TIF-IA polypeptide, its encoding nucleic acid and pharmaceutical use

Abstract

The present invention belongs to the field of bioengineering technology, and discloses a TIF-IA polypeptide, its encoding nucleic acid and pharmaceutical uses. The TIF-IA polypeptide has the amino acid sequence shown in SEQ ID NO.1, and its encoding nucleic acid is the sequence shown in SEQ ID NO.2. Experiments show that the TIF-IA polypeptide and its encoding nucleic acid can inhibit lipid synthesis by promoting nucleolar stress, thereby reducing lipid accumulation, and can be applied to the preparation of drugs for preventing and / or treating non-alcoholic fatty liver disease, and can also be used for non-alcoholic fatty liver disease prediction scoring.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a TIF-IA polypeptide, its encoding nucleic acid, and pharmaceutical uses thereof. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic syndrome mainly characterized by excessive lipid accumulation, and is mainly induced by multiple factors such as obesity, type 2 diabetes, hyperlipidemia and other diseases. It represents a series of diseases ranging from simple lipid accumulation in the liver (hepatic steatosis) to more severe forms, and may further progress to cirrhosis or even liver cancer as the disease progresses. Fatty liver disease has increasingly become a common disease globally. The prevalence of NAFLD is about 15% in China and about 20% in Europe and America. The pathogenesis of NAFLD involves insulin resistance, hepatic lipid accumulation, endoplasmic reticulum stress, mitochondrial dysfunction, alteration of intestinal flora, and activation of inflammatory pathways. Therefore, if hepatic lipid accumulation can be effectively reduced, it may help prevent the progression of NAFLD to more severe liver diseases. For the treatment of NAFLD, currently there are mainly non-drug treatments, drug treatments, surgical treatments and other means. For early-stage patients, non-drug treatments are mostly adopted, such as lifestyle interventions and weight loss. As the disease progresses, drug symptomatic treatment needs to be comprehensively considered according to its etiology, such as lipid-lowering drugs, insulin-sensitizing drugs, regulating oxidative stress and inflammation, anti-fibrotic drugs, and drugs regulating intestinal flora and immune function. For patients with advanced cirrhosis or even liver cancer, surgical treatments such as liver transplantation can be considered. Therefore, early and precise intervention is the key to the treatment of NAFLD. Designing therapeutic drugs with more targets may be the research trend of future drug development.

[0003] TIF-IA is a transcription factor of RNA polymerase I and is the mammalian homolog of yeast Rrn3p, playing a central role in the transcription of ribosomal DNA genes. TIF-IA is highly expressed in bone marrow, thyroid, testis, ovary, liver, lung and other tissues. Its expression is also crucial for maintaining the nucleolar structure and cell viability. Activating liver RNA transcription can promote liver energy consumption, thereby reducing lipid accumulation in the liver. Therefore, TIF-IA can promote the transcription of rRNA, thereby promoting nucleolar stress, and to a certain extent affects cell fate, such as apoptosis, senescence, autophagy and differentiation, in response to stress-induced damage. Through the implementation of the present invention, exploring the occurrence mechanism of TIF-IA therein provides a new precise treatment plan for NAFLD. Summary of the Invention

[0004] Aiming at the deficiencies in the prior art, the purpose of the present invention is to provide a TIF-IA polypeptide, its encoding nucleic acid, and pharmaceutical uses thereof, which have great prospects in the preparation of potential predictive indicators and candidate drugs for NAFLD.

[0005] In the first aspect of the present invention, there is provided the use of a TIF-IA polypeptide having the amino acid sequence shown in SEQ ID NO.1, a nucleic acid encoding the TIF-IA polypeptide, a vector containing the nucleic acid, or a composition containing the TIF-IA polypeptide in the preparation of a drug for preventing and / or treating non-alcoholic fatty liver disease.

[0006] In the second aspect of the present invention, there is provided the use of a TIF-IA polypeptide having the amino acid sequence shown in SEQ ID NO.1, a nucleic acid encoding the TIF-IA polypeptide, a vector containing the nucleic acid, or a composition containing the TIF-IA polypeptide in the prediction score of non-alcoholic fatty liver disease.

[0007] Furthermore, the nucleotide sequence of the nucleic acid encoding the TIF-IA polypeptide is as shown in SEQ ID NO.2.

[0008] In the third aspect of the present invention, there is provided a nucleic acid which is a nucleic acid encoding a TIF-IA polypeptide, the amino acid sequence of the TIF-IA polypeptide is as shown in SEQ ID NO.1, and the nucleotide sequence of the nucleic acid is as shown in SEQ ID NO.2.

[0009] Beneficial effects: The present invention utilizes bioengineering technology to recombinantly insert a DNA sequence corresponding to a polypeptide into the eukaryotic expression vector pcDNA3.1(+). After digestion with enzymes and sequence analysis prove successful recombination, this eukaryotic expression recombinant polypeptide is transfected into hepatocytes, and immunoblotting proves the protein expression of the polypeptide, realizing the recombination of the polypeptide. The lipid metabolism-promoting function of the polypeptide is studied. Experiments show that TIF-IA can inhibit lipid synthesis by promoting nucleolar stress, thereby reducing lipid accumulation. It has very important development prospects for clinical treatment applied to NAFLD. Brief Description of the Drawings

[0010] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0011] Figure 1It is the immunohistochemical result diagram of TIF-IA polypeptide in the liver tissues of normal people and NAFLD patients in Example 1 of the present invention. Among them, the upper figure is the representative immunohistochemical staining result of TIF-IA in normal liver and NAFLD liver tissues, and the lower figure is the statistical analysis result of the positive ratio of TIF-IA in normal and NAFLD liver tissues (**, P<0.01, with statistical significance);

[0012] Figure 2 It is the immunoblot detection result diagram of the expression of polypeptide molecules in hepatocytes after transfection with the recombinant vector of TIF-IA polypeptide in Example 2 of the present invention;

[0013] Figure 3 It is the oil red staining diagram that the TIF-IA polypeptide in Example 3 of the present invention reduces the fat accumulation in the palmitic acid-stimulated group of HepG2; the upper figure is the representative illustration result of lipid deposition in control and HepG2 cells transfected with the recombinant vector of TIF-IA polypeptide, and the lower figure is the statistical analysis result of the lipid accumulation level in HepG2 after control and transfection with the recombinant vector of TIF-IA polypeptide (***, P<0.001, with statistical significance). Detailed implementation manners

[0014] The present application will be further elaborated below in combination with the implementation manners and examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. In addition, it should be understood that after reading the content taught in the present application, those skilled in the art can make various changes or modifications to the present application, and these equivalent forms also fall within the protection scope of the appended claims of the present application.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present application belongs. The terms used in the description of the present application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0016] The amino acid sequence of the TIF-IA polypeptide in the present application is shown in SEQ ID NO.1 in the sequence listing, specifically as follows:

[0017] 1 maapllhtrl pgdaaasssa vkklgasrtg isnmralend ffnspprktv rfggtvtevl

[0018] 61 lkykkgetnd fellknqlld pdikrlpwln rsqtvveeyl aflgnlvsaq tvflrpclsm

[0019] 121 iashfvpprv iikegdvdvs dsddeddnlp anfdtchral qiiaryvpst pwflmpilve

[0020] 181 kfpfvrkser

[0021] The cDNA sequence of the TIF-IA polypeptide in this application is shown in SEQ ID NO.2 of the sequence listing, as follows:

[0022] 1 ATGGCGGCGC CTCTCCTCCA CACCCGGTTG CCTGGTGACG CTGCCGCCTC CTCAAGCGCA

[0023] 61 GTAAAAAAGC TCGGCGCTAG TAGGACCGGC ATCTCTAATA TGAGGGCACT CGAGAACGAT

[0024] 121 TTCTTTAATA GCCCTCCGCG AAAAACTGTA CGATTTGGTG GAACAGTCAC AGAGGTCCTG

[0025] 181 TTGAAGTATA AGAAAGGTGA GACCAACGAC TTTGAATTGC TCAAAAACCA ACTCCTTGAC

[0026] 241 CCTGACATAA AGAGGTTGCC ATGGCTTAAT AGGAGTCAAA CGGTCGTAGA AGAATATTTG

[0027] 301 GCATTTTTGG GCAACCTTGT ATCTGCACAG ACAGTGTTCC TGCGCCCTTG CTTGAGTATG

[0028] 361 ATCGCGTCCC ACTTCGTGCC GCCAAGAGTT ATCATTAAAG AGGGGGATGT TGATGTGTCC

[0029] 421 GATAGCGATG ATGAGGACGA CAATCTGCCG GCTAACTTTG ATACATGTCA CCGGGCGCTG

[0030] 481 CAAATCATTG CGAGATATGT CCCCTCTACC CCGTGGTTCT TGATGCCCAT CTTGGTAGAG

[0031] 541 AAATTCCCAT TCGTCAGAAA ATCCGAACGA

[0032] It was found through experiments in this application that the polypeptide of the sequence shown in SEQ ID NO.1 can inhibit lipid synthesis by promoting nucleolar stress, thereby reducing lipid accumulation.

[0033] The following are some specific examples.

[0034] Example 1: Expression of TIF-IA polypeptide in immunohistochemistry of liver tissues of normal people and NAFLD patients

[0035] The liver tissue was sectioned and dried. It was repeatedly soaked in xylene and absolute ethanol and then washed with water. Antigen retrieval; after blocking with hydrogen peroxide for 20 min, it was washed three times with PBS; the enzyme-labeled goat anti-rabbit IgG complex was added for serum blocking, incubated at 37 °C for 20 min, and washed 3 times with PBS; incubated with the primary antibody at 4 °C overnight; incubated with the secondary antibody at room temperature for 1 h, and washed three times with PBS; developed with DAB; counterstained the cell nucleus; dehydrated and sealed the slide; observed under the stain. The immunohistochemical results showed that the expression of TIFIA was significantly decreased in the liver tissues of the NAFLD patient group compared with the normal population, suggesting that the downregulation of nucleolar and nucleolar stress functions is associated with the abnormal accumulation of fat in the livers of NAFLD patients ( Figure 1 ).

[0036] Example 2: Construction of recombinant cDNA of TIF-IA polypeptide and detection of its expression in hepatoma cells

[0037] 1. Construction of eukaryotic expression vector of the polypeptide

[0038] The nucleic acid sequence (SEQ ID NO.2) encoding the polypeptide of the sequence shown in SEQ ID NO.1 was obtained by commissioning Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis service. After purification, it was double digested with ECOR1 and AgeI, and the fragment was recovered using a Gel DNA Recovery Kit. The plasmid pcDNA3.1(+) was digested with the same enzymes and then the DNA was recovered simultaneously. Then, the recovered polypeptide nucleic acid DNA and plasmid DNA were mixed and incubated with T4 DNA ligase at 16 °C overnight. The obtained recombinant plasmid was transformed into competent Escherichia coli, and then the bacteria were picked, shaken, and the plasmid was extracted, namely the plasmid DNA expressing the polypeptide of the sequence shown in SEQ ID NO.1.

[0039] 2. Expression identification of TIF-IA polypeptide

[0040] HepG2 cells were seeded into six-well plates. When the cells adhered and reached a density of about 70%-90%, the cell medium was changed to 2 ml of Opti-DMEM without serum and antibiotics. Two 1.5-ml EP tube solutions were prepared (1 tube contained 100 μl of Opti-DMEM + 10 μl of Lip2000; 1 tube contained 1.2 μg of plasmid + 1.2 μg of PAX2 + 0.6 μg of PMD2G + 100 μl of Opti-DMEM) and left standing for about 15 min. The two tube solutions were mixed and added to the cells. After 6 hours, the medium was changed. After 48 hours, the cells were collected, washed 3 times with PBS, and lysed with immunoprecipitation lysis buffer (RIPA + CM + PI). After standing for 30 min, ground on ice and centrifuged in a centrifuge, and then Loading Buffer (about 1 / 4 of the volume of the lysis buffer) was added, and heated at 100 °C for about 7 min for Western Blotting verification. After steps of electrophoresis (80 V), membrane transfer (300 mA), blocking for 2 h, incubation with primary antibody, membrane washing, incubation with secondary antibody, and membrane washing, finally, the target band was developed and photographed with a Bio-Rad imager. The immunoblotting results showed that this polypeptide was successfully expressed in hepatocytes, with a molecular weight of 22 kDa, which was consistent with the prediction ( Figure 2 ).

[0041] Example 3: Oil red staining showed that TIF-IA polypeptide reduced lipid accumulation in the HepG2 palmitic acid-stimulated group

[0042] HepG2 cells were cultured in six-well plates. After stimulating the cells with palmitic acid (0.4 mM) for 24 h, the culture medium was discarded, and the cells were washed 3 times with PBS. The cells were fixed with 4% paraformaldehyde for 30 min. After discarding the paraformaldehyde, the cells were stained with oil red dye in the dark for 20 min, rinsed with isopropanol for several seconds and then washed 3 times with PBS, and observed under a microscope. As Figure 3 shown, the results indicated that the introduction of TIF-IA polypeptide could significantly inhibit the accumulation of lipids in hepatocytes induced by palmitic acid stimulation, suggesting its ability to alleviate abnormal lipid metabolism and abnormal lipid accumulation in hepatocytes ( Figure 3 ).

Claims

1. Use of a TIF-IA polypeptide having an amino acid sequence of SEQ ID NO.1, a nucleic acid encoding the TIF-IA polypeptide, a vector comprising the nucleic acid, or a composition comprising the TIF-IA polypeptide in the preparation of a drug for preventing and / or treating non-alcoholic fatty liver disease.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the nucleic acid encoding the TIF-IA polypeptide is shown in SEQ ID NO.

2.

3. A nucleic acid, characterized in that The nucleic acid is a nucleic acid encoding a TIF-IA polypeptide, the amino acid sequence of the TIF-IA polypeptide is shown in SEQ ID NO.1, and the nucleotide sequence of the nucleic acid is shown in SEQ ID NO.2.

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