Biomarkers and applications of diabetic cataract

Abstract

This application relates to the technical field of diabetic cataract detection, specifically to biomarkers for diabetic cataracts and their applications. This application demonstrates that lncRNA-HIF1A-AS2 is significantly upregulated in the anterior lens capsule and aqueous humor of patients with diabetic cataracts. Based on the detection of lncRNA-HIF1A-AS2 expression in aqueous humor samples, diabetic cataracts can be effectively diagnosed. ROC curve verification shows a diagnostic accuracy of 88.0%, a sensitivity of 92.0%, and an AUC value of 0.878. Therefore, lncRNA-HIF1A-AS2 can serve as a biomarker for detecting diabetic cataracts and has significant application potential.

Description

Technical Field

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

[0002] Diabetic cataract (DC) is a common ocular complication of diabetes. Its pathogenesis is closely related to the abnormal activation of the polyol pathway induced by hyperglycemia. Under prolonged hyperglycemic conditions, the activity of aldose reductase in the lens increases, promoting the conversion of glucose to sorbitol, which accumulates in the lens. This leads to osmotic imbalance, oxidative stress, and non-enzymatic glycosylation of proteins, ultimately causing apoptosis and clouding of lens fibroblasts. Epidemiological studies show that the incidence of cataracts in diabetic patients is 10-15 years earlier than in non-diabetic individuals, and the disease progresses significantly faster.

[0003] Aqueous humor, as intraocular fluid, exists within a complex, dynamically circulating system, and its composition is closely linked to the local physiological and pathological states of the eye. Compared to blood, aqueous humor can reflect changes in the intraocular microenvironment more promptly and accurately. Therefore, the detection and analysis of secretory proteins and biomarkers in aqueous humor has become an important means of revealing the state of eye diseases.

[0004] In recent years, long noncoding RNA (lncRNA) has gradually become a research hotspot in the biomedical field. These RNA molecules, exceeding 200 nucleotides in length and lacking protein-coding function, are deeply involved in the development and progression of diabetic complications through various complex mechanisms such as epigenetic regulation and chromatin remodeling. However, research on lncRNA biomarkers for diabetic cataracts is still in its early stages, and no biomarkers that meet both the needs of early diagnosis and possess good specificity have yet been discovered. This situation, to some extent, restricts the development of precise detection technologies based on molecular biomarkers and hinders the formulation and implementation of targeted intervention strategies. Therefore, developing effective lncRNA biomarkers for diabetic cataracts has significant clinical and research value. Summary of the Invention

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

[0006] Therefore, this application discloses at least the following technical solutions:

[0007] The embodiments disclose the application of reagents for detecting biomarkers in samples in the preparation of products for detecting diabetic cataracts, wherein the biomarker is lncRNA-HIF1A-AS2, and the nucleotide sequence of lncRNA-HIF1A-AS2 is SEQ ID NO: 1.

[0008] Furthermore, the reagents used to detect biomarkers in the test samples may be selected from at least one of the following: preparing a kit for detecting diabetic cataracts; preparing a kit to assist in displaying biological information related to diabetic cataracts, especially early diabetic cataracts; preparing a kit to assist in the diagnosis of diabetic cataracts; and screening candidate drugs for treating diabetic cataracts or reducing the risk of early diabetic cataracts.

[0009] Furthermore, the reagent was used to detect the expression level of lncRNA-HIF1A-AS2 using nucleic acid amplification technology.

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

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

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

[0013] Furthermore, the sample was collected from the aqueous humor.

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

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

[0016] Figure 1 The box plot for quality assessment of RNA chip data from the anterior capsule of the lens provided in the example is 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 This is a graph showing the results of high-throughput analysis screening for lncRNAs related to diabetic cataract development, provided in the example; wherein:

[0018] Figure 2 A is a hierarchical clustering heatmap of lncRNA expression profiles for samples from the DC and NC groups;

[0019] Figure 2 B is a hierarchical clustering heatmap of lncRNA expression profiles for samples from the DC and ARC groups;

[0020] Figure 2 C and Figure 2 D is the Venn diagram of the intersection of differentially expressed lncRNAs;

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

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

[0023] Figure 3 The example provides the verification of high-throughput chip screening results by quantitative PCR, detecting the relative expression level of lncRNA-HIF1A-AS2 in anterior lens capsule samples from DC, ARC, and NC groups.

[0024] Figure 4 This is a statistical graph showing the expression levels of lncRNA-HIF1A-AS2 in aqueous humor samples from the DC and ARC groups provided in the examples.

[0025] Figure 5 The results are ROC curve analysis of lncRNA-HIF1A-AS2 content in 50 DC aqueous humor samples and 50 ARC aqueous humor samples provided in the examples. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0027] This application screened differentially expressed lncRNAs in anterior lens capsule samples clinically diagnosed with diabetic cataract, age-related cataract, and a control group. After verification by quantitative PCR and signal pathway analysis, it was found that the expression level of lncRNA-HI1A-AS2 was significantly upregulated in diabetic cataract, which can effectively diagnose diabetic cataract.

[0028] In one screening process, anterior capsule samples were collected from patients clinically diagnosed with diabetic cataract (DC), age-related cataract (ARC), and control (NC) patients from an eye bank. Differentially expressed lncRNAs were screened: the Arraystar Human lncRNA Microarray platform was used to analyze lncRNAs related to the occurrence of diabetic cataract. After verifying the experimental results of sequencing analysis with quantitative PCR, combined with GO and KEGG signaling pathway analysis, a target was finally identified for subsequent assays. The expression difference of target lncRNA-HI1A-AS2 in the aqueous humor of diabetic cataract patients and age-related cataract patients was verified.

[0029] Sequence Information Table

[0030]

[0031]

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

[0033] Screening process for lncRNA-HIF1A-AS2

[0034] The examples disclose a more detailed screening process for lncRNA-HIF1A-AS2. Specifically, it includes:

[0035] 1. Sample preparation:

[0036] Anterior 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 for differentially expressed lncRNAs:

[0038] After the samples passed the initial testing, lncRNA was detected using the Arraystar human lncRNA microarray V4.0; after removing rRNA from the total RNA, mRNA was obtained (mRNA-ONLY). TMThe Eukaryotic mRNA Isolation Kit (Epicentre) was used to amplify and transcribe each sample into fluorescent cRNA using random primers. The labeled cRNAs were then purified using the RNeasy Mini Kit (Qiagen), and their concentration and activity were detected using a NanoDrop ND-1000. The labeled RNA was then hybridized to a microarray, washed, fixed, and scanned using an Agilent DNA Microarray Scanner (partnumber G2505C). The microarray image was obtained and values ​​were read using Agilent Feature Extraction software (v11.0.1.1), yielding raw data. The raw data were then Quantile-normalized and processed using GeneSpring GX v12.1 software (Agilent Technologies). After normalization, high-quality probes were screened for differential expression analysis of lncRNAs. The distribution of each sample is shown below. Figure 1 As shown. Differential expression analysis of lncRNAs was performed on the standardized data (see...). Figure 2 (A and 2B).

[0039] Further, in order to identify upregulated lncRNAs in DCs, such as Figure 2 As shown in C, the red circle in "DC VS NC" represents the comparison with the NC group, where 279 upregulated lncRNAs were expressed only in the DC group. The blue circle in "DC VS ARC" represents the comparison with the ARC group, where 11 upregulated lncRNAs were expressed only in the DC group. The overlapping shaded area indicates that 70 of the same upregulated lncRNAs were expressed in both the red and blue circles, which is also the number of upregulated lncRNAs we are looking for.

[0040] Further, in order to identify downregulated lncRNAs in DCs, such as Figure 2 As shown in D, the green circle in "DC VS NC" represents the 153 downregulated lncRNAs expressed only in the DC group compared to the NC group, and the orange circle in "DC VS ARC" represents the 349 downregulated lncRNAs expressed only in the DC group compared to the ARC group. The overlapping shaded area indicates that 489 of the same downregulated lncRNAs are expressed in the green and orange circles, which is also the number of downregulated lncRNAs we are looking for.

[0041] Quantitative PCR validation of high-throughput microarray analysis revealed that, for example Figure 3 As shown, lncRNA-HIF1A-AS2 showed significant differences among the DC, ARC, and NC groups, with 20 typical cases selected from each group.

[0042] Detection of lncRNA-HIF1A-AS2 expression in aqueous humor

[0043] This section describes the detection of lncRNA-HIF1A-AS2 expression levels in the aqueous humor using RT-PCR. The specific steps include:

[0044] 1) Sample separation

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

[0046] 2) RNA extraction

[0047] Add Trizol to the aqueous humor sample and incubate at room temperature for 10 min to allow for complete lysis (Note: If no further steps are required, the sample can be stored at -70℃ for long-term storage). Add 200 μL of chloroform to each 1 mL of Trizol, vortex vigorously to mix, and incubate at room temperature for 3-5 min to allow for natural phase separation. Centrifuge at 12000 rpm for 15 min at 4℃. The sample will separate into three layers: an orange organic phase, an intermediate layer, and a 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 incubate at room temperature for 15 min. Centrifuge at 12000 rpm for 10 min at 4℃, discard the supernatant, and the RNA will precipitate at the bottom of the tube. Add 1 mL of 75% ethanol (prepared with RNase-free water) to the RNA precipitate, gently vortex the centrifuge tube, and resuspend the precipitate. Add 1 mL of 75% ethanol to each 1 mL of Trizol. Centrifuge at 8000 rpm for 5 min at 4℃, and discard the supernatant. After air-drying at room temperature, add 50 μL of RNase-free water to the precipitate to fully dissolve the RNA, and store at -70°C.

[0048] 3) RNA quality testing

[0049] The concentration of RNA was determined using a UV spectrophotometer at absorbances of 260 nm and 280 nm; the A concentration of the RNA solution was... 260 / A 280 The ratio of the two values ​​represents the RNA purity, ranging from 1.8 to 2.1. Simultaneously, RNA quality is assessed using agarose gel electrophoresis, and the RNA is observed and photographed under ultraviolet transmitted light.

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

[0051] Reverse transcription was performed using HiFiScript gDNARemoval RTMasterMix from Kangwei Century Biotechnology Co., Ltd. The following reagents were added according to the kit specifications (40 μL):

[0052] Table 1 Reaction system for removing genomic DNA

[0053]

[0054]

[0055] Table 2 Reverse Transcription Reaction System

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

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

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

[0059] The quantitative PCR reaction system (20 μL) was prepared using the MagicSYBR Mixture kit from Kangwei Century Biotechnology Co., Ltd., according to the instructions: 10 μL of 2×Magic SYBR Mixture, 0.4 μL each of the specific qRT-PCR upstream and downstream primers (specifically recognizing lncRNA-HIF1A-AS2 or actin), 1 μL of cDNA from the sample to be tested, and RNase-free ddH2O to a final volume of 20 μL. The PCR amplification steps were: 95℃ for 15 s, 60℃ for 1 min; 95℃ for 15 s, 50℃ for 30 s; 40 cycles.

[0060] 6) Data Analysis

[0061] Real-time quantitative PCR was performed on the same sample to detect the target RNA and internal reference RNA separately. The expression level of the internal reference RNA was used as a baseline, and the target RNA was normalized. The relative expression level of the target RNA was then analyzed using the DeltaCt method, a standard technique in this field. The Delta Ct value (ΔCt) for all samples was obtained by subtracting the Ct value of the internal reference gene actin from the Ct value of the target gene in all samples. The formula is expressed as ΔCt = Ct(target gene) - Ct(internal reference gene actin). Finally, the disease susceptibility of the tested patients was determined by comparing the expression differences of lncRNA-HIF1A-AS2 between the test samples and the target samples (e.g., ...). Figure 4 As shown, Figure 4 (15 representative cases were selected from each group).

[0062] 7) Result Interpretation

[0063] The expression level of the target lncRNA-HIF1A-AS2 was detected by real-time quantitative PCR using the SYBR dye method, and the ΔCt range of each group of control samples was obtained (see Table 3). The ΔCt values ​​of the test samples were compared to analyze the differences between the experimental and control groups. Further analysis was conducted to determine whether the values ​​of each sample were within the range of the control group. If the ΔCt of the test sample was within the range of the control group, or less than the ΔCt range, the test sample was considered negative for diabetic cataracts; if the ΔCt was greater than the ΔCt range, it was considered positive for diabetic cataracts.

[0064] Table 3. Range of ΔCt for each group of samples

[0065] |HIF1A-AS2Ct mean - internal parameter Ct mean| Aqueous humor in control group patients 6.06-7.21 Aqueous humor in experimental group patients 8.06-9.59

[0066] Based on the above results, ROC curves were plotted using GraphPad Prism 9.0. Aqueous humor samples were collected from 50 clinically diagnosed patients with diabetic cataracts and 50 patients with age-related cataracts, and the lncRNA-HIF1A-AS2 content in the aqueous humor was analyzed. ROC curves were plotted to evaluate the ability of the lncRNA-HIF1A-AS2-based method to distinguish between patients with diabetic cataracts and those with age-related cataracts.

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

[0068]

[0069] The results are as follows Figure 5 As shown in Table 4, when using lncRNA-HIF1A-AS2 to detect diabetic cataracts and age-related cataracts, the diagnostic value (the cutoff value being the RNA content with the highest Youden index) was 0.78, the AUC value of the ROC curve was 0.878, the accuracy was 88.0%, and the sensitivity was 92.0%. These results indicate that lncRNA-HIF1A-AS2 can serve as a biomarker for the diagnosis of diabetic cataracts, 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 description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. The application of reagents for detecting biomarkers in samples in the preparation of products for detecting diabetic cataracts, characterized in that, The biomarker is lncRNA-HIF1A-AS2, and the nucleotide sequence of lncRNA-HIF1A-AS2 is SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The reagent was used to detect the expression level of lncRNA-HIF1A-AS2 using nucleic acid amplification technology.

3. The application according to claim 1, characterized in that, The products include primers, probes, nucleic acid strips, or kits for detecting the expression level of lncRNA-HIF1A-AS2 in samples.

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

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

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

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