Smurf1-targeting micro RNA inhibitor sequence combination and application thereof

Abstract

The invention provides a micro RNA (Ribonucleic Acid) inhibitor sequence combination for targeting Smurf1, which is characterized in that the micro RNA inhibitor sequence combination for targeting Smurf1 comprises a mimic sequence (SEQ ID NO.1-2) and an inhibitor sequence (SEQ ID NO.3) corresponding to the design of a miR-154-5p sequence. The invention also provides an application. The Smurf1-targeted micro RNA inhibitor sequence combination is used for preparing a medicine for treating fibrosis diseases. The Smurf1-targeting micro RNA inhibitor sequence combination designed by the invention has good specificity and relatively high transfection efficiency, has good performance in computer simulation, tool cell bioinformatics transfection verification and in-vitro experimental disease model verification, can inhibit a TGF beta / Smad pathway mediated fibrosis process by increasing the Smurf1 level, and has good application prospects. The compound has good treatment potential and application prospects for fibrosis diseases such as kidney fibrosis and can be used for preparing fibrosis disease related drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a combination of microRNA inhibitor sequences targeting Smurf1 and its application. Background Art

[0002] Fibrotic diseases refer to the excessive deposition of extracellular matrix caused by chronic tissue damage, and this pathological process can affect multiple organs and tissues. Fibrotic diseases include, but are not limited to, idiopathic pulmonary fibrosis, liver fibrosis, cardiac fibrosis, and renal fibrosis, etc. The occurrence of fibrosis involves a variety of harmful stimuli, including toxins, infectious pathogens, autoimmune responses, and mechanical stress, which induce myofibroblasts to excessively deposit extracellular matrix proteins, resulting in tissue sclerosis. During the fibrotic process, cytokines and growth factors such as members of the transforming growth factor-β (TGFβ) family play important roles. They bind to stem cell surface receptors and initiate downstream signal transduction, leading to the nuclear translocation of Smad2 / 3 transcriptional regulators, and further enhancing myofibroblast differentiation and the production and secretion of extracellular matrix proteins. The TGFβ / Smad signaling pathway is a key regulatory pathway in fibrotic diseases. After TGFβ1 binds to its membrane receptor TGFβ receptor 1 (TRI), it activates another receptor TRII. TRII phosphorylates and activates intracellular Smad2 and Smad3, which form a heteropolymer with Smad4. After this complex enters the nucleus, it binds to transcriptional co-activators or co-repressors, and then regulates the transcription of downstream target genes. In contrast, Smad7 binds to TRI and TRII, and can inhibit the phosphorylation of Smad2 and Smad3, thereby inhibiting the conduction of the TGFβ1 pathway. Smads participate in the pathological process of fibrosis through these two positive and negative regulatory effects in the TGFβ1 pathway. Therefore, fibrotic diseases involve multiple organs, their pathogenesis is complex, and the TGFβ / Smad pathway plays a key role in it. Targeted gene research on this pathway has important clinical significance for the development of new targeted drugs.

[0003] Non-coding RNAs (ncRNAs) refer to RNA molecules transcribed from the genome that do not encode proteins, including microRNAs (miRNAs), small interfering RNAs (siRNAs), Piwi-interacting RNAs (piRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), long interspersed nuclear elements (LINEs), short interspersed nuclear elements (SINEs), enhancer RNAs (eRNAs), etc. Among them, miRNA is a highly conserved non-coding RNA with a length of 18-25 nucleotides. It regulates gene expression through the imperfect complementary base sequence in the 3'-untranslated region of the target mRNA, thereby affecting multiple cellular processes from growth and development to disease occurrence. Transcription factor binding analysis shows that during the growth and development stage, miR-154 is rich in Smad3 binding elements (SBEs) that conduct the TGFβ1 pathway. Therefore, miR-154 may have the potential to regulate fibrotic diseases through the TGFβ / Smad pathway, but the related technologies and drug development applications of the microRNA mechanism are currently unknown. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a combination of microRNA inhibitor sequences targeting Smurf1 and its application in view of the deficiencies of the above-mentioned prior art. The combination of microRNA inhibitor sequences targeting Smurf1 has good specificity and high transfection efficiency, and shows good performance in computer simulation, bioinformatics transfection verification of tool cells, and verification of in vitro experimental disease models. It can inhibit the fibrosis process mediated by the TGFβ / Smad pathway by increasing the level of Smurf1, has good therapeutic potential and application prospects for fibrotic diseases such as kidney fibrosis, and can be used for the preparation of drugs related to fibrotic diseases.

[0005] To solve the above technical problem, the technical solution adopted by the present invention is: a combination of microRNA inhibitor sequences targeting Smurf1, the combination of microRNA inhibitor sequences targeting Smurf1 includes a mimic sequence and an inhibitor sequence designed corresponding to the miR-154-5p sequence;

[0006] The nucleotide sequence of the sense strand of the mimic sequence is as shown in SEQ ID NO.1;

[0007] The nucleotide sequence of the antisense strand of the mimic sequence is as shown in SEQ ID NO.2;

[0008] The nucleotide sequence of the inhibitor sequence is as shown in SEQ ID NO.3.

[0009] The present invention also provides the use of the above-mentioned combination of microRNA inhibitor sequences targeting Smurf1, and the combination of microRNA inhibitor sequences targeting Smurf1 is used for preparing a medicament for fibrotic diseases.

[0010] Preferably, the combination of microRNA inhibitor sequences targeting Smurf1 is used for preparing a medicament for renal fibrosis.

[0011] The mode of action of the combination of microRNA inhibitor sequences targeting Smurf1 for treating fibrotic diseases is to inhibit the mature microRNA miR-154-5p that binds to the 3'UTR of Smurf1, thereby increasing the expression level of Smurf1, inhibiting the fibrotic process mediated by the TGFβ / Smad pathway, and having good therapeutic potential and application prospects for fibrotic diseases exemplified by renal fibrosis, providing a possibility for the clinical application of the combination of microRNA inhibitor sequences targeting Smurf1 in treating fibrotic diseases.

[0012] The present invention has the following advantages compared with the prior art:

[0013] 1. The combination of microRNA inhibitor sequences targeting Smurf1 is used for preparing a medicament for treating fibrotic diseases, has a clear and distinct binding mechanism, and has good specificity and high transfection efficiency in vivo.

[0014] 2. The combination of microRNA inhibitor sequences targeting Smurf1 has good performance in computer simulation, bioinformatics transfection verification of tool cells, and verification of in vitro experimental disease models, can inhibit the fibrotic process mediated by the TGFβ / Smad pathway by increasing the level of Smurf1, has good potential for drug development and application prospects for fibrotic diseases such as renal fibrosis, provides a possibility for the clinical application of the combination of microRNA inhibitor sequences targeting Smurf1 as a medicament for fibrotic diseases, and can be used for the preparation of medicaments related to fibrotic diseases.

[0015] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Description of the Drawings

[0016] Figure 1 is the ubiquitination binding site analysis known in Example 1 of the present invention.

[0017] Figure 2 is the inBio_Map interaction analysis in Example 1 of the present invention.

[0018] Figure 3 is the IntAct interaction analysis in Example 1 of the present invention.

[0019] Figure 4Analysis of the recognition characteristics of Ubibrowser ubiquitin ligase in Example 1 of the present invention.

[0020] Figure 5 Analysis of the sequence alignment between human and rat in the UniProtKB / Swiss-Prot database in Example 1 of the present invention.

[0021] Figure 6 Analysis of the prediction of human and rat Smurf1 target genes by RNA22 V2 in Example 1 of the present invention.

[0022] Figure 7 Verification of the transfection efficiency of the sequence combination in Example 2 of the present invention.

[0023] Figure 8 Mechanism diagram of miR-154-5p regulating renal fibrosis by interfering with Smad3 phosphorylation through Smurf1 and jointly binding Smad7 and TRI in Example 2 of the present invention. Figure 9 Application of the sequence combination as a drug for renal fibrosis in Example 2 of the present invention. Detailed implementation manners

[0024] Example 1

[0025] This example describes the design scheme of the microRNA inhibitor sequence combination of Smurf1.

[0026] 1. Analyze the ubiquitination site of Smurf1 using the Netpath / Netslim database

[0027] As a member of the HECT family of E3 ubiquitin ligases, Smurf1 is a key enzyme that determines substrate specificity in the ubiquitination modification pathway. It can recognize ubiquitinated protein substrates and selectively regulate the ubiquitination degradation process of effector molecule Smads. The mechanism of action is shown in Figure 1 A. Through the analysis of known ubiquitination binding sites using the Netpath / Netslim database, it is found that the known ubiquitination binding sites of Smurf1 are RhoA, Smad7, and TRI ( Figure 1 B).

[0028] 2. Interaction analysis of Smurf1

[0029] Using inBio_Map (v2016_09_12) and IntAct Molecular Int for protein interaction analysis, it is found that Smurf1 interacts with multiple Smads in the TGFβ1 pathway, such as TGFβ receptors, Smads, RhoA, and Smurf2, etc. ( Figure 2 and Figure 3 ).

[0030] 3. Identification Feature Analysis of Smurf1

[0031] The Ubibrowser database was used to verify the recognition features of ubiquitin ligases, and it was found that Smurf1 had a high ubiquitination binding ability with Smad2, Smad3, Smad4, and Smurf2 respectively ( Figure 4 ), and Smad3 had potential binding sites with the C2 and HECT regions of Smurf1 (MH1: position 31 - 131, length 101; MH2: position 226 - 403, length 178). It was speculated that Smurf1 and Smad3 might have a potential ubiquitination binding mode.

[0032] 4. Sequence Alignment of Human and Rat Smurf1 and Smad3 in the UniProtKB / Swiss-Prot Database

[0033] The structures of human and rat Smurf1 and Smad3 collected from the UniProtKB / Swiss-Prot database were aligned. The sequence alignment results showed that in the C2 and HECT regions of Smurf1 and the MH1 and MH2 regions of Smad3, the corresponding sequences of humans and rats were exactly the same ( Figure 5 ), and it was speculated that humans and rats had a highly similar binding mode. Therefore, using rats as the research object could well simulate the binding situation of human Smurf1 and Smad3, facilitating the next experimental verification of protein binding and ubiquitination.

[0034] 5. Prediction of Human and Rat Smurf1 Target Genes by RNA22 V2 Computational Algorithm

[0035] To further study the Smurf1 target genes in depth, this study used the RNA22 V2 computational algorithm to predict the target gene miR-154-5p of human and rat Smurf1. The Smurf1 gene was downloaded from the NCBI Gene database, and the mature miRNA sequences were downloaded from the miRBase database. The results of bioinformatics analysis showed that there was a target gene miR-154-5p that bound to multiple binding sites of human and rat SMURF1. The 985 site of human SMURF1 and the 72, 327, and 1715 sites of rat SMURF1 could bind to human and rat miR-154-5p respectively ( Figure 6 ), and it was speculated that humans and rats had a highly similar binding mode, facilitating the next verification experiment of miRNA-protein binding.

[0036] 6. Design and Verification of miR-154-5p Interference Sequences Based on Smurf1 Target Genes

[0037] miR-154 is located in the miRNA-enriched region of the single-stranded chromosome of mammalian chromosome 14q32. The 5'-arm end of the precursor miR-154 (sequence: 5'-UAGGUUAUCCGUGUUGCCUUCG-3', which forms miR-154-5p after maturation) has been confirmed to be controlled by a differentially methylated region (DMR) of approximately 200 kb in the Dlk1-Gtl2 (rodent) / Dlk-Dio3 (human) domain upstream of the miRNA cluster. Transcription factor binding analysis shows that this miRNA is rich in SBEs that conduct the TGFβ1 pathway. The corresponding mimics designed according to the mature miR-154-5p sequence of miR-154 are SEQ ID NO.1-2, and the inhibitor is SEQ ID NO.3. The negative control of the interference sequence designed for detection is 5'-CAGUACUUUUGUGUAGUACAA-3'.

[0038] Sense sequence of the mimic: 5'-UAGGUUAUCCGUGUUGCCUUCG-3' (SEQ ID NO.1)

[0039] Antisense sequence of the mimic: 5'-AAGGCAACACGGAUAACCUAUU-3' (SEQ ID NO.2)

[0040] Inhibitor sequence: 5'-CGAAGGCAACACGGAUAACCUA-3' (SEQ ID NO.3)

[0041] SEQ ID NO.1-3 are RNA sequences. When inputting 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 sequences is actually uracil "U".

[0042] According to this example Figures 1 to 6Analysis results show that Smurf1 has multiple binding sites with RhoA, Smad7, and TRI in the TGFβ / Smad pathway, and has potential interactions with TGFβ / Smad pathway-related proteins such as TGFβ receptors, Smads, RhoA, and Smurf2. It has a high ubiquitination binding ability with Smad2, Smad3, Smad4, and Smurf2 respectively. The corresponding sequences of Smurf1 and Smad3 in humans and rats are exactly the same, and there are potential binding sites for interaction, indicating that Smurf1 can bind to TGFβ / Smad pathway proteins mainly Smad3, leading to its ubiquitination. In addition, humans and rats have a highly similar binding mode, and there are multiple Smurf1 binding sites that can bind to miR-154-5p. The designed microRNA inhibitor sequence combination targeting Smurf1 has a clear and distinct binding mechanism, which can theoretically effectively improve the specificity of the drug.

[0043] Example 2

[0044] This example describes the application of the microRNA inhibitor sequence combination targeting Smurf1 in Example 1 as a drug for fibrotic diseases, including verification of the transfection efficiency of the interference sequence combination and its application as a drug for kidney fibrosis.

[0045] 1. Verification of the transfection efficiency of the interference sequence combination

[0046] To verify the transfection efficiency of the microRNA inhibitor sequence combination targeting Smurf1, BLAST was used to test the efficiency of the designed interference sequence combination. The test results showed that the designed interference sequence combination in this invention could only simulate Smurf1-related sequences in the BLAST calculation simulation, and no other mRNA sequences were generated, indicating that Smurf1 designed in this invention has extremely high specificity, which can theoretically ensure the specificity of Smurf1 after entering cells or the body. Therefore, it is proved from the computer level that Smurf1 has the possibility of efficiently entering the body to work.

[0047] In actual in vitro experiments, after transfecting the interference sequence into the tool cell 293T, the transfection efficiency of the microRNA inhibitor sequence combination targeting Smurf1 was verified, and at the same time, the luciferase reporter gene was used to verify the binding situation between miR-154-5p and Smurf1. Figure 7The results in A showed that among the miR-154-5p inhibitor combinations, there was no statistical significance between the control sequence and the blank sequence. There were significant increases and decreases in the mimic sequences (SEQ ID NO.1-2) and the inhibitor sequence (SEQ ID NO.3) compared with the control sequence. The relative expression level of the mimic sequences (SEQ ID NO.1-2) increased to 1486.67% of the control sequence, and the relative expression level of the inhibitor sequence SEQ ID NO.3 decreased to 14.67% of the control sequence, proving that the combination of microRNA inhibitor sequences targeting Smurf1 had a high transfection efficiency. Figure 7 The results in B showed that the combination of microRNA inhibitor sequences targeting Smurf1 could significantly reduce the fluorescence ratio of firefly luciferase / renilla luciferase, that is, it could efficiently reduce the fluorescence intensity of the binding to the 3’UTR region of SMURF1 mRNA, thus indicating that miR-154-5p could specifically bind to the 3’UTR region of SMURF1 mRNA. The above two experimental results both proved the specificity and high efficiency of the combination of microRNA inhibitor sequences targeting Smurf1 in cell experiments.

[0048] 2. Application of the interference sequence combination as a drug for kidney fibrosis

[0049] In this example, kidney fibrosis disease involves the injury and necrosis of renal resident cells, leading to the gradual loss of renal function, which is one of the fibrosis diseases. The team explored the mechanism by which miR-154-5p regulates kidney fibrosis by interfering with Smad3 phosphorylation through Smurf1 and the co-binding of Smad7 and TRI in previous theoretical studies ( Figure 8 ). To explore the drug application of the combination of microRNA inhibitor sequences targeting Smurf1 in kidney fibrosis, we constructed a glucotoxic kidney fibrosis model with glomerular mesangial cells cultured in high glucose, added the inhibitor sequence SEQ ID NO.3 of miR-154-5p and the control sequence to the cells respectively, and detected the expression of fibrosis factors by adding Smurf1 inhibitor at the same time. The results showed that compared with the control sequence, the combination of microRNA inhibitor sequences targeting Smurf1 first reduced the expression level of miR-154-5p in glomerular mesangial cells cultured in high glucose ( Figure 9 A), and at the same time could significantly increase the level of Smurf1 ( Figure 9 B). In addition, it also reduced the levels of fibrosis factors (pSmad3 / Smad3) ( Figure 9 C) and ubiquitination (Ubiquitin) ( Figure 9 D), and significantly reduced the fibrotic nephropathy phenotype (excessive proliferation of mesangial cells) ( Figure 9(E) It is demonstrated that the combination of microRNA inhibitor sequences targeting Smurf1 has therapeutic potential as a drug for kidney fibrosis.

[0050] The above two examples show that the combination of microRNA inhibitor sequences targeting Smurf1 designed by the present invention has good specificity and high transfection efficiency, and has good performance in computer simulation, transfection verification of tool cell bioinformatics, and verification of in vitro experimental disease models. It can inhibit the fibrosis process mediated by the TGFβ / Smad pathway by increasing the level of Smurf1, and has good therapeutic potential and application prospects as a drug for fibrosis diseases such as kidney fibrosis.

[0051] The above is only a preferred embodiment of the present invention, and does not impose any limitation on the present invention. Any simple modification, change, and equivalent change made to the above embodiments according to the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A microRNA inhibitor sequence combination targeting Smurf1, characterized in that: The microRNA inhibitor sequence combination targeting Smurf1 includes a miR-154-5p sequence designed corresponding mimic sequence and inhibitor sequence; The nucleotide sequence of the sense sequence of the mimetic sequence is shown in SEQ ID NO.1; The nucleotide sequence of the antisense sequence of the mimetic sequence is shown in SEQ ID NO.2; The nucleotide sequence of the inhibitor sequence is shown in SEQ ID NO.

3.

2. A use of the microRNA inhibitor sequence combination targeting Smurf1 as claimed in claim 1, characterized in that: The microRNA inhibitor sequence combination targeting Smurf1 is used for preparing medicine for fibrotic diseases.

3. The use according to claim 2, characterized in that: The microRNA inhibitor sequence combination targeting Smurf1 is used to prepare a drug for renal fibrosis.

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