A bioinformatics-based method for analyzing common targets of the miR-106a-363 cluster

Abstract

The present invention relates to the field of bioinformatics and discloses a method for analyzing the common targets of the miR-106a-363 cluster based on bioinformatics, comprising the following steps: correlation analysis between the miR-106-363 cluster and tuberculosis; target gene and pathway analysis of tuberculosis-related members in the miR-106-363 cluster; and verification of the interaction between members of the miR-106-363 cluster and key target genes in tuberculosis infection. The present invention adopts the above-mentioned method to predict the common targets of the miR-106a-363 cluster, locates the potential key targets of the miRNA cluster affecting tuberculosis immunity through literature analysis combined with the prediction results, and verifies the regulatory relationship between the bovine miR-106a-363 cluster and the target genes, revealing its potential role in tuberculosis immunity in dairy cows, and providing a new theoretical basis and research ideas for further research on tuberculosis and anti-tuberculosis gene editing breeding targets.

Description

Technical Field

[0001] The present invention relates to the technical field of bioinformatics, and in particular to a method for analyzing common targets of a miR-106a-363 cluster based on bioinformatics. Background Art

[0002] MicroRNAs (miRNAs) are a class of small RNA molecules, typically 20 to 24 nucleotides in length, that can bind to the 3' untranslated region of target mRNAs through pairing, leading to mRNA degradation or translational inhibition, thereby regulating target gene expression. A miRNA cluster consists of multiple similar or identical miRNA genes. Member genes often share similar sequences, though the sequences of their mature miRNAs may differ slightly. MiRNA genes within the same cluster are often regulated by similar mechanisms and function synergistically in specific biological processes. Therefore, miRNA clusters can provide a more efficient and accurate mechanism for regulating gene expression. The regulatory functions of miRNA genes within a cluster are synergistic, allowing them to regulate the same biological process through different target genes. By regulating the expression of multiple target genes, miRNA clusters play an important role in the regulation of physiological and pathological processes, including cell proliferation, apoptosis, cell cycle regulation, cell differentiation, immune responses, and tumor metastasis. Functional studies of miRNA clusters are crucial for understanding gene expression regulatory networks and their role in disease development. Currently, most studies on the relationship between miRNA and tuberculosis are limited to certain specific single miRNAs. The present invention focuses on how clustered miRNAs jointly regulate the physiological processes during tuberculosis infection.

[0003] This study uses bioinformatics methods to predict the common targets of the miR-106a-363 cluster, locates the core common targets of the miRNAs in the cluster in the regulatory mechanism, and further analyzes the main pathways affected by them in tuberculosis-related physiological processes. Literature analysis combined with the above prediction results locates the potential key targets of this miRNA cluster influencing tuberculosis immunity. Various molecular biological methods are used to verify the regulatory relationship between the bovine miR-106a-363 cluster and target genes, revealing its potential role in tuberculosis immunity in dairy cows. This provides a new theoretical basis and research ideas for further tuberculosis research and the exploration of anti-tuberculosis gene editing breeding targets. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for analyzing the common targets of the miR-106a-363 cluster based on bioinformatics, predict the common targets of the miR-106a-363 cluster using bioinformatics methods, and further analyze the main pathways affected by it in tuberculosis-related physiological processes. By combining literature analysis with prediction results, the potential key targets of the miRNA cluster affecting tuberculosis immunity are located, and the regulatory relationship between the bovine miR-106a-363 cluster and the target gene is verified by molecular biology methods, revealing its potential role in tuberculosis immunity in dairy cows, and providing a new theoretical basis and research ideas for further research on tuberculosis and anti-tuberculosis gene editing breeding targets.

[0005] To achieve the above objectives, the present invention provides a method for analyzing common targets of the miR-106a-363 cluster based on bioinformatics, comprising the following steps:

[0006] Step 1: Correlation analysis between the miR-106-363 cluster and tuberculosis. We searched the database for tuberculosis-related miRNA and mRNA sequencing data and analyzed the expression trends of the main members of the miR-106-363 cluster and their correlation with key genes in tuberculosis-related regulatory processes and tuberculosis phenotypes.

[0007] Step 2: Target gene and pathway analysis of tuberculosis-related members of the miR-106-363 cluster. Target genes of tuberculosis-related members of the miR-106-363 cluster were predicted and screened using relevant software. These genes were then used for GO and KEGG analysis to map the main pathways in which they participate, and to identify key genes regulated by miRNAs in the cluster.

[0008] Step 3: Verify the interaction between members of the miR-106-363 cluster and key target genes in tuberculosis infection. Use various molecular biological techniques to confirm the interaction relationship between tuberculosis-related members and target genes in the miR-106-363 cluster, and verify whether the target genes regulated by microRNAs in the cluster can affect the infection process of Mycobacterium tuberculosis through bacterial challenge experiments.

[0009] Furthermore, the specific operations of step 1 are:

[0010] ① Through literature search and gene function annotation, we located a miRNA cluster related to tuberculosis: miR-106-363;

[0011] ② GEO data analysis of cattle-related omics data showed that the genes of the miR-106-363 cluster members were downregulated during the process of tuberculosis infection, which is consistent with the research content in the literature. The miR-106-363 cluster was used as the object to further study its regulatory function on tuberculosis infection.

[0012] Furthermore, the specific operations of step 2 are:

[0013] ① Screening of key members in the cluster: The miR-106-363 cluster includes six members. Three members were selected as the main research objects through molecular feature analysis, target prediction and pathway enrichment analysis;

[0014] ② Common target prediction: To explore the common targets of the main members of the cluster, we first predicted the targets by integrating multiple databases. We then combined disease databases, pathway enrichment analysis, and literature search to obtain target genes as research objects.

[0015] ③Verify the relationship between the target gene and the main members in the cluster: The dual luciferase reporter enzyme experiment verified that the 3'UTR region of the target gene is the target of the main members in the cluster. At the same time, the qPRC combined with Western blot experiment verified the inhibitory effect of the main members in the cluster on the target gene at the transcription and protein levels.

[0016] Furthermore, ① the molecular characteristics analysis included conservation analysis and minimum free energy analysis; the three members screened out were 18b, 20b, and 106a.

[0017] Furthermore, the specific operations of step 3 are:

[0018] ① Verify that the main members of the cluster regulate autophagy in tuberculosis infection by inhibiting target genes: After transfecting miRNA / siRNA and attacking the bacteria, detect the expression of the autophagy marker LC3 to verify the effect of the main members of the cluster on autophagy. The CFU experiment also verifies the role of the main members of the cluster in the infection process.

[0019] Furthermore, in step 3, the target gene is Rab5.

[0020] Furthermore, the miR-106a-363 cluster affects the infection process of Mycobacterium tuberculosis by targeting Rab5.

[0021] The advantages and positive effects of the method of the present invention based on bioinformatics analysis of common targets of the miR-106a-363 cluster are:

[0022] This study uses bioinformatics methods to predict the common targets of the miR-106a-363 cluster, locates the core common targets of the miRNAs in the cluster in the regulatory mechanism, and further analyzes the main pathways affected by them in tuberculosis-related physiological processes. Literature analysis combined with the above prediction results locates the potential key targets of this miRNA cluster influencing tuberculosis immunity. Various molecular biological methods are used to verify the regulatory relationship between the bovine miR-106a-363 cluster and target genes, revealing its potential role in tuberculosis immunity in dairy cows. This provides a new theoretical basis and research ideas for further tuberculosis research and the exploration of anti-tuberculosis gene editing breeding targets.

[0023] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The process in the embodiment of the method for analyzing the common targets of the miR-106a-363 cluster based on bioinformatics of the present invention Figure 1 ;

[0025] Figure 2 The process in the embodiment of the method for analyzing the common targets of the miR-106a-363 cluster based on bioinformatics of the present invention Figure 2 . DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0027] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0028] Example

[0029] The method for analyzing the common targets of the miR-106a-363 cluster based on bioinformatics includes the following steps: Figure 1 shown):

[0030] Step 1: Correlation analysis between the miR-106-363 cluster and tuberculosis. We searched the database for tuberculosis-related miRNA and mRNA sequencing data and analyzed the expression trends of the main members of the miR-106-363 cluster and their correlation with key genes in tuberculosis-related regulatory processes and tuberculosis phenotypes.

[0031] Step 2: Target gene and pathway analysis of tuberculosis-related members of the miR-106-363 cluster. Target genes of tuberculosis-related members of the miR-106-363 cluster were predicted and screened using relevant software. These genes were then used for GO and KEGG analysis to map the main pathways in which they participate, and to identify key genes regulated by miRNAs in the cluster.

[0032] Step 3: Verify the interaction between members of the miR-106-363 cluster and key target genes in tuberculosis infection. Use various molecular biological techniques to confirm the interaction relationship between tuberculosis-related members and target genes in the miR-106-363 cluster, and verify whether the target genes regulated by microRNAs in the cluster can affect the infection process of Mycobacterium tuberculosis through bacterial challenge experiments.

[0033] More specifically, the detailed steps of the method for analyzing the common targets of the miR-106a-363 cluster based on bioinformatics are as follows Figure 2 As shown,

[0034] 1. Preliminary work: Because miRNAs exist in clusters and miRNAs in the same cluster often work synergistically, a literature search combined with gene function annotation led to the localization of the tuberculosis-associated miRNA cluster miR-106-363.

[0035] 2. GEO data analysis: First, existing literature has shown that members of the miR-106-363 cluster play a regulatory role in the process of tuberculosis infection. Then, we downloaded and analyzed relevant bovine omics data. The analysis results showed that the genes of this cluster member were downregulated during the process of tuberculosis infection, which is consistent with the research content of the literature. This provides a theoretical basis for further studying this cluster as an object to regulate its function in tuberculosis infection.

[0036] 3. Identification of Key Cluster Members: Since the miR-106-363 cluster contains six members, each with distinct regulatory functions on the TB disease process, we first sought to exclude members with weak TB associations and identify genes with key regulatory functions as targets for investigation. Through molecular signature analysis (conservation, minimum free energy), target prediction, and pathway enrichment analysis, we identified three members (18b, 20b, and 106a) as key members of the cluster regulating TB infection and focused our investigation on them.

[0037] 4. Common target prediction: In order to explore the common targets of the main members of the cluster, we first predicted the targets by integrating multiple databases. Combining disease databases, pathway enrichment analysis and literature retrieval, we obtained a group of target genes as research objects.

[0038] 5. Verification of the relationship between target genes and key cluster members: Dual-luciferase reporter assays confirmed that the 3'UTR region of the target gene was the target of the key cluster member. qPCR coupled with Western blot assays also verified the inhibitory effect of the key cluster members on the target gene at both the transcriptional and protein levels. These experiments identified Rab5 as the most promising candidate for further investigation. Literature review revealed its association with autophagy in tuberculosis infection, leading to the hypothesis that key cluster members regulate the progression of tuberculosis infection by influencing autophagy.

[0039] 6. Verify that the main members of the cluster regulate autophagy in tuberculosis infection by inhibiting target genes: verify the effect of the main members of the cluster on autophagy by transfecting miRNA / siRNA and detecting the expression of the autophagy marker LC3 after infection, and verify the role of the main members of the cluster in the infection process through CFU experiments.

[0040] This study uses bioinformatics methods to predict the common targets of the miR-106a-363 cluster, locates the core common targets of the miRNAs in the cluster in the regulatory mechanism, and further analyzes the main pathways affected by them in tuberculosis-related physiological processes. Literature analysis combined with the above prediction results locates the potential key targets of this miRNA cluster influencing tuberculosis immunity. Various molecular biological methods are used to verify the regulatory relationship between the bovine miR-106a-363 cluster and target genes, revealing its potential role in tuberculosis immunity in dairy cows. This provides a new theoretical basis and research ideas for further tuberculosis research and the exploration of anti-tuberculosis gene editing breeding targets.

[0041] Therefore, the present invention adopts the above-mentioned method of analyzing the common targets of the miR-106a-363 cluster based on bioinformatics, uses bioinformatics methods to predict the common targets of the miR-106a-363 cluster, and further analyzes the main pathways affected by it in tuberculosis-related physiological processes. Through literature analysis combined with the prediction results, the potential key targets of the miRNA cluster affecting tuberculosis immunity are located, and the regulatory relationship between the bovine miR-106a-363 cluster and the target gene is verified by molecular biology methods, revealing its potential role in tuberculosis immunity in dairy cows, providing a new theoretical basis and research ideas for further research on tuberculosis and anti-tuberculosis gene editing breeding targets.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for analyzing common targets of the miR-106a-363 cluster based on bioinformatics, characterized in that: The following steps are involved: Step 1: Correlation analysis between the miR-106-363 cluster and tuberculosis. We searched the database for tuberculosis-related miRNA and mRNA sequencing data and analyzed the expression trends of the main members of the miR-106-363 cluster, as well as their correlation with key genes in tuberculosis-related regulatory processes and tuberculosis phenotypes. Step 2: Target gene and pathway analysis of tuberculosis-related members of the miR-106-363 cluster. Target genes of tuberculosis-related members of the miR-106-363 cluster were predicted and screened using relevant software. GO and KEGG analyses were used to map the main pathways in which they participated, and to screen the key genes regulated by miRNAs in the cluster. Step 3: Verify the interactions between members of the miR-106-363 cluster and key target genes in tuberculosis infection. Molecular biology techniques will be used to confirm the interactions between tuberculosis-related members of the miR-106-363 cluster and target genes. Furthermore, bacterial challenge experiments will be conducted to verify whether target genes regulated by cluster microRNAs can affect the progression of Mycobacterium tuberculosis infection. The specific operations of step 3 are: ① Verify that the main members of the cluster regulate autophagy in tuberculosis infection by inhibiting target genes: After transfection with miRNA / siRNA and infection, the expression of the autophagy marker LC3 was detected to verify the effect of the main members of the cluster on autophagy. The role of the main members of the cluster in the infection process was also verified by CFU assay. In step 3, the target gene is Rab5; The miR-106a-363 cluster affects the infection process of Mycobacterium tuberculosis by targeting Rab5.

2. The method for analyzing common targets of the miR-106a-363 cluster based on bioinformatics according to claim 1, characterized in that: The specific operations of step 1 are: ① Through literature search and gene function annotation, we located a miRNA cluster related to tuberculosis: miR-106-363; ② GEO data analysis of cattle-related omics data showed that the genes of the miR-106-363 cluster members were downregulated during the process of tuberculosis infection, which is consistent with the research content in the literature. The miR-106-363 cluster was used as the object to further study its regulatory function on tuberculosis infection.

3. The method for analyzing common targets of the miR-106a-363 cluster based on bioinformatics according to claim 1, characterized in that: The specific operations of step 2 are: ① Screening of key members in the cluster: The miR-106-363 cluster includes six members. Three members were selected as the main research objects through molecular feature analysis, target prediction and pathway enrichment analysis; ② Common target prediction: To explore the common targets of the main members of the cluster, we first predicted the targets by integrating multiple databases. We then combined disease databases, pathway enrichment analysis, and literature search to obtain target genes as research objects. ③Verify the relationship between the target gene and the main members in the cluster: The dual luciferase reporter enzyme experiment verified that the 3'UTR region of the target gene is the target of the main members in the cluster. At the same time, the qPRC combined with Western blot experiment verified the inhibitory effect of the main members in the cluster on the target gene at the transcription and protein levels.

4. The method for analyzing common targets of the miR-106a-363 cluster based on bioinformatics according to claim 3, characterized in that: ① The molecular characteristics analysis included conservation analysis and minimum free energy analysis; the three members screened out were 18b, 20b, and 106a.

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