A miRNA and its application in regulating C1QTNF6 gene expression and preparing regulatory products

Abstract

This invention discloses a miRNA belonging to the field of biotechnology. The miRNA is pal-miR-133a-3p, and its nucleotide sequence is shown in SEQ ID NO. 1. The target gene of pal-miR-133a-3p is C1QTNF6, and the nucleotide sequence of the target gene C1QTNF6 is shown in SEQ ID NO. 2. This invention also discloses the application of the miRNA in regulating C1QTNF6 gene expression and its application in preparing products that regulate the expression of this gene. The pal-miR-133a-3p of this invention can inhibit the expression of C1QTNF6, affect adipocyte differentiation, and thus regulate lipid synthesis, showing potential application value in the regulation of duck lipid metabolism and differentiation. Furthermore, the regulatory product prepared using this miRNA can achieve targeted regulation of the C1QTNF6 gene with precise and efficient regulatory effects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a miRNA and its application in regulating C1QTNF6 gene expression and preparing products that regulate C1QTNF6 gene expression. Background Technology

[0002] miRNAs (microRNAs) are a class of short non-coding RNAs that can regulate gene expression at the post-transcriptional level and participate in various biological processes such as cell proliferation, differentiation, and lipid metabolism. Studies have shown that miRNAs mainly regulate gene expression by binding to the 3′ junction of target genes. UTR binding enables negative or positive regulation of target gene expression. Currently, the luciferase reporter gene assay is a commonly used technique for verifying the interaction between miRNAs and target genes. This method involves transcoding the 3′ segment of the target gene... The UTR sequence was amplified and cloned into a dual-luciferase reporter vector, co-transfected with the target miRNA into tool cells, and then the changes in luciferase activity were detected to directly determine whether the miRNA acted on the target site.

[0003] C1q / TNF-related protein family member CTRP6 (C1QTNF6) plays a crucial role in lipid metabolism and inflammation regulation. Previous studies have shown that knocking down CTRP6 inhibits adipocyte differentiation, enhances lipolysis, and reduces lipid synthesis. Simultaneously, it significantly downregulates the expression of key adipogenic transcription factors PPARγ and C / EBPα, while upregulating the levels of KLF2 and PRDM1, thereby improving lipid metabolism. Furthermore, as an adipokine, CTRP6 can promote the production of pro-inflammatory factors such as TNF-α through autocrine mechanisms and inhibit adipocyte insulin signaling via paracrine mechanisms. These effects can exacerbate chronic low-grade inflammation and insulin resistance in adipose tissue, thereby affecting glucose and lipid metabolism and are closely related to the pathogenesis of metabolic diseases such as type 2 diabetes, thus possessing potential regulatory research value. Summary of the Invention

[0004] In response to one or more of the above-mentioned defects or improvement needs of the prior art, the present invention provides a miRNA and its application in regulating C1QTNF6 gene expression and preparing regulatory products, so as to affect adipocyte differentiation by intervening in the expression of this gene, thereby regulating lipid synthesis.

[0005] To achieve the above objectives, according to a first aspect of the present invention, a miRNA is provided, said miRNA being pal-miR-133a-3p, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] As a further improvement of the present invention, the target gene of pal-miR-133a-3p is C1QTNF6, and the nucleotide sequence of the target gene C1QTNF6 is shown in SEQ ID NO.2.

[0007] According to a second aspect of the present invention, there is an application of the miRNA in regulating the expression of the C1QTNF6 gene, wherein the miRNA inhibits the expression of the C1QTNF6 gene by binding to the 3′-UTR region of the target gene C1QTNF6.

[0008] As a further improvement of the present invention, the application includes regulating adipocyte differentiation, thereby regulating fat synthesis.

[0009] As a further improvement of the present invention, the fat cells are derived from ducks.

[0010] According to a third aspect of the present invention, there is an application of the miRNA described above in the preparation of a product that regulates the expression of the C1QTNF6 gene, wherein the miRNA inhibits the expression of the C1QTNF6 gene by binding to the 3′-UTR region of the target gene C1QTNF6.

[0011] As a further improvement of the present invention, the application includes regulating adipocyte differentiation, thereby regulating fat synthesis.

[0012] As a further improvement of the present invention, the fat cells are derived from ducks.

[0013] This invention identifies a novel miRNA based on high-throughput sequencing of duck subcutaneous preadipocytes. The differential expression of this miRNA during adipocyte-induced differentiation was verified using miRanda and RNA-hybrid prediction, as well as quantitative real-time PCR, and its negative correlation with the target gene C1QTNF6 was confirmed. Furthermore, a mimicry was synthesized based on the mature pal-miR-133a-3p sequence, and dual-luciferase reporter vectors for wild-type and mutant C1QTNF6 were constructed and transfected into HEK cells. 293T cell assays showed that it significantly inhibited the activity of wild-type 3′UTR luciferase, while having no effect on mutant cells, confirming the target relationship between the two. This invention lays the foundation for elucidating the molecular mechanism by which pal-miR-133a-3p regulates C1QTNF6's involvement in duck fat deposition, and has potential application value in the regulation of duck fat metabolism and differentiation, as well as in the preparation of regulatory products.

[0014] In summary, the technical solutions conceived by this invention have the following beneficial effects compared with the prior art: (1) This invention obtained a novel miRNA (pal-miR-133a-3p) from duck subcutaneous adipose tissue through high-throughput sequencing screening. Its target gene is C1QTNF6 (i.e., CTRP6). C1QTNF6 can promote the release of pro-inflammatory factors such as TNF-α through autocrine action and inhibit insulin signaling through paracrine action, thereby aggravating chronic low-grade inflammation in adipose tissue and reducing the insulin sensitivity of adipocytes. The pal-miR-133a-3p of this invention can inhibit the expression of C1QTNF6, affect adipocyte differentiation, and thus regulate adipocyte synthesis, and has potential application value in the regulation of duck fat metabolism and adipocyte differentiation.

[0015] (2) Based on the mature sequence of pal-miR-133a-3p, the present invention chemically synthesizes the corresponding mimics and constructs wild-type and mutant dual-luciferase reporter vectors of C1QTNF6 gene respectively. The target relationship between pal-miR-133a-3p and C1QTNF6 is verified by co-transfection experiment, laying an experimental foundation for further elucidating the molecular mechanism by which pal-miR-133a-3p regulates C1QTNF6 gene to achieve fat deposition in duck.

[0016] (3) The miRNA of the present invention can specifically bind to the 3′-UTR region of the C1QTNF6 gene to precisely inhibit the expression of the C1QTNF6 gene. The target of action is clear. The regulatory product prepared in this way can achieve targeted regulation of the C1QTNF6 gene, and the regulatory effect is precise and efficient. Attached Figure Description

[0017] Figure 1 This is the morphological observation result of duck subcutaneous preadipocytes obtained in this embodiment of the invention after 1 day of culture; Figure 2 This is a schematic diagram illustrating the analysis of the binding site of pal-miR-133a-3p to the C1QTNF6 gene in an embodiment of the present invention; Figure 3 This represents the relative expression level of pal-miR-133a-3p in the control group and experimental group in this embodiment of the invention. Figure 4 The relative expression levels of C1QTNF6 in the control group and experimental group in this embodiment of the invention; Figure 5 The results show the activity detection of pal-miR-133a-3p and C1QTNF6-3′UTR dual-luciferase reporter vectors in the embodiments of the present invention. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0019] Example 1: Isolation, culture, induction of differentiation, and screening and identification of pal-miR-133a-3p of duck subcutaneous preadipocytes 1. Laboratory animal and sample processing and isolation and culture of precursor adipocytes Six healthy 13-day-old miniature meat ducks were selected, and subcutaneous adipose tissue was collected from their abdomens under aseptic conditions. Preadipocytes were isolated using type I collagenase digestion; the tissue was then minced to approximately 1 mm. 3 Cells were sized and treated with 0.1% type I collagenase, then incubated at 37°C with shaking for 60 min. The cells were then filtered through a 70 μm cell sieve and centrifuged to collect the cell pellet. The cells were resuspended in DMEM / F12 complete medium containing 10% fetal bovine serum and antibiotics and cultured (morphological observations after 1 day of culture are shown in the figure). Figure 1 (As shown). All experimental procedures have been approved by the animal ethics committee.

[0020] 2. Cell differentiation induction The experiment was divided into two groups: an undifferentiated group (cultured for 0 days) serving as the control group; and an induced differentiation group (cultured for 4 days) which underwent differentiation induction with 300 μM oleic acid added to the culture medium, serving as the experimental group. Each group was configured with 3 biological replicates to evaluate the effect of oleic acid on adipocyte differentiation.

[0021] 3. High-throughput sequencing and miRNA screening Transcriptome small RNA sequencing technology was used to screen and identify differentially expressed genes and miRNAs between the two groups; combined with mRNA-miRNA joint analysis, candidate genes and miRNAs related to fat formation in ducks were identified. Through comparative analysis, a novel miRNA with significant differential expression in the experimental group was obtained, named pal-miR-133a-3p, and its nucleotide sequence is shown in SEQ ID NO.1.

[0022] Example 2: Validation of the correlation between pal-miR-133a-3p and C1QTNF6 gene expression. 1. Target gene prediction and functional background The target genes of pal-miR-133a-3p were predicted using the miRanda and RNAhybrid software programs. Candidate gene C1QTNF6 (nucleotide sequence shown in SEQ ID NO. 2) associated with adipogenesis was screened for further experimental validation. It is known that knockdown of C1QTNF6 (i.e., CTRP6) inhibits adipogenesis in 3T3-L1 cells and activates AMPK in C2C12 cells to promote fatty acid oxidation, thereby regulating lipid metabolism. Analysis of the binding sites of pal-miR-133a-3p and C1QTNF6 genes is shown below. Figure 2 As shown.

[0023] 2. Quantitative Real-Time PCR Detection The expression levels of pal-miR-133a-3p and C1QTNF6 in the duck control and experimental groups were detected using the following kits and reaction systems. The kits used included: miRNA / mRNA reverse transcription kit, specific primers for the target miRNA / mRNA, internal control primers, and a quantitative real-time PCR kit.

[0024] mRNA reverse transcription: HiScriptII RT SuperMix for qPCR (+gDNAwipe), catalog number R223-01, was purchased from Novizan. The reverse transcription system is shown in Table 1, and the reaction program was: 42℃ for 2 min; 50℃ for 15 min; 85℃ for 5 s.

[0025] Table 1 mRNA reverse transcription system

[0026] miRNA reverse transcription: The miRNA 1st Strand cDNA Synthesis Kit (bytailingA), catalog number MR201, was used. The reaction system is shown in Table 2, and the program was 37℃ for 60 min and 85℃ for 5 min.

[0027] Table 2 miRNA reverse transcription system

[0028] Quantitative real-time PCR reaction solution: TaqPro Universal SYBR qPCR MasterMix (catalog number Q712-02) was purchased from Novizan. The reaction system is shown in Table 3. The program was: 95℃ for 15 min; 94℃ for 15 s, 55℃ for 30 s, 70℃ for 30 s, for a total of 40 cycles.

[0029] Table 3 Real-time quantitative PCR reaction system

[0030] Internal control gene selection: U6 was used as the internal control for miRNA, and β-actin was used as the internal control for mRNA. Primer sequences are shown in Table 4.

[0031] Table 4. Primer sequence list for qRT quantification

[0032] 3. Statistical Analysis All data were statistically analyzed and plotted using GraphPadPrism 9.5 software. Data were processed using t-tests and one-way ANOVA, and results are expressed as mean ± standard deviation (Mean ± SD). Statistical significance was defined as P < 0.05. " indicates P < 0.05, " " indicates P < 0.01, " " indicates P < 0.001, " "Indicates P < 0.0001".

[0033] 4. Results qRT-PCR results showed that pal-miR-133a-3p was significantly different between the control and experimental groups (see...). Figure 3 (p<0.01); C1QTNF6 showed a highly significant difference between the two groups (see [reference needed]). Figure 4 (P<0.001).

[0034] The results showed that during duck adipocyte differentiation, pal miR 133a The increase in 3p expression and the decrease in C1QTNF6 expression occurred simultaneously, and the two were significantly negatively correlated. This suggests that palladium... miR 133a 3p is likely involved in the negative regulation of C1QTNF6, providing a logical basis and necessity for subsequent verification of the target relationship.

[0035] Example 3: Dual-luciferase reporter assay to verify the relationship between pal-miR-133a-3p and C1QTNF6 gene targeting. To verify the target relationship between pal-miR-133a-3p and C1QTNF6, this embodiment constructed wild-type and mutant dual-luciferase reporter vectors for the C1QTNF6 gene. Based on the mature sequence of pal-miR-133a-3p (SEQ ID NO.1), corresponding mimics (pal-miR-133a-3pmimics) and a negative control (NC) were chemically synthesized and co-transfected with the reporter vectors into HEK-293T cells for detection. The specific methods are as follows: 1. Construction of the pmirGLO-C1QTNF6-3′UTR recombinant plasmid A 3′-UTR fragment (360 bp, SEQ ID NO. 2) of the duck C1QTNF6 gene was amplified by PCR, digested with enzymes, and cloned into the NheI / SalI restriction site of the dual-luciferase reporter vector pmirGLO to construct a reporter vector containing the wild-type C1QTNF6-3′UTR sequence (SEQ ID NO. 3). Simultaneously, an expression vector containing the mutant C1QTNF6-3′UTR sequence (SEQ ID NO. 4) was constructed using site-directed mutagenesis, in which the target site of pal-miR-133a-3p was mutated.

[0036] 2. Dual-luciferase reporter gene analysis (1) HEK-293T cell culture: cultured to the required density under normal conditions.

[0037] (2) Synthesis of miRNA mimics: mimics and NC were synthesized based on the mature pal-miR-133a-3p sequence. Primer sequences are shown in Table 4.

[0038] Table 4 miRNA synthesis primer sequences

[0039] (3) Transfection: HEK-293T cells were co-transfected with the C1QTNF6 wild-type or mutant recombinant vector and pal-miR-133a-3pmimics / NC, and 4 groups were set up: ①C1QTNF6-WT+pal-miR-133a-3pmimics (Experimental Group 1) ②C1QTNF6-WT+NC (Control Group 1) ③C1QTNF6-Mut+pal-miR-133a-3pmimics (Experimental Group 2) ④C1QTNF6-Mut+NC (Control Group 2) Transfection procedure: Seed cells into 24-well plates using complete medium without antibiotics. When confluence reaches approximately 60%, wash cells with PBS and replace with serum-free DMEM medium; follow the Lipofectamine protocol. ™ Prepare the transfection complex according to the 3000 instructions: Solution A (1 μL Lipofectamine™ 3000 + 25 μL opti-MEM) and let stand at room temperature for 5 min; Solution B (25 μL opti-MEM + 0.5 μg plasmid + 1 μL 20 μM mimics / NC) and let stand at room temperature for 5 min; Mix equal volumes of solutions A and B, let stand at room temperature for 20 min, and then add to each group of wells. After culturing for 6 h, replace with complete medium without antibiotics and continue culturing for 24 h before collecting cells.

[0040] (4) Detection of relative activity of dual-luciferase reporter gene The test results show (see) Figure 5 Compared with the C1QTNF6-WT / NC group, the luciferase activity in the pal-miR-133a-3pmimics / C1QTNF6-WT group was significantly reduced (P<0.01); while there was no significant difference between the pal-miR-133a-3pmimics / C1QTNF6-Mut group and the C1QTNF6-Mut / NC group.

[0041] The results showed that pal-miR-133a-3p could specifically inhibit the luciferase activity of wild-type C1QTNF6-3′UTR, proving that there is a pal-miR-133a-3p binding site in the 3′-UTR region of the C1QTNF6 gene, and that C1QTNF6 is its target gene.

[0042] C1QTNF6 (CTRP6), an adipokine, regulates adipocyte differentiation, glucose metabolism, and insulin resistance through autocrine and paracrine pathways. This invention validates the targeted inhibitory effect of pal-miR-133a-3p on C1QTNF6, suggesting that it can influence adipocyte differentiation and thus regulate lipid synthesis by modulating the expression of this gene, indicating potential application value in the regulation of duck lipid metabolism and differentiation, as well as in the preparation of regulatory products.

[0043] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A miRNA, characterized in that, The miRNA is pal-miR-133a-3p, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The miRNA according to claim 1, characterized in that, The target gene of pal-miR-133a-3p is C1QTNF6, and the nucleotide sequence of the target gene C1QTNF6 is shown in SEQ ID NO.

2.

3. The application of the miRNA according to claim 2 in regulating C1QTNF6 gene expression, characterized in that, The miRNA inhibits the expression of the C1QTNF6 gene by binding to the 3′-UTR region of the target gene C1QTNF6.

4. The application of the miRNA according to claim 3 in regulating C1QTNF6 gene expression, characterized in that, The applications include regulating adipocyte differentiation, thereby regulating fat synthesis.

5. The application of the miRNA according to claim 4 in regulating C1QTNF6 gene expression, characterized in that, The fat cells were derived from ducks.

6. The application of the miRNA according to claim 2 in the preparation of a product regulating C1QTNF6 gene expression, characterized in that, The miRNA inhibits the expression of the C1QTNF6 gene by binding to the 3′-UTR region of the target gene C1QTNF6.

7. The application of the miRNA according to claim 6 in the preparation of products regulating C1QTNF6 gene expression, characterized in that, The applications include regulating adipocyte differentiation, thereby regulating fat synthesis.

8. The application of the miRNA according to claim 7 in the preparation of products regulating C1QTNF6 gene expression, characterized in that, The fat cells were derived from ducks.

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