophotometer; the ratio of A 260 / A 280 is the purity of RNA, and the ratio ranges from 1.8 to 2.1. At the same time, the quality of RNA was detected by agarose gel electrophoresis, and observations and photographs were taken under ultraviolet transillumination.

[0050] 4) Obtain cDNA samples by reverse transcription of RNA

[0051] For reverse transcription, HiFiScript gDNARemoval RTMasterMix from CWBiotech Co., Ltd. was used, and the following components were added according to the system (40 μL) provided by the kit:

[0052] Table 1 Reaction system for removing genomic DNA

[0053]

[0054]

[0055] Table 2 Reverse transcription reaction system

[0056] Reagent Reaction system (40 μL) Reaction solution after removing genomic DNA 20 μL 5×HiFiScript RT Master Mix 8 μL RNase-Free Water 12 μL

[0057] After preparing the above reaction system for removing genomic DNA, incubate the system in a PCR instrument at 42 °C for 2 min, and then perform the reverse transcription system; after thoroughly mixing the reverse transcription system, centrifuge it and place it in the PCR instrument, react at 37 °C for 15 min, and then react at 85 °C for 5 s. Store the obtained cDNA at -20 °C.

[0058] 5) SYBR Green dye method for real-time fluorescence quantitative PCR

[0059] For the quantitative PCR reaction system (20 μL), use the MagicSYBR Mixture kit from CWBIO Co., Ltd. and configure the system according to the instructions as follows: 2×Magic SYBR Mixure 10 μL, 0.4 μL each of the specific qRT-PCR upstream and downstream primers (specifically recognizing lncRNA-HIF1A-AS2 or actin), 1 μL of the cDNA of the sample to be tested, and make up to 20 μL with RNase free ddH2O. PCR amplification steps: 95 °C for 15 s, 60 °C for 1 min; 95 °C for 15 s, 50 °C for 30 s; 40 cycles.

[0060] 6) Data analysis

[0061] Perform real-time fluorescence quantitative PCR detection on the target RNA and internal reference RNA of the same sample respectively. Based on the expression level of the internal reference, normalize the target RNA. Subsequently, analyze the relative expression level of the target RNA using the commonly used DeltaCt method in the field. Subtract the Ct value of the internal reference gene actin from the Ct value of the target gene of all samples to obtain the Delta Ct value (ΔCt) of all samples. The formula is expressed as ΔCt = Ct(target gene) - Ct(internal reference gene actin). Finally, by comparing the expression differences of lncRNA-HIF1A-AS2 between the samples to be tested and the target samples, judge the disease susceptibility of the patients to be tested (as Figure 4 shown, Figure 4 15 representative cases selected from each group).

[0062] 7) Result interpretation

[0063] The expression level of the target lncRNA-HIF1A-AS2 was detected by SYBR Green dye-based real-time fluorescence quantitative PCR, and the ΔCt ranges of the control samples in each group were obtained, as shown in Table 3. The ΔCt of the test samples was compared to analyze the differences between the experimental group and the control group. Further analyze whether the values of each sample are within the range of the control group. If the ΔCt of the test sample is within the ΔCt range of the control group or less than this ΔCt range, the test sample is considered negative for diabetic cataract; if the ΔCt is greater than this ΔCt range, it is considered positive for diabetic cataract.

[0064] Table 3 ΔCt ranges of samples in each group

[0065] |Mean Ct of HIF1A-AS2 - Mean Ct of internal reference| Aqueous humor of control group patients 6.06-7.21 Aqueous humor of experimental group patients 8.06-9.59

[0066] According to the above results, a ROC curve was plotted using GraphPad Prism 9.0. Aqueous humor samples from 50 patients with diabetic cataract and 50 patients with age-related cataract diagnosed clinically were collected respectively for the analysis of the aqueous humor content of lncRNA-HIF1A-AS2. A ROC curve was plotted to evaluate the ability of the method based on lncRNA-HIF1A-AS2 to distinguish between patients with diabetic cataract and those with age-related cataract.

[0067] Table 4 Results of using lncRNA-HIF1A-AS2 as a biomarker for the diagnosis of diabetic cataract

[0068]

[0069] The results are as Figure 5 shown in Table 4. When using lncRNA-HIF1A-AS2 to detect aqueous humor samples to distinguish between patients with diabetic cataract and those with age-related cataract, the diagnostic value (the RNA content with the largest Youden index was used as the cut-off value) was 0.78, the AUC value of its ROC curve was 0.878, the accuracy rate was 88.0%, and the sensitivity was 92.0%. The results indicate that lncRNA-HIF1A-AS2 can be used as a biological biomarker for the diagnosis of diabetic cataract, and the expression level of lncRNA-HIF1A-AS2 in human aqueous humor has very high specificity, accuracy, and sensitivity in the diagnosis of diabetic retinopathy.

[0070] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. Use of a reagent for detecting a biomarker in a sample in the preparation of a product for detecting diabetic cataract, characterized in that, The biomarker is lncRNA-HIF1A-AS2, and the nucleotide sequence of the lncRNA-HIF1A-AS2 is SEQ ID NO:

1.

2. The application according to claim 1, wherein The reagent uses nucleic acid amplification technology to detect the expression level of lncRNA-HIF1A-AS2.

3. The application according to claim 1, wherein The product includes primers, probes, nucleic acid membrane strips or kits for detecting the expression level of lncRNA-HIF1A-AS2 in a sample.

4. The application according to claim 3, characterized in that The product includes the primer pair shown in SEQ ID NO: 2 and 3.

5. The application according to claim 4, characterized in that, The product includes the primer pair shown in SEQ ID NO: 2 and 3, gDNA Remover Mix, and 5×HiFiScript RT Master Mix.

6. The application according to any one of claims 1-5, characterized in that The sample is collected from aqueous humor.

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