TGF [beta]-related miRNA sequence combination and application thereof

Abstract

The invention provides a TGF (Transforming Growth Factor) beta-related miRNA (Micro Ribonucleic Acid) sequence combination, which comprises a simulant sequence and an inhibitor sequence which are designed according to a miR-192 mature body miR-192-5p, and the sense sequence of the simulant sequence is SEQ ID NO.1; the antisense sequence of the simulant sequence is SEQ ID NO.2 or an antisense strengthening sequence of the simulant sequence designed based on the SEQ ID NO.2; the sequence of the inhibitor is SEQ ID NO.3 or a reinforcing sequence of the sequence of the inhibitor designed based on the SEQ ID NO.3. The TGF [beta]-related miRNA sequence combination is used for preparing medicines for treating diabetic kidney diseases. The TGF beta related miRNA sequence has high expression consistency and reaction consistency, is stable in drug species, has high specificity and transfection efficiency, has good treatment potential and application prospects for early diabetic kidney diseases, has good expressions in in-vivo and in-vitro experiments, and has good application prospects. The possibility is provided for clinical application of the TGF beta related miRNA sequence combination for treating the diabetic kidney diseases, and the TGF beta related miRNA sequence combination can be used for preparing related medicines for treating the diabetic kidney diseases.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a TGFβ-related miRNA sequence combination and application thereof. Background Art

[0002] Diabetic kidney disease (DKD) is a chronic kidney disease caused by diabetes. It is a common complication of type 1 and type 2 diabetes and one of the main causes of end-stage renal disease (ESRD). The clinical characteristics of DKD include persistent proteinuria and / or a gradual decrease in glomerular filtration rate (GFR). The pathogenesis of DKD is complex, involving major pathophysiological mechanisms such as oxidative stress, inflammation, and autophagy. Among them, activation of the transforming growth factor β (TGFβ) / Smad signaling pathway can promote renal fibrosis, which is an important sign of DKD progression. The TGFβ / Smad signaling pathway plays a key role in the onset and progression of DKD. Receptor-regulated Smads (R-Smads) include Smad1, Smad2, Smad3, Smad5 and Smad8, which directly interact with TGFβ family receptors and transmit signals; co-Smad is Smad4, which serves as a common mediator in the signal transduction process; inhibitory Smads (I-Smads) include Smad6 and Smad7, which negatively regulate the TGF-β signaling pathway. The activation of TGFβ and its downstream signaling protein Smads plays a vital role in diabetic kidney damage.

[0003] RNA interference (RNAi) technology is a post-transcriptional gene silencing mechanism that silences the expression of specific genes by degrading specific mRNA transcripts. Small interfering RNA (siRNA) is a type of RNAi technology that recognizes and degrades target mRNA by binding to RNA-induced silencing complexes. It has the potential to target any mRNA and improve patient compliance through infrequent dosing. However, siRNA therapy faces challenges including targeted administration, poor tissue permeability, nuclease inactivation, rapid renal elimination, immune activation, and off-target effects. Although bioavailability and stability have been improved by chemical modification of siRNA and the use of delivery systems, these problems have not been fully resolved.

[0004] MicroRNA (miRNA) is a class of non-coding single-stranded RNA molecules with a length of about 22 nucleotides encoded by endogenous genes. They participate in the post-transcriptional regulation of gene expression in animals and plants. Their main function is to regulate gene expression by base pairing with target gene mRNA to degrade mRNA or hinder its translation. They have high sequence conservation, expression timing and tissue specificity, and therefore have begun to become another new choice for studying RNAi technology drugs in recent years. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a TGFβ-related miRNA sequence combination and its application in view of the deficiencies of the above-mentioned prior art. The TGFβ-related miRNA sequence combination has high expression consistency and reaction consistency, stable drug species, high specificity and transfection efficiency, can inhibit the excessive proliferation of glomerular mesangial cells under high-glucose culture through the TGFβ / Smad pathway, has good therapeutic potential and application prospects for early diabetic kidney disease, and has good performance in both in vivo and in vitro experiments. It is possible for the clinical application of the TGFβ-related miRNA sequence combination to treat diabetic kidney disease, and can be used to prepare related drugs for diabetic kidney disease.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a TGFβ-related miRNA sequence combination, wherein the TGFβ-related miRNA sequence combination includes a mimic sequence and an inhibitor sequence designed according to the mature form of miR-192, miR-192-5p;

[0007] The nucleotide sequence of the sense sequence of the mimetic sequence is shown in SEQ ID NO.1;

[0008] The antisense sequence of the mimetic sequence is a nucleotide sequence such as the sequence shown in SEQ ID NO.2 or an antisense enhancement sequence of the mimetic sequence designed based on SEQ ID NO.2;

[0009] The inhibitor sequence is a nucleotide sequence such as the sequence shown in SEQ ID NO.3 or a strengthening sequence of the inhibitor sequence designed based on SEQ ID NO.3;

[0010] The antisense enhancement sequence of the mimetic sequence designed based on SEQ ID NO. 2 is: 5'-ACCUGUCAAUUCAUAGGUCAGUUAAUUChol-3';

[0011] The enhancing sequence of the inhibitor sequence designed based on SEQ ID NO. 3 is: 5'-CUGGCUGUCAAUUCAUAGGUCAGAUAAChol-3'.

[0012] The present invention also provides an application of the above-mentioned TGFβ-related miRNA sequence combination, characterized in that the TGFβ-related miRNA sequence combination is used to prepare a drug for diabetic kidney disease.

[0013] The mode of action of using the TGFβ-related miRNA sequence combination as a drug for treating diabetic kidney disease is to inhibit the excessive proliferation of mesangial cells in diabetic kidney disease by inhibiting the TGFβ / Smad pathway. It has good drug potential and application prospects for the early stages of diabetic kidney disease, and provides a possibility for the clinical application of TGFβ-related miRNA sequence combinations in the treatment of diabetic kidney disease.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] 1. The TGFβ-related miRNA sequence combination of the present invention is screened from four miRNAs. The four miRNAs may be closely related to the production of urinary protein in diabetic kidney disease. The target genes of the miRNA sequences are all related to the TGFβ pathway, which theoretically determines the feasibility of the invention. In vitro experimental model screening was performed using high glucose, TGFβ1 recombinant protein and inhibitors, and four miRNA inhibitors to select miRNA sequences with high expression consistency and reaction consistency, which can ensure the specificity of the drug and the precise regulation of cellular mechanisms. The TGFβ-related miRNA sequence combination described in the present invention is designed based on the common miR-192-5p sequence of humans and rats, and can form a similar hairpin structure to ensure the species stability of the drug.

[0016] 2. The TGFβ-related miRNA sequence described in the present invention has been verified to have high specificity and transfection efficiency, and can inhibit the excessive proliferation of glomerular mesangial cells under high-glucose culture through the TGFβ / Smad pathway. It has good drug potential and application prospects for early diabetic kidney disease, and has good performance in both in vivo and in vitro experiments, providing the possibility for the clinical application of TGFβ-related miRNA sequence combinations in the treatment of diabetic kidney disease, and can be used to prepare drugs for the treatment of diabetic kidney disease.

[0017] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the miRNA sequencing result of clinical patients in Example 1 of the present invention.

[0019] Figure 2 This is the Cytoscape software miRNA-target gene mRNA analysis in Example 1 of the present invention.

[0020] Figure 3 It is a Venn diagram of target gene prediction in the TargetScan database in Example 1 of the present invention.

[0021] Figure 4This is the change of TGFβ1 regulating miRNA in the in vitro experiment in Example 1 of the present invention. *: statistically significant compared with the NG group, #: statistically significant compared with the HG group.

[0022] Figure 5 These are the changes of the four miRNA inhibitors in the in vitro experiment in Example 1 of the present invention. *: Statistically significant compared with the NG+NC or HG+NC group.

[0023] Figure 6 It is the hairpin structure of human and rat miR-192 in Example 1 of the present invention.

[0024] Figure 7 This is the in vivo and in vitro experimental expression in Example 2 of the present invention.

[0025] Figure 8 This is the application of the drug for in vitro and in vivo experiments on diabetic kidney disease in Example 2 of the present invention. DETAILED DESCRIPTION

[0026] Example 1

[0027] This example describes the screening and design of TGFβ-related miRNAs.

[0028] 1. Clinical sequencing to screen miRNA genes related to diabetic kidney disease

[0029] In order to explore the expression level of miRNA in diabetic kidney disease, we collected clinical type 2 diabetes patients and age-matched physical examination center subjects of the same period to detect serum related miRNA levels. Type 2 diabetes patients were grouped according to different urinary albumin excretion rates. The primer sequences of related miRNAs are shown in Table 1.

[0030] Table 1 miRNA primer sequence information

[0031] sequence name Accession number sequence Length (nt) Hsa-miR-192-5p MIMAT0000222 5'-CUGACCUAUGAAUUGACAGCC-3' 21 Hsa-miR-216a-5p MIMAT0000273 5'-UAAUCUCAGCUGGCAACUGUGA-3' 22 Hsa-miR-217-5p MIMAT0000274 5'-UACUGCAUCAGGAACUGAUUGGA-3' 23 Hsa-miR-224-5p MIMAT0000281 5'-UCAAGUCACUAGUGGUUCCGUUUAG-3' 25

[0032] Figure 1 The miRNA expression levels of type 2 diabetic patients with different urinary albumin excretion rates and their normal control groups are shown. Compared with the normal control group, miR-217 and miR-224 in type 2 diabetic patients increased significantly, while miR-192 and miR-216a decreased significantly (P<0.05). In type 2 diabetic patients, miR-217 and miR-224 increased with the increase of urinary albumin excretion rate, while miR-192 and miR-216a decreased with the increase of urinary albumin excretion rate, and the differences between the groups were statistically significant (P<0.05). The above results indicate that the four miRNAs may be closely related to the production of urinary protein in diabetic kidney disease.

[0033] 2. Bioinformatics screening of target genes and related pathways of miRNA sequences

[0034] Target gene prediction was performed using the TargetScan database, and miRNA-target gene mRNA visualization was performed using Cytoscape. Figure 2 It can be seen that the TGFβ pathway-related factors as target genes have a lot of intersections with the four miRNAs. The Venn diagram is used to intersect the target genes related to the four miRNAs, and the results are as follows: Figure 3 As shown, it can be seen that there are 2 identical predicted target genes among the 4 miRNAs, and there are 0, 17, 19 and 32 identical target genes among the 3 miRNAs, and most of these genes are related to the TGFβ pathway.

[0035] 3. In vitro screening of miRNA sequences

[0036] In order to verify the effectiveness of miRNA sequences in in vivo experiments and to screen out miRNA sequences that meet the requirements, the present invention designed relevant miRNA inhibitors to explore specific interaction mechanisms. The primers and inhibitor sequences required for the designed relevant miRNA screening are shown in Table 2.

[0037] Table 2 Information on primers and inhibitors for relevant miRNA screening

[0038]

[0039]

[0040] The early pathophysiological mechanism of diabetic kidney disease is related to the proliferation of glomerular mesangial cells. Based on our previous research, a rat glomerular mesangial cell model cultured in high glucose was established, and miRNA detection was used to explore the changes of TGFβ1-regulated miRNAs in diabetic kidney damage. Figure 4 The treatment results showed that high glucose (HG group) increased the expression of miR-192 (mature form is miR-192-5p), miR-216a (mature form is miR-216a-5p), miR-217 (mature form is miR-217-5p) and TGFβ1, but decreased miR-224 (mature form is miR-224-5p), which was consistent with the results of treatment with TGFβ1 recombinant protein (TGFβ1+ group, 2ng / ml, model #8915, Cell Signaling Technology), but opposite to the results of treatment with TGFβ1 inhibitor (TGFβ1- group, SB431542, 10μmol / L, model #14775, Cell Signaling Technology) (P<0.05).

[0041] For further in vitro experimental screening, rat glomerular mesangial cells with or without high glucose (HG group and NG group) were treated with miRNA inhibitors (hereinafter described as specific miRNA-groups) to explore the specific interaction mechanism. Figure 5 The treatment results showed that the relative expressions of miR-192 (mature form is miR-192-5p), miR-216a (mature form is miR-216a-5p) and miR-217 (mature form is miR-217-5p) were significantly decreased in the miRNA inhibitors of miR-192 / 216 / 217a, and significantly increased in the miR-224 inhibitor; while miR-224 (mature form is miR-224-5p) and TGFβ1 proteins were significantly increased in the miRNA inhibitors of miR-192 / 216 / 217a, and significantly decreased in the miR-224 inhibitor (P<0.05). Using high glucose, TGFβ1 recombinant protein and inhibitors, and four miRNA inhibitors for in vitro experimental model screening, the mature form of miR-192, miR-192-5p, was selected, which has a miRNA sequence combination with high expression consistency and response consistency, and can ensure the specificity of the drug and the precise regulation of cellular mechanisms.

[0042] 4. TGFβ-related miRNA sequence design

[0043] The hairpin structures of human and rat miR-192 are shown in Figure 6 As shown, the mature miRNA sequences were downloaded from the miRBase database, hsa-miR-192-5p, Accession number: MIMAT0000222, sequence: 5'-CUGACCUAUGAAUUGACAGCC-3'; rno-miR-192-5p, Accession number: MIMAT0000867, sequence: 5'-CUGACCUAUGAAUUGACAGCC-3'; the sequence similarity between the two is 100%, so rats can be used as a mammalian species for studying humans.

[0044] Through the QIAGEN GeneGlobe database and miRNA RT primer and primer3plus software, the mimic sequence designed based on the mature miR-192 miR-192-5p is SEQ ID NO.1-2, and the inhibitor sequence is SEQ ID NO.3. At the same time, the antisense enhancer sequence of the mimic sequence designed based on SEQ ID NO.2 and the enhancer sequence of the inhibitor sequence designed based on SEQ ID NO.3 were designed.

[0045] Sense sequence of the analog sequence:

[0046] 5'-CUGACCUAUGAAUUGACAGCC-3' (SEQ ID NO. 1);

[0047] Antisense sequence of the mimetic sequence:

[0048] 5'-CUGUCAAUUCAUAGGUCAGUU-3' (SEQ ID NO. 2);

[0049] Inhibitor sequence:

[0050] 5'-GGCUGUCAAUUCAUAGGUCAG-3' (SEQ ID NO. 3);

[0051] SEQ ID NO.1-3 is an RNA sequence. When input into the WIPOSequence software, the nucleotide sequence must only contain the symbols listed in Part 1 of Annex I of WIPOST.26. When the symbol "t" is used without further explanation, it will be interpreted as thymine in DNA and uracil in RNA. The t in the SEQ ID NO:1-3 sequence is actually uracil "U".

[0052] The antisense enhanced sequence of the mimetic sequence designed based on SEQ ID NO.2 has 2 thio-backbone modifications (AC) at the 5' end, 4 thio-backbone modifications (AAUU) at the 3' end, cholesterol modification (Chol) and full-chain methoxy modification:

[0053] 5'-ACCUGUCAAUUCAUAGGUCAGUUAAUUChol-3';

[0054] The enhanced sequence of the inhibitor sequence designed based on SEQ ID NO.3 has 2 thiolate backbone modifications (CU) at the 5' end, 4 thiolate backbone modifications (AUAA) at the 3' end, cholesterol modification (Chol) and full-chain methoxy modification:

[0055] 5'-CUGGCUGUCAAUUCAUAGGUCAGAUAAChol-3';

[0056] The TGFβ-related miRNA sequence combination of the present invention is designed based on the common miR-192-5p sequence of humans and rats, and can form a similar hairpin structure to ensure the species stability of the drug. Compared with the universal sequence, the two enhanced sequences have higher cell membrane affinity and higher transfection efficiency, and are suitable for in vivo and in vitro interference experiments, with higher stability and efficiency.

[0057] 5. Specificity and transfection efficiency of TGFβ-related miRNA sequences

[0058] In order to verify the specificity and transfection efficiency of the TGFβ-related miRNA sequence combination of the present invention, the mimic sequences SEQ ID NO.1-2 and the inhibitor sequence SEQ ID NO.3 were transfected into the rat glomerular mesangial cell line, respectively. Figure 5 A and 5B are the expression of miR-192-5p detected using rat-specific primer sequence 5'-UAAUCUCAGCUGGCAACUGUGA-3'. The results showed that compared with the negative control, the relative expression of miR-192-5p in cells treated with the mimic sequence (SEQ ID NO.1-2) increased by 34.56% (normal glucose-treated cells, NG+192+ group) and 37.21% (high glucose-treated cells, HG+192+ group), respectively, and the relative expression of miR-192-5p in cells treated with the inhibitor sequence (SEQ ID NO.3) decreased by 39.26% (normal glucose-treated cells, NG+192- group) and 37.54% (high glucose-treated cells, HG+192- group), respectively, both of which were statistically significant compared with the negative control (P<0.05), indicating that the TGFβ-related miRNA sequence of the present invention has been verified to have high specificity and transfection efficiency.

[0059] According to the results of Example 1, the four miRNAs may be closely related to the production of urinary protein in diabetic kidney disease, and the target genes of the miRNA sequences are all related to the TGFβ pathway, which theoretically determines the feasibility of the invention. Using high glucose, TGFβ1 recombinant protein and inhibitors and four miRNA inhibitors to screen in vitro experimental models, miRNA sequences with high expression consistency and reaction consistency are selected to ensure the specificity of the drug and the precise regulation of cellular mechanisms. The TGFβ-related miRNA sequence combination described in the present invention is designed based on the common miR-192-5p sequence of humans and rats, and can form a similar hairpin structure to ensure the species stability of the drug. The TGFβ-related miRNA sequence described in the present invention has been verified to have high specificity and transfection efficiency.

[0060] Example 2

[0061] This example describes the use of the TGFβ-related miRNA sequence combination in Example 1 as a drug for diabetic kidney disease, including expression status and therapeutic application.

[0062] 1. Expression of TGFβ-related miRNA sequences in in vitro experiments.

[0063] The activation of TGFβ and its downstream signaling protein Smads induces fibrosis, which plays a vital role in diabetic kidney damage. In order to study the therapeutic application of TGFβ-related miRNA sequences in diabetic kidney disease in vivo and in vitro experiments, we first transfected the mimic sense sequence SEQ ID NO.1, the antisense enhancer sequence designed based on the mimic sequence SEQ ID NO.2, and the enhancer sequence designed based on the inhibitor sequence SEQ ID NO.3 in the glomerular mesangial cell line, and used the rat-specific primer sequence 5'-UAAUCUCAGCUGGCAACUGUGA-3' to detect the expression of miR-192-5p. After the authentication drug entered, the expression of TGFβ / Smad-induced fibrosis factors was detected ( Figure 7 ). The results showed that in primary glomerular mesangial cells and cell lines transfected with mimic sequences (enhanced sequences of SEQ ID NO.1 and SEQ ID NO.2), fibrosis factors were significantly reduced, while transfection with inhibitor sequences (enhanced sequences of SEQ ID NO.3) significantly increased fibrosis factors, indicating that the miRNA sequence of the present invention can affect the expression level of fibrosis factors induced by TGFβ / Smad.

[0064] 2. Application of TGFβ-related miRNA sequences as drugs for diabetic kidney disease in in vivo and in vitro experiments.

[0065] The early pathophysiological mechanism of diabetic kidney disease is related to the proliferation of mesangial cells, and its pathophysiological characteristics are related to increased proteinuria and decreased renal function. In order to study the therapeutic application of TGFβ-related miRNA sequences in diabetic kidney disease in vivo and in vitro experiments, we constructed a high-glucose cultured mesangial cell line and a high-fat diet + streptozotocin-induced diabetic kidney injury rat primary mesangial cell culture, which were used as in vivo and in vitro models for the treatment of diabetic kidney disease, respectively. The mimic sequence (enhanced sequence of SEQ ID NO.1 and SEQ ID NO.2) and the inhibitor sequence (enhanced sequence of SEQ ID NO.3) were transfected, and the expression of miR-192-5p was detected using the rat-specific primer sequence 5'-UAAUCUCAGCUGGCAACUGUGA-3'. After the authentication drug entered, the cell proliferation was detected using the CCK-8 assay ( Figure 8). The results showed that the cell proliferation in primary mesangial cells and cell lines transfected with the mimic sequence (enhanced sequence of SEQ ID NO.1 and SEQ ID NO.2) was significantly reduced, while the cell proliferation was significantly increased by transfection with the inhibitor sequence (enhanced sequence of SEQ ID NO.3), indicating that the TGFβ-related miRNA sequence of the present invention can inhibit the excessive proliferation of mesangial cells in in vitro and in vivo experiments of diabetic kidney disease.

[0066] The above two examples show that the four miRNAs may be closely related to the production of urinary protein in diabetic kidney disease, and the target genes of the miRNA sequences are all related to the TGFβ pathway, which theoretically determines the feasibility of the invention. Using high glucose, TGFβ1 recombinant protein and inhibitors and four miRNA inhibitors to screen in vitro experimental models, miRNA sequences with high expression consistency and reaction consistency are selected, which can ensure the specificity of the drug and the precise regulation of cellular mechanisms. The TGFβ-related miRNA sequence combination described in the present invention is designed based on the common miR-192-5p sequence of humans and rats, and can form a similar hairpin structure to ensure the species stability of the drug. The TGFβ-related miRNA sequence described in the present invention has been verified to have high specificity and transfection efficiency, can inhibit the excessive proliferation of glomerular mesangial cells under high glucose culture through the TGFβ / Smad pathway, has good drug potential and application prospects for early diabetic kidney disease, and has good performance in both in vivo and in vitro experiments, which provides a possibility for the drug application of TGFβ-related miRNA sequence combination in the treatment of diabetic kidney disease. The TGFβ-related miRNA sequence combination in the present invention can be used to prepare a drug for diabetic kidney disease.

[0067] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A TGFβ-related miRNA sequence combination, characterized in that: The TGFβ-related miRNA sequence combination includes a mimic sequence and an inhibitor sequence designed based on the mature miR-192 miR-192-5p; The nucleotide sequence of the sense sequence of the mimetic sequence is shown in SEQ ID NO.1; The antisense sequence of the mimetic sequence is a nucleotide sequence such as the sequence shown in SEQ ID NO.2 or an antisense enhancement sequence of the mimetic sequence designed based on SEQ ID NO.2; The inhibitor sequence is a nucleotide sequence such as the sequence shown in SEQ ID NO.3 or a strengthening sequence of the inhibitor sequence designed based on SEQ ID NO.3; The antisense enhancement sequence of the mimetic sequence designed based on SEQ ID NO. 2 is: 5'-ACCUGUCAAUUCAUAGGUCAGUUAAUUChol-3'; The enhancing sequence of the inhibitor sequence designed based on SEQ ID NO. 3 is: 5'-CUGGCUGUCAAUUCAUAGGUCAGAUAAChol-3'.

2. A use of the TGFβ-related miRNA sequence combination as claimed in claim 1, characterized in that: The TGFβ-related miRNA sequence combination is used to prepare a medicine for diabetic kidney disease.

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