Polypeptide and application of siRNA thereof in prevention and / or treatment of pulmonary arterial hypertension

Abstract

The invention provides a polypeptide and application of siRNA thereof in prevention and / or treatment of pulmonary arterial hypertension. The amino acid sequence of the polypeptide is as shown in SEQ ID NO: 2, the polypeptide is encoded by a part of nucleotide sequence of LINC02426 host gene, the polypeptide can improve the proliferation capacity of pulmonary artery smooth muscle cells and promote the smooth muscle cells to be converted from a contraction type to a synthetic type so as to cause pulmonary vascular remodeling, and the polypeptide is expected to serve as a new target of pulmonary hypertension diseases and can be used as a new target of the pulmonary hypertension diseases. And a novel medicine capable of reversing pulmonary vascular remodeling is developed. The invention further designs siRNA for inhibiting the expression of the polypeptide, and experiments prove that the siRNA can reduce the proliferation capacity of smooth muscle cells and inhibit the conversion of the cells from a contraction type to a synthetic type through endogenous knock-down of the polypeptide, so that pulmonary vascular remodeling is improved, and the pulmonary vascular remodeling is expected to be fundamentally inhibited or reversed. The survival rate of the pulmonary arterial hypertension patient is prolonged.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more specifically, to the use of a polypeptide and its siRNA in preventing and / or treating pulmonary hypertension. Background Art

[0002] Pulmonary hypertension (PH) is a progressive, extremely malignant and high-mortality pulmonary vascular disease with pulmonary vascular remodeling as its fundamental pathological feature. It is reported that the global prevalence of PH is about 1%, and the prevalence rate in the elderly population over 65 years old can reach 10%. Pulmonary hypertension has become one of the major diseases worldwide.

[0003] The pathogenesis of pulmonary hypertension is complex, and the key pathological feature is the irreversible remodeling of pulmonary blood vessels under the stimulation of genetic factors, hypoxia, inflammation, etc. Inflammatory response is the key to the vicious cycle formed by the interaction between immune cells and pulmonary artery smooth muscle cells, which ultimately leads to irreversible pathological pulmonary vascular remodeling. However, there is no drug that can effectively reverse pulmonary vascular remodeling in the clinical treatment of pulmonary hypertension. At present, the commonly used drugs for pulmonary hypertension are prostacyclin analogs or receptor agonists (such as treprostinil), endothelin receptor antagonists (such as macitentan), phosphodiesterase 5 inhibitors (such as sildenafil) and soluble guanylate cyclase agonists (such as riociguat). However, these drugs are mainly used for the dilation of pulmonary blood vessels and the relief of clinical symptoms of patients with pulmonary hypertension, and cannot fundamentally inhibit or reverse the problem of pathological vascular remodeling. Therefore, even with the use of these drugs, the 5-year survival rate of patients is less than 50%.

[0004] Therefore, exploring precise inflammatory regulation mechanisms and targets and finding new drugs that can reverse pulmonary vascular remodeling are of great significance for the clinical prevention and treatment of pulmonary hypertension. Summary of the invention

[0005] The purpose of the present invention is to provide a polypeptide and its siRNA for use in the prevention and / or treatment of pulmonary hypertension. The polypeptide is a novel polypeptide PEP14 encoded by the long non-coding RNA LINC02426 host gene, which can increase the proliferation ability of pulmonary arterial smooth muscle cells and promote the conversion of smooth muscle cells from contractile to synthetic type, thereby leading to pulmonary vascular remodeling. The PEP14 polypeptide can be used as a new target for pulmonary hypertension to develop new drugs that can reverse pulmonary vascular remodeling. Further, a siRNA that inhibits the expression of the polypeptide is designed for the polypeptide, and it is verified by experiments that the siRNA can reduce the proliferation ability of smooth muscle cells and inhibit the conversion of cells from contractile to synthetic type by endogenously knocking down the aforementioned polypeptide, thereby improving pulmonary vascular remodeling, and is expected to fundamentally inhibit or reverse pulmonary vascular remodeling.

[0006] Based on the above, the first object of the present invention is to provide an isolated polypeptide, the amino acid sequence of the polypeptide is shown in SEQ ID NO:2.

[0007] Preferably, the polypeptide is encoded by the host gene of long non-coding RNA LINC02426.

[0008] Another object of the present invention is to provide the use of the aforementioned polypeptide as a drug target in screening drugs for preventing and / or treating pulmonary arterial hypertension.

[0009] Another object of the present invention is to provide the use of the aforementioned polypeptide inhibitor in the preparation of a medicament for preventing and / or treating pulmonary arterial hypertension.

[0010] Preferably, the inhibitor of the polypeptide includes: siRNA or gene knockout vector of the gene encoding the polypeptide, or protein antibody of the polypeptide, or one or more of other inhibitors capable of inhibiting the expression of the polypeptide.

[0011] Another object of the present invention is to provide a siRNA for inhibiting the expression of the aforementioned polypeptide, wherein the siRNA comprises a sense chain and an antisense chain, wherein the nucleotide sequence of the sense chain is 5'-UUUUAUCUCCAAUUAAAGCUGdTdT-3', and the nucleotide sequence of the antisense chain is 5'-GCUUUAAUUGGAGAUAAAAGCdTdT-3'.

[0012] Another object of the present invention is to provide a recombinant vector comprising a sequence capable of transcribing the aforementioned siRNA.

[0013] Another object of the present invention is to provide a recombinant cell, wherein the recombinant cell contains the aforementioned recombinant vector.

[0014] Another object of the present invention is to provide the use of the aforementioned siRNA, or the aforementioned recombinant vector, or the aforementioned recombinant cell in the preparation of a drug for preventing and / or treating pulmonary hypertension.

[0015] Optionally, the drug further comprises a pharmaceutically acceptable carrier.

[0016] Compared with the prior art, the beneficial effects of the present invention include at least:

[0017] 1. The present invention, through proteomics analysis, identified for the first time a new isolated polypeptide, called PEP14, with an amino acid sequence of MTEIKLSALIGDKS. Experiments have also shown that the PEP14 polypeptide can activate the Sting pathway, trigger an inflammatory response, and can also increase the proliferation of pulmonary artery smooth muscle cells, and promote the conversion of pulmonary artery smooth muscle cells from a contractile type to a synthetic type, thereby leading to pulmonary vascular remodeling and promoting the progression of pulmonary hypertension. This suggests that the PEP14 polypeptide provided by the present invention is expected to be used as a new target for pulmonary hypertension and the development of new drugs that can reverse pulmonary vascular remodeling is of great significance for the clinical prevention and treatment of pulmonary hypertension.

[0018] 2. Furthermore, for the PEP14 polypeptide, the present invention designs and provides an siRNA for inhibiting the expression of the polypeptide, called si-PEP14, and experimentally verified that the si-PEP14 can reduce the proliferation ability of HPASMCs cells and inhibit the conversion of HPASMCs cells from a contractile type to a synthetic type by endogenous knockdown of PEP14, thereby improving pulmonary vascular remodeling, and is expected to fundamentally inhibit or reverse pulmonary vascular remodeling, improve the prevention and treatment effects of pulmonary arteries, and prolong the survival rate of patients with pulmonary hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the detection and analysis diagram after PEP14 was obtained; among them:

[0020] A is the mass spectrometry analysis of PEP14;

[0021] B is the HPLC purity analysis chart of PEP14.

[0022] Figure 2 This is the result of detecting cell proliferation by CCK8 in Example 1.

[0023] Figure 3 The results of the EdU experiment to detect cell proliferation in Example 1; wherein:

[0024] A is a picture taken with a fluorescence microscope; B is the numerical statistics of A.

[0025] Figure 4 The results of the cell scratch test in Example 1, wherein:

[0026] A is a picture taken during the scratch test; B is the numerical statistics of A.

[0027] Figure 5 The results of Western blotting in Example 1 to detect the expression levels of proliferation proteins PCNA, Cyclin D1 and collagen synthesis-related protein Col1a1, wherein:

[0028] A is the electrophoresis band diagram; B is the numerical statistics of A.

[0029] Figure 6 These are the cardiac ultrasound measurement results of the rats in each group in Example 1.

[0030] Figure 7 The following are the hemodynamic test results of rats in each group in Example 1.

[0031] Figure 8 The HE staining results of the pulmonary vessels of each group of rats in Example 1; wherein:

[0032] A is the HE-stained image; B and C are the numerical statistics of A.

[0033] Fig. 9 The HE staining results of myocardial tissues of rats in each group in Example 1 are shown in FIG. 1 ; wherein:

[0034] A is the HE staining image; B is the numerical statistics of A.

[0035] Fig.10 The Sirius red staining results of the heart tissues of the rats in each group in Example 1; wherein:

[0036] A is the Sirius red stained image; B is the numerical statistics of A.

[0037] Fig.11 The results of Western blotting for detecting the expression levels of Sting pathway-related proteins P-Sting, P-TBK1, and P-IRF3 in Example 1, wherein:

[0038] A is the electrophoresis band diagram; B is the numerical statistics of A.

[0039] Fig.12 This is the result of detecting cell proliferation by CCK8 in Example 2.

[0040] Fig.13 The results of the EdU experiment to detect cell proliferation in Example 2; wherein:

[0041] A is a picture taken with a fluorescence microscope; B is the numerical statistics of A.

[0042] Fig.14 The results of Western blotting in Example 2 for detecting the expression levels of proliferation protein Cyclin D1, PCNA and collagen synthesis-related protein Col1a1, wherein:

[0043] A is the electrophoresis band diagram; B is the numerical statistics of A.

[0044] In addition, the above Figure 9-11The DMSO group indicated that no MCT modeling was performed and no PEP14 was applied; the PEP14 group indicated that no MCT modeling was performed and PEP14 was applied; the MCT group indicated that MCT modeling was performed and PEP14 was not applied; the MCT+PEP14 group indicated that MCT modeling was performed and PEP14 was applied; Figure 12-14 In the figure, the Scramble group indicates that the cells were not transfected with si-PEP14, and the si-PEP14 group indicates that the cells were transfected with si-PEP14. DETAILED DESCRIPTION

[0045] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0046] As described in the background art, existing therapeutic drugs for pulmonary hypertension mainly act on pulmonary vasodilation and cannot fundamentally inhibit or reverse the problem of pathological pulmonary vascular remodeling, resulting in a 5-year survival rate of less than 50% for patients. Therefore, this field urgently needs to explore precise inflammation regulation mechanisms and targets, and find new drugs that can reverse pulmonary vascular remodeling to improve the treatment effect and survival rate of patients with pulmonary hypertension.

[0047] To achieve the above purpose, the present invention has identified a new isolated polypeptide, called PEP14, through proteomics analysis. The PEP14 polypeptide is encoded by a partial nucleotide sequence of the open reading frame of the long non-coding RNA LINC 02426 hostgene derived from human pulmonary artery smooth muscle cells, and has 14 amino acids. Crucially, the present invention has found that the PEP14 polypeptide can promote the proliferation of pulmonary artery smooth muscle cells and the conversion of pulmonary artery smooth muscle cells from contractile to synthetic, thereby leading to pulmonary vascular remodeling and promoting the progression of pulmonary hypertension, suggesting that it plays an important role in the phenotypic conversion of pulmonary artery smooth muscle cells. Furthermore, siRNA designed for the PEP14 polypeptide sequence can inhibit vascular inflammation and the conversion of pulmonary artery smooth muscle cells from contractile to synthetic, thereby improving pulmonary vascular remodeling, and is expected to fundamentally inhibit or reverse pulmonary vascular remodeling, improve the prevention and treatment effects of pulmonary arteries, and prolong the survival rate of patients with pulmonary hypertension.

[0048] Based on the above, the present invention first provides an isolated polypeptide, which is named PEP14 in the present invention, and the amino acid sequence of the PEP14 polypeptide is: MTEIKLSALIGDKS (SEQ ID NO: 2). In some embodiments, the PEP14 polypeptide can be synthesized in vitro; in other embodiments, it can also be endogenously expressed in human cells and encoded by a partial nucleotide sequence of the host gene open reading frame of the long non-coding RNA LINC 02426.

[0049] Further, based on the important discovery of the present invention that the PEP14 polypeptide is involved in the key pathological process of the occurrence and development of pulmonary hypertension, the present invention provides the use of the PEP14 polypeptide as a drug target in screening drugs for preventing and / or treating pulmonary hypertension, and the use of the inhibitor of the PEP14 polypeptide in preparing drugs for preventing and / or treating pulmonary hypertension. The polypeptide inhibitor includes: one or more of the siRNA or gene knockout vector of the polypeptide encoding gene, or the polypeptide protein antibody, or other inhibitors capable of inhibiting the expression of the polypeptide.

[0050] Furthermore, the present invention also provides a siRNA for inhibiting the expression of the PEP14 polypeptide, wherein the siRNA comprises a sense chain and an antisense chain, the nucleotide sequence of the sense chain is 5'-UUUUAUCUCCAAUUAAAGCUGdTdT-3', and the nucleotide sequence of the antisense chain is 5'-GCUUUAAUUGGAGAUAAAAGCdTdT-3'.

[0051] Also provided is a recombinant vector, the recombinant vector includes a sequence capable of transcribing the aforementioned siRNA, and a recombinant cell, the recombinant cell contains the aforementioned recombinant vector. "Recombinant vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In short, as long as they can replicate and be stable in the host, any plasmid and vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0052] Furthermore, the present invention also provides the use of the aforementioned siRNA, or the aforementioned recombinant vector, or the aforementioned recombinant cell in the preparation of a drug for preventing and / or treating pulmonary arterial hypertension.

[0053] In some embodiments, the drug further includes a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not necessary active ingredients themselves and are not overly toxic after administration. Suitable pharmaceutically acceptable carriers are well known to those of ordinary skill in the art. A full description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences. The pharmaceutically acceptable carrier in the composition may contain a liquid, such as water, phosphate buffer, Ringer solution, physiological saline, balanced salt solution, glycerol or sorbitol, etc. In addition, auxiliary substances may also be present in these carriers, such as lubricants, glidants, wetting agents or emulsifiers, pH buffer substances and stabilizers, such as albumin, etc.

[0054] The following will be combined with the attached Figures 1 to 14 And specific examples, detailed description of the acquisition of the PEP14 polypeptide of the present invention and its function and application.

[0055] Example 1: Sequence analysis and in vitro synthesis of PEP14 and its role in promoting the development of pulmonary hypertension

[0056] (I) PEP14 sequence analysis

[0057] The sequence of the host gene of LINC 02426 is as follows (SEQ ID NO: 1):

[0058]

[0059]

[0060] In the above sequence, the underlined bold part is the PEP14 nucleotide sequence. The sequence translated into amino acids is: MTEIKLSALIGDKS (SEQ ID NO: 2).

[0061] (II) In vitro synthesis of PEP14

[0062] A conventional solid phase peptide synthesis method was used to synthesize a peptide according to the amino acid sequence shown in SEQ ID NO: 2, and the accuracy and purity of the obtained peptide were analyzed by mass spectrometry and HPLC. Figure 1 As shown, mass spectrometry confirmed that the amino acid was correct and its molecular weight was 1505.77 ( Figure 1 A); the purity of the obtained polypeptide was 95.54% ( Figure 1 B) Dissolve in DMSO before use.

[0063] (III) PEP14 promotes the proliferation of human pulmonary artery smooth muscle cells (HPASMCs) and promotes the conversion of HPASMCs from contractile to synthetic

[0064] The effects of PEP14 obtained by the aforementioned solid phase peptide synthesis method on the proliferation and phenotypic differentiation of human pulmonary artery smooth muscle cells (HPASMCs) were evaluated experimentally.

[0065] 1. CCK8 assay to detect cell proliferation

[0066] Human HPASMCs cells in logarithmic growth phase were seeded in 24-well plates, with 1×10 5 After the cells were completely attached, different final concentrations (1 μM, 5 μM, 10 μM) of PEP14 peptide (dissolved in DMSO) were added to the experimental group under normoxic conditions, and the same volume of DMSO solvent was added to the control group under normoxic conditions and cultured for 24 h.

[0067] After the culture, the control group and the three experimental groups were added with 10-fold diluted CCK8 solution and incubated for 2 h, and the absorbance at 450 nm was measured using a microplate reader.

[0068] The results are as follows Figure 2 As shown, compared with the control group, the OD450 values ​​of the cell culture fluids of the three experimental groups of the present invention were significantly increased, that is, the cell density of the cell culture fluids of the experimental groups was significantly increased, indicating that the PEP14 polypeptide provided by the present invention promoted the proliferation of HPASMCs cells.

[0069] 2. EdU staining to detect cell proliferation

[0070] HPASMCs in logarithmic growth phase were seeded in 24-well plates, with 1×10 5 After the cells were completely attached, the experimental group was added with PEP14 polypeptide (dissolved in DMSO) at a final concentration of 5 μM under normoxic conditions, and the control group was added with an equal volume of DMSO solvent under normoxic conditions and cultured for another 24 h.

[0071] After the culture, the control group and the experimental group were treated as follows: 2×EdU solution was added for incubation for 2 h, then the cells were washed, 4% paraformaldehyde fixative was added to fix the cells, Triton X-100 was added to permeabilize the cells, and then EdU click reaction solution was added for 30 min. After washing the cells, Hoechst3342 was used to stain the nucleus for 10 min, and the cells were photographed after washing.

[0072] The results are as follows Figure 3 As shown in A and B, compared with the control group (DMSO), the fluorescence value of the cell culture fluid of the experimental group (PEP14) was significantly increased, indicating that the PEP14 polypeptide provided by the present invention promoted the proliferation of HPASMCs cells.

[0073] 3. Scratch assay to detect cell migration

[0074] HPASMCs in logarithmic growth phase were seeded in 6-well plates, with 1×10 5 After the cells are completely attached, use a 1000μL pipette tip to scratch the bottom of the plate vertically, wash twice with PBS and take pictures. After taking pictures, add serum-free medium containing equal amounts of DMSO and PEP14, and take pictures again after 24 hours of treatment. Use ImageJ to calculate the width of the scratch to evaluate cell migration.

[0075] The results are as follows Figure 4 As shown in A and B, compared with the control group, the scratch width of the experimental group was significantly reduced, indicating that the PEP14 polypeptide provided by the present invention can significantly improve the proliferation and migration ability of HPASMCs cells.

[0076] 4. WB detection of cell proliferation and phenotype conversion proteins

[0077] HPASMCs in logarithmic growth phase were seeded in 6-well plates, with 1×10 6 After the cells were completely attached, the experimental group was added with PEP14 polypeptide (dissolved in DMSO) at a final concentration of 5 μM under normoxic conditions, and the control group was added with an equal volume of DMSO solvent under normoxic conditions and cultured for another 48 h.

[0078] After the culture, the control group and the experimental group were treated as follows: the culture medium was discarded and PBS was added for washing twice; pre-cooled RIPA strong lysis buffer (protease inhibitors were added before use) was dripped, and the cells were collected by scraping and aspirated into a centrifuge tube. After lysis on ice for 10 min, the supernatant was collected by centrifugation at 12000 rpm; after BCA quantification, loading buffer was added, boiled at 100 °C for 10 min, and stored at -20 °C; the proteins were separated using 10% SDS-PAGE gel, transferred to a 2.5 μm pore size PVDF membrane, blocked with 5% skim milk at room temperature for 1.5 h, and incubated with specific primary antibodies overnight. The next day, the membranes were incubated with HRP-labeled secondary antibodies at room temperature for 1.5 h, and then developed using the A1600 color development system.

[0079] The results are as follows Figure 5 As shown in the figure, compared with the control group (DMSO), the expression levels of proliferation proteins Cyclin D1 and PCNA in the HPASMCs cells of the experimental group (PEP14) were significantly increased ( Figure 5 A), indicating that the PEP14 polypeptide provided by the present invention is beneficial to promoting the proliferation of HPASMCs cells; at the same time, the expression level of collagen synthesis-related protein Col1a1 in the experimental group cells also increased significantly, indicating that the PEP14 polypeptide provided by the present invention promotes the conversion of HPASMCs cells from contractile type to synthetic type, thereby leading to and aggravating pulmonary vascular remodeling.

[0080] (IV) PEP14 exacerbates the deterioration of pulmonary vascular remodeling in the MCT-induced PAH rat model

[0081] Animal experiments were further conducted to verify the role of the PEP14 polypeptide provided by the present invention in the occurrence and development of pulmonary hypertension.

[0082] Thirty 8-week-old male SD rats were randomly divided into 4 groups: blank control group (DMSO), drug control group (PEP14), MCT model group (MCT), and MCT drug group (MCT+PEP14). Among them, the blank control group and drug control group did not undergo MCT-induced modeling, while the MCT model group and MCT drug group underwent modeling treatment: rats were anesthetized with 3% isoflurane gas and given a single subcutaneous injection of monocrotaline (MCT) 60 mg / kg for four weeks to induce rat pulmonary hypertension model. PEP14 (25 mg / kg) was instilled once every 3 days after MCT injection for 1 day in the drug control group and MCT drug group until the end of the experiment.

[0083] After the experiment, the following tests were performed:

[0084] 1. Cardiac ultrasound measurement: After giving mice anesthesia with chloral hydrate, use High-resolution small animal ultrasound imaging system measures the ratio of pulmonary artery acceleration and ejection time in rats.

[0085] The results are as follows Figure 6 As shown, PEP14 promoted the increase of pulmonary artery acceleration and ejection time ratio in MCT-induced pulmonary hypertension rats and normal rats.

[0086] 2. Hemodynamic testing: After anesthetizing mice with chloral hydrate, carefully cut the chest wall to expose the heart, insert the tip of the 1.4F micro-pressure sensor catheter into the right ventricle, and use the PowerLab data acquisition system to stably and continuously monitor the right ventricular pressure value of the rat.

[0087] The results are as follows Figure 7 As shown, PEP14 promoted the increase in pulmonary artery pressure in MCT-induced pulmonary hypertension rats and normal rats.

[0088] 3. Histopathology: Rat lung tissues were fixed with 4% paraformaldehyde for 24 h, embedded in paraffin, sectioned, and then stained with H&E and Sirius red.

[0089] The results showed that PEP14 promoted pulmonary vascular thickening in MCT-induced pulmonary hypertension rats and normal rats ( Figure 8 AC), myocardial cell area increased ( Fig. 9 AB), and cardiac fibrosis ( Fig.10 AB).

[0090] (V) PEP14 can activate the Sting pathway and trigger inflammatory response

[0091] HPASMCs in logarithmic growth phase were seeded in 6-well plates, with 1×10 6After the cells were completely attached, the experimental group was added with PEP14 polypeptide (dissolved in DMSO) at a final concentration of 5 μM under normoxic conditions, and the control group was added with an equal volume of DMSO solvent under normoxic conditions and cultured for another 48 h.

[0092] After the culture was completed, the culture medium was discarded and PBS was added for washing twice; pre-cooled RIPA strong lysis buffer (protease inhibitors were added before use) was added, and the mixture was collected and aspirated into a centrifuge tube. After lysis on ice for 10 minutes, the supernatant was collected by centrifugation at 12000rpm; after BCA quantification, loading buffer was added, boiled at 100℃ for 10 minutes, and stored at -20℃. The proteins were separated using 10% SDS-PAGE gel, transferred to a 2.5μm pore size PVDF membrane, blocked with 5% skim milk at room temperature for 1.5 hours, and incubated with a specific primary antibody overnight. The next day, the HRP-labeled secondary antibody was incubated at room temperature for 1.5 hours, and the color was developed using the A1600 color development system.

[0093] The results are as follows Fig.11 As shown, compared with the control group (DMSO), the activities of various proteins in the Sting pathway (including P-Sting, P-TBK1, and P-IRF3) in the experimental group (PEP14) were significantly increased, which promoted the inflammatory response and led to pulmonary vascular remodeling.

[0094] The above results indicate that the PEP14 polypeptide provided by the present invention is involved in the key pathological process of the occurrence and development of pulmonary hypertension. PEP14 promotes inflammatory response, enhances the proliferation and migration ability of pulmonary artery smooth muscle cells, and promotes the phenotypic conversion of pulmonary artery smooth muscle cells from contractile to synthetic type, leading to pulmonary vascular remodeling and deterioration.

[0095] Example 2: Endogenous knockdown of PEP14 can inhibit the proliferation of HPASMCs and inhibit the conversion of HPASMCs cells from contractile to synthetic

[0096] 1. Design and synthesis of si-PEP14

[0097] The siRNA targeting the PEP14 sequence was designed and named si-PEP14 in the present invention, and the targeting sequence was: ATGACAGAGATCAAGCTGTCAGCTTTAATTGGAGATAAAAGCTAA.

[0098] The sense strand sequence of si-PEP14 is: 5′-UUUUAUCUCCAAUUAAAGCUGdTdT-3′ (SEQ ID NO: 3);

[0099] The antisense sequence of si-PEP14 is 5′-GCUUUAAUUGGAGAUAAAAGCdTdT-3′ (SEQ ID NO: 4).

[0100] In the above two sequences, dT represents deoxythymine.

[0101] The RNA sequence was entrusted to a sequence synthesis company for chemical synthesis and subsequent purification to obtain si-PEP14 dry powder. The si-PEP14 dry powder was centrifuged to the bottom of the tube, 1OD was added with 125μL DEPC water to fully dissolve, and gently pipetted to mix to prepare a 20μM stock solution, which was stored at -20℃ after aliquoting.

[0102] 2. si-PEP14 transfection of pulmonary artery smooth muscle cells

[0103] HPASMCs in logarithmic growth phase were seeded in 24-well plates, with 1×10 5 After culturing cells overnight, transfection was performed when the cell density was between 50% and 60%.

[0104] Add 50 pmoles of si-PEP14 to 200 μL of jetPRIME buffer, gently invert the tube to mix, and then centrifuge; add 4 μL of transfection reagent to the system. transfection reagent), gently blow several times with a pipette to mix, and let it stand after slight centrifugation; after incubation at room temperature for 10-15 minutes, the growth medium was aspirated and the medium containing 5% FBS was added; after the incubation, the transfection system was slowly dripped into the medium and placed in an incubator for continued cultivation; after 6 hours of transfection, the cell state was observed, the medium containing the transfection reagent was aspirated and discarded, and fresh medium was added for continued cultivation for 36 hours to complete the transfection culture of si-PEP14.

[0105] 3. si-PEP14 inhibits the proliferation of HPASMCs cells and inhibits the transformation of HPASMCs cells from contractile to synthetic

[0106] The human pulmonary artery smooth muscle cells (HPASMCs) cultured with si-PEP14 transfection were further cultured and CCK8, EdU and Western Blot detection were performed as follows:

[0107] 3.1 CCK8 detection of cell proliferation

[0108] The HPASMCs that had completed si-PEP14 transfection and culture were divided into two groups: a normoxic group and a hypoxic group, and were cultured under normoxic and hypoxic conditions for 24 hours, respectively. At the same time, HPASMCs that had not undergone si-PEP14 transfection and culture were included as a control group (Scramble), which was also divided into two groups and cultured under normoxic and hypoxic conditions, respectively. After the culture, 10-fold diluted CCK8 solution was added and incubated for 2 hours, and the absorbance at 450nm was measured using an enzyme reader.

[0109] The results are as follows Fig.12 As shown, compared with the control group cultured under normoxic conditions, the cell density (OD450 value) of the control group cultured under hypoxic conditions increased significantly, indicating that hypoxic conditions promote the proliferation of HPASMCs cells and lead to pulmonary vascular remodeling. The si-PEP14 provided by the present invention can reduce the proliferation ability of HPASMCs cells cultured under normoxic / hypoxic conditions by endogenously knocking down PEP14.

[0110] 3.2 EdU detection of cell proliferation

[0111] HPASMCs that completed si-PEP14 transfection and culture were divided into two groups: normoxic group and hypoxic group, and were cultured under normoxic and hypoxic conditions for 24 hours, respectively. At the same time, HPASMCs that were not transfected with si-PEP14 were included as the control group (Scramble), which was also divided into two groups and cultured under normoxic and hypoxic conditions, respectively. After the culture, 2×EdU solution was added and incubated for 2 hours, then the cells were washed, 4% paraformaldehyde fixative was added to fix the cells, Triton X-100 was added to permeabilize the cells, and then EdU click reaction solution was added to react for 30 minutes. After washing the cells, Hoechst3342 was used to stain the nucleus for 10 minutes, and the cells were photographed after washing.

[0112] The results are as follows Fig.13 As shown in A and B, by comparing the fluorescence detection results of the two control groups, it can be seen that hypoxic conditions significantly promoted the proliferation of HPASMCs cells, leading to pulmonary vascular remodeling. However, after transfection with si-PEP14, the fluorescence value of the cell culture fluid of the experimental group (si-PEP14) was significantly lower than that of the control group (Scramble) under the same culture conditions (hypoxia or normoxia), indicating that the si-PEP14 provided by the present invention can reduce the proliferation ability of HPASMCs cells by endogenously knocking down PEP14.

[0113] 3.3WB detection of cell proliferation and phenotypic conversion proteins

[0114] HPASMCs after transfection and culture were divided into two groups: normoxic group and hypoxic group, which were cultured under normoxic and hypoxic conditions for 36 hours respectively. HPASMCs without si-PEP14 transfection and culture were included as control group (Scramble), which were also divided into two groups and cultured under normoxic and hypoxic conditions respectively. After culture, the culture medium was discarded and washed twice with PBS. Pre-cooled RIPA strong lysis buffer (protease inhibitors were added before use) was dripped, and the cells were collected by cell scraping and aspirated into a centrifuge tube. After lysis on ice for 10 minutes, the supernatant was collected by centrifugation at 12000rpm. After BCA quantification, loading buffer was added, boiled at 100℃ for 10 minutes, and stored at -20℃. The proteins were separated using 10% SDS-PAGE gel, transferred to a 2.5μm pore size PVDF membrane, blocked with 5% skim milk at room temperature for 1.5 hours, and incubated with specific primary antibodies overnight. The next day, the HRP-labeled secondary antibody was incubated at room temperature for 1.5 hours, and the color was developed using the A1600 color development system.

[0115] The results are as follows Fig.14 As shown in A and B, by comparing the WB test results of the two control groups, it can be seen that hypoxic conditions significantly increased the expression of cell proliferation proteins Cyclin D1 and PCNA and the expression of collagen synthesis-related protein Col1a1, that is, promoted the proliferation of HPASMCs cells, and promoted the conversion of HPASMCs cells from contractile to synthetic, thereby leading to pulmonary vascular remodeling. However, after transfection with si-PEP14, the expression levels of Cyclin D1, PCNA and Col1a1 of the experimental group (si-PEP14) cells were significantly lower than those of the control group (Scramble) under the same culture conditions (hypoxia or normoxia), indicating that the si-PEP14 provided by the present invention can reduce the proliferation ability of HPASMCs cells by endogenously knocking down PEP14, and inhibit the conversion of HPASMCs cells from contractile to synthetic, thereby improving pulmonary vascular remodeling.

[0116] In summary, the PEP14 provided by the present invention can improve the proliferation capacity of human pulmonary artery smooth muscle cells and promote the conversion of smooth muscle cells from contractile to synthetic type, thereby leading to pulmonary vascular remodeling, and the polypeptide is expected to be used as a new target for pulmonary hypertension disease, and develop a new drug that can reverse pulmonary vascular remodeling. Further, the siRNA (si-PEP14) designed for the polypeptide can reduce the proliferation capacity of smooth muscle cells by endogenous knockdown of PEP14, and inhibit the conversion of smooth muscle cells from contractile to synthetic type, thereby improving pulmonary vascular remodeling, and is expected to fundamentally inhibit or reverse pulmonary vascular remodeling and prolong the survival rate of patients with pulmonary hypertension.

[0117] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. An isolated polypeptide, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO:

2.

2. The isolated polypeptide according to claim 1, characterized in that The polypeptide is encoded by the host gene of long non-coding RNA LINC02426.

3. Use of the polypeptide according to claim 1 or 2 as a drug target in screening drugs for preventing and / or treating pulmonary arterial hypertension.

4. Use of the polypeptide inhibitor according to claim 1 or 2 in the preparation of a medicament for preventing and / or treating pulmonary arterial hypertension.

5. The use according to claim 4, characterized in that The inhibitor of the polypeptide includes: siRNA or gene knockout vector of the gene encoding the polypeptide, or protein antibody of the polypeptide, or one or more of other inhibitors capable of inhibiting the expression of the polypeptide.

6. An siRNA for inhibiting the expression of the polypeptide according to claim 1, characterized in that: The siRNA comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand is 5'-UUUUAUCUCCAAUUAAAGCUGdTdT-3', and the nucleotide sequence of the antisense strand is 5'-GCUUUAAUUGGAGAUAAAAGCdTdT-3'.

7. A recombinant vector, characterized in that: The recombinant vector comprises a sequence capable of transcribing the siRNA according to claim 6.

8. A recombinant cell, characterized in that The recombinant cell contains the recombinant vector according to claim 7.

9. Use of the siRNA according to claim 6, or the recombinant vector according to claim 7, or the recombinant cell according to claim 8 in the preparation of a medicament for preventing and / or treating pulmonary arterial hypertension.

10. The use according to claim 9, characterized in that The medicament further includes a pharmaceutically acceptable carrier.

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