Application of Pxdc1 gene in regulation and control of exosome secretion

Abstract

The invention relates to the technical field of biomedicine, in particular to application of a Pxdc1 gene in regulation and control of exosome secretion. The key effect of Pxdc1 in induction of cell exosome formation and secretion is disclosed for the first time, and a new strategy is provided for treatment of cardiovascular diseases. By constructing Pxdc1 overexpression plasmids or virus vectors, secretion of exosomes in 293T cells and primary rat myocardial cells is successfully promoted, and a foundation is laid for preparing reagents or drugs for regulating and controlling exosome secretion. Meanwhile, RNA interference is utilized to reduce Pxdc1 expression, exosome secretion is effectively inhibited, and the core regulation effect of Pxdc1 is further verified. The invention not only deepens the understanding of the Pxdc1 gene function, but also provides an innovative thought for the cell-free therapy and drug delivery system of cardiovascular diseases, and has important scientific value and application potential.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of the Pxdc1 gene in regulating exosome secretion. Background Art

[0002] Extracellular vesicles (EVs), especially exosomes, as key mediators of intercellular communication, have occupied an increasingly important position in biomedical research. Exosomes are formed from multivesicular endosomes (MVEs) inside cells and are released into the extracellular environment by fusing with the cell membrane. These tiny lipid bilayer membrane particles carry a variety of bioactive molecules, such as proteins, nucleic acids, and lipids, thereby transmitting information between cells and participating in various physiological and pathological processes.

[0003] In recent years, with the in-depth study of exosomes, people have gradually recognized their great potential in disease diagnosis, treatment, and regenerative medicine. However, despite the broad functions and application prospects of exosomes, the fine regulatory mechanism of their secretion is still poorly understood. In particular, which genes are involved in the regulation of exosome secretion, and how these genes affect the generation, secretion, and function of exosomes, remain the hotspots and difficulties in current research. In clinical fields such as cardiovascular diseases, the progress of traditional treatment methods is slow. In particular, the treatment effect of heart failure drugs is not good, and the clinical trials of myocardial protective drugs have also frequently failed. Therefore, finding new therapeutic targets and drug carriers to improve the treatment effect and quality of life of patients has become an urgent need in current medical research. Exosomes, due to their unique properties and functions, have become an ideal choice for potential new therapeutic agents and drug carriers. However, to achieve this goal, it is first necessary to deeply understand the regulatory mechanism of exosome secretion.

[0004] Currently, although some studies have begun to explore the influence of genes on exosome secretion, most of these studies focus on known genes related to exosome generation and secretion, and the exploration of other new genes that may be involved in this process is still insufficient. Therefore, discovering new genes that regulate exosome secretion and deeply studying their mechanisms of action are of great significance for promoting the application of exosomes in the biomedical field. Although exosomes show great potential in intercellular communication and disease treatment, the fine regulatory mechanism of their secretion, especially the discovery of new regulatory genes and the study of their mechanisms of action, still face many challenges and unknowns. Summary of the Invention

[0005] In view of this, the present invention proposes the application of the Pxdc1 gene in regulating exosome secretion.

[0006] The technical solution of the present invention is realized as follows:

[0007] In a first aspect, the present invention provides the use of the Pxdc1 gene in regulating exosome secretion, and said use is not for the purpose of disease treatment.

[0008] In some specific embodiments, the Pxdc1 gene is of human or rat origin; the full-length cDNA sequence of human Pxdc1 has the ID of NM_183373.4 in the NCBI database; the full-length cDNA sequence of rat-derived Pxdc1 has the ID of NM_001025719.1 in the NCBI database.

[0009] In some specific embodiments, overexpression of the Pxdc1 gene promotes exosome secretion.

[0010] In some specific embodiments, overexpression of the Pxdc1 gene is achieved by adenovirus infection of primary rat cardiomyocytes. In other specific embodiments, overexpression of the Pxdc1 gene is achieved by plasmid transfection of 293T cells.

[0011] In some specific embodiments, knockdown of the expression of the Pxdc1 gene inhibits exosome secretion. Further, knockdown of the expression of the Pxdc1 gene is achieved by an RNA interference molecule targeting the Pxdc1 gene, and said RNA interference molecule includes at least one of siRNA, shRNA, and miRNA mimics.

[0012] In a second aspect, the present invention provides the use of the Pxdc1 gene as a target in the preparation of a reagent for regulating exosome secretion.

[0013] In some specific embodiments, the Pxdc1 gene is of human or rat origin; the full-length cDNA sequence of human Pxdc1 has the ID of NM_183373.4 in the NCBI database; the full-length cDNA sequence of rat-derived Pxdc1 has the ID of NM_001025719.1 in the NCBI database. The reagent regulates exosome secretion through the following route (1) or (2): (1) promoting exosome secretion by upregulating the expression of the Pxdc1 gene; (2) inhibiting exosome secretion by knocking down the expression of the Pxdc1 gene.

[0014] In a third aspect, the present invention provides a method for regulating exosome secretion in vitro. In cells cultured in vitro, exosome secretion is regulated through the following route (1) or (2): (1) promoting exosome secretion by upregulating the expression of the Pxdc1 gene; (2) inhibiting exosome secretion by knocking down the expression of the Pxdc1 gene.

[0015] The beneficial effects of the present invention at least include the following:

[0016] The present invention discloses the key role of Pxdc1 in inducing the formation and secretion of extracellular exosomes. The present invention firstly proposes the Pxdc1 gene as a brand-new molecular action target, and by precisely regulating the molecular function of this gene, it can effectively regulate the secretion level of exosomes in 293T cells and primary rat ventricular myocytes (Neonatal Rat Ventricular Myocytes, NRVMs). This discovery provides an innovative clinical strategy for cell-free therapy and drug delivery systems for cardiovascular diseases.

[0017] By means of constructing plasmids or viral vectors with overexpressed Pxdc1, etc., the present invention realizes the overexpression of the Pxdc1 gene in cells, and further promotes the formation and secretion of exosomes; it lays a solid foundation for preparing reagents or drugs capable of regulating exosome secretion. On the other hand, the present invention also explores a method for reducing the expression level of the Pxdc1 gene through gene knockdown technology, successfully inhibits the secretion of exosomes, and further verifies the core role of Pxdc1 in exosome regulation. The present invention not only deepens the understanding of the function of the Pxdc1 gene and its mechanism of action in the regulation of exosome secretion in cells, but also opens up a new research direction for the field of cardiovascular disease treatment, and has great scientific value and application prospects. Brief Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 This is the change of overexpressing Pxdc1 in 293T cells in the embodiments of the present invention, including the following:

[0020] Figure 1 a shows the results of detecting the mRNA level of overexpressed Pxdc1 in 293T cells by qRT-PCR technology;

[0021] Figure 1 b shows the results of detecting the protein level of 293T cells after overexpressing Pxdc1 by Western blot method, and the right figure is the quantitative analysis;

[0022] Figure 1 c shows the influence of overexpressing Pxdc1 in 293T cells on the expression levels of exosome secretion-related markers in whole cell lysate (WCL) and exosomes detected by Western blot;

[0023] Figure 2 The changes in overexpression of Pxdc1 in primary rat ventricular myocytes (NRVMs) in the embodiments of the present invention are as follows:

[0024] Figure 2 a shows the results of detecting the mRNA level of overexpressed Pxdc1 in NRVMs cells by qRT-PCR technology;

[0025] Figure 2 b shows the results of detecting the protein level in NRVMs cells after overexpression of Pxdc1 by Western blot method, and the right figure is the quantitative analysis;

[0026] Figure 2 c shows the effect of overexpression of Pxdc1 in NRVMs cells on the expression levels of exosome secretion-related markers in whole cell lysate (WCL) and exosomes detected by Western blot;

[0027] Figure 3 The changes in knockdown of Pxdc1 by siRNA in primary rat ventricular myocytes (NRVMs) in the embodiments of the present invention are as follows:

[0028] Figure 3 a shows the results of detecting the mRNA level of Pxdc1 knocked down by siRNA in NRVMs cells by qRT-PCR technology;

[0029] Figure 3 b shows the results of detecting the protein level in NRVMs cells after knockdown of Pxdc1 by siRNA by Western blot method, and the right figure is the quantitative analysis;

[0030] Figure 3 c shows the effect of knockdown of Pxdc1 by siRNA in NRVMs cells on the expression levels of exosome secretion-related markers in whole cell lysate (WCL) and exosomes detected by Western blot. Detailed implementation manners

[0031] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention. For those conditions not specified in the embodiments, they shall be carried out according to the conventional conditions or the conditions recommended by the manufacturer. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0032] The methods used in the following examples are all conventional methods unless otherwise specified. For specific steps, please refer to: 《Molecular Cloning: A Laboratory Manual》(Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor). The acquisition routes of various biological materials described in the examples are only provided as an experimental acquisition route to achieve the specific disclosure purpose, and should not be a limitation on the source of biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the prompts in the examples. The genes, proteins or their fragments involved in the present invention can be natural purified products, chemically synthesized products, or products produced using recombinant techniques from prokaryotic or eukaryotic hosts (such as bacteria, yeast, plants, etc.).

[0033]

Glossary Explanation

[0034] In some specific embodiments of the present invention, the meanings of relevant terms are as follows:

[0035] 293T cells: The 293T cell line is derived from human embryonic kidney cells HEK-293 and has been inserted with the temperature-sensitive gene of SV40 T-antigen through genetic engineering technology, thus obtaining the characteristic of high transfection efficiency. 293T cells carry the SV40 replication origin site (SV40-ORI), can autonomously replicate and amplify plasmids, and significantly increase the copy number of exogenous gene expression vectors and the yield of target proteins. In addition, this cell line has the transfection characteristic of adenovirus E1A gene, so it is widely used in fields such as virus vector packaging, signal pathway research, and drug screening.

[0036] Primary rat ventricular myocytes (NRVMs): NRVMs are isolated from rat heart tissue by the combined digestion method of collagenase-trypsin and differential adhesion method, and belong to terminally differentiated cells. Under in vitro culture conditions, NRVMs can maintain physiological characteristics such as spontaneous rhythmic beating. According to functional classification, NRVMs include working cardiomyocytes and conductive cardiomyocytes. The former constitutes the main body of the heart wall and is responsible for mechanical contraction function, while the latter participates in electrical signal transmission to coordinate heart beating. Primary rat ventricular myocytes are mainly used to study problems related to the mechanisms of cardiovascular diseases.

[0037] The Pxdc1 gene, also known as C6orf145. In humans (Homo sapiens), the Pxdc1 gene is characterized by encoding PX domain-containing protein 1. This gene exhibits genetic diversity, specifically manifested as having 4 transcripts and 214 orthologous genes. In the human gene bank, the full-length cDNA sequence hPxdc1, with the GenBank reference sequence number NM_183373.4, corresponds to the mRNA transcript of the Pxdc1 gene and is a linear mRNA, with a total length of 1878 base pairs (bp).

[0038] Overexpression: Increasing the expression level of a specific gene in cells through genetic engineering techniques. In some specific embodiments of the present invention, it is achieved by constructing an overexpression vector (such as a plasmid or viral vector) containing the target gene and introducing it into cells.

[0039] Adenovirus infection: A method of introducing foreign genes into cells using adenovirus as a vector. Adenovirus is an enveloped DNA virus that can efficiently transfer genes into various types of cells.

[0040] Knockdown: Knockdown refers to reducing the expression level of a specific gene in cells through gene silencing techniques. In some specific embodiments of the present invention, it is achieved through RNA interference (RNAi) technology, including the use of small interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA) mimics, etc.

[0041] Sequence information table

[0042]

[0043]

[0044] In the embodiments of this application, all animal experimental procedures were reviewed and approved by the Animal Ethics Committee of Shenzhen Hospital of Fuwai Hospital, Chinese Academy of Medical Sciences, and were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (8th Edition) published by the National Institutes of Health (USA).

[0045] I. Experimental methods

[0046] 1. Isolation and culture of primary rat ventricular myocytes (NRVMs)

[0047] Disinfect the chest skin of neonatal SD rats (within 3 days after birth, purchased from Zhuhai Beston Biotechnology Co., Ltd.) with 75% (v / v) ethanol. Use curved scissors to cut open the chest skin and ribs, and use curved forceps to take out the heart, and place it in a petri dish (diameter 6 mm) containing ADS buffer (NaCl 120 mM, HEPES (pH 7.4) 20 mM, NaH2PO4 8 mM, glucose 6 mM, KCl 5 mM, MgSO4 0.8 mM) placed on ice. Remove the large blood vessels, auricles and atria attached to the surface of the heart, and cut the ventricular tissue into pieces of 1 mm 2 . Add a digestive solution containing 0.08% (w / v) type II collagenase (Sigma) and 0.125% (w / v) trypsin (Sigma) to the minced ventricular tissue, and digest at 37 °C for 20 min each time. Discard the supernatant for the first time and start collecting the supernatant from the second time. This process continues until the heart tissue is completely digested. Use Percoll (GE Healthcare) density gradient centrifugation to separate and remove fibroblasts, and culture cardiomyocytes in high-glucose DMEM (Hyclone) medium containing 10% (v / v) fetal bovine serum (Gbico) and 1% (v / v) penicillin / streptomycin. After culturing for 24 h, change the medium to high-glucose DMEM medium containing 1% (v / v) ITS supplement, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0048] 2. Plasmid construction

[0049] The full-length cDNA sequence of human Pxdc1, hPxdc1 (NM_183373.4), and the full-length cDNA sequence of rat-derived Pxdc1, rPxdc1 (NM_001025719.1), were obtained from the National Center for Biotechnology Information (NCBI) database. According to the conventional methods in the field of molecular biology, the entire coding regions of human Pxdc1 and rat-derived were cloned into the NheI and XhoI sites of the pcDNA3.1-3xFLAG plasmid to construct the recombinant plasmids pcDNA3.1-hPxdc1-3xFLAG and pcDNA3.1-pcDNA3.1-rPxdc1-3xFLAG.

[0050] The cDNA fragments of mature hPxdc1 and rPxdc1 were amplified using primers hPxdc1 CZF and hPxdc1 CZR as well as rPxdc1 CZF and rPxdc1 CZR (sequences are shown in the sequence information table). The pcDNA3.1-hPxdc1-3xFLAG and pcDNA3.1-rPxdc1-3xFLAG were transferred into Escherichia coli DH-5α by conventional methods in the art for large-scale replication of the plasmids. To overexpress Pxdc1 in NRVMs, the pcDNA3.1-rPxdc1-3xFLAG plasmid was then sent to Shanghai Hanheng Biotechnology Co., Ltd. (Project No.: HH20240803WHZL-ADP01) for viral packaging. An adenovirus vector containing an empty plasmid was used as a control.

[0051] Overexpression of human Pxdc1 in 293T cells. The plasmid transfection steps are as follows: First, 293T cells were cultured in a six-well plate and transfected when the cell density reached 70-80%. According to the manufacturer's instructions, JetPRIME transfection reagent was used for transfection, and the transfection ratio was IetPRIME: plasmid = 6 (ul / well): 3 (ug / well). After 12 hours of transfection, the culture medium was replaced with high-glucose DMEM medium containing 10% FBS and 1% penicillin / streptomycin to remove the plasmid. After continuing to culture for 36 hours, the cells were harvested.

[0052] 3. Adenovirus infection of primary rat cardiomyocytes (NRVMs)

[0053] In the overexpression experiment of NRVMs, Pxdc1 was overexpressed by infecting NRVMs with adenovirus. The steps of virus infection are as follows: First, the cells were cultured in six-well plates. After 24 hours of serum-free culture of NRVMs, 1 μL of virus was added to each well of the six-well plate according to the optimal amount of virus used in the preliminary experiment. After 12 hours of virus infection of NRVMs, the culture medium was replaced with high-glucose DMEM medium containing 1% (v / v) ITS supplement and 100U / mL penicillin and 100μg / mL streptomycin to remove the virus. After continuing to culture for 36 hours, the cells were harvested.

[0054] 4. siRNA knockdown of Pxdc1 in NRVMs cells

[0055] The expression of certain genes in cells was inhibited by siRNA transfection. The rPxdc1-specific siRNA oligonucleotides were purchased from GenePharma. The target sequences of the siRNAs used are shown in SEQ ID NO: 5 - 6. According to the manufacturer's instructions, siRNA transfection was performed using Lipofectamine iMAX (Invitrogen) at a transfection ratio of iMAX:siRNA = 3:2. After 12 h of transfection of NRVMs, the culture medium was replaced with high-glucose DMEM medium containing 1% ITS and 1% penicillin / streptomycin to remove the siRNA. After continued culture for 36 h, the cells were harvested.

[0056] 5. Exosome collection by ultracentrifugation

[0057] After transfection, the cells were cultured in serum-free cell medium for 36 h. The culture medium was collected, centrifuged at 500 g at low temperature for 10 min to remove the precipitate, and the supernatant was centrifuged at 20000 g at low temperature for 20 min to remove the precipitate again. The collected supernatant was centrifuged at 140000 g at low temperature for 70 min, and the exosomes sedimented at the bottom of the tube. The exosomes were resuspended in 20 mL of PBS solution, centrifuged at 140000 g at low temperature for 70 min for washing, and finally resuspended in an appropriate amount of PBS and stored at -20 °C in the refrigerator for later use.

[0058] 6. Western blot

[0059] Total cell lysates (WCL) were extracted from cells. Cells were lysed in RIPA lysis buffer (Beyotime, Nanjing, China). The cell lysates were centrifuged at 12,000 g for 15 mins at 4°C. Protein concentrations were determined using a BCA protein quantification kit, and equal amounts of protein extracts were subjected to gel electrophoresis (SDS-PAGE) for protein separation and then transferred to PVDF membranes. The PVDF membranes were blocked with 5% skim milk for 1 h at room temperature and then incubated overnight with specific primary antibodies at 4°C. The next day, after washing three times with TBST, the membranes were incubated with secondary antibodies for 1 h at room temperature and then washed three times with TBST. Finally, protein bands on the membranes were detected using a chemiluminescent reagent (Beyotime). Chemiluminescent signals were quantified using an ECL imager and analyzed using QuantityOne software (Bio-Rad, Hercules). The specific primary antibodies were: anti-Pxdc1 (customized from Nanjing Genscript Biotech Co., Ltd.), anti-ALIX (Proteintech, #12422-1-AP), anti-TSG101 (ATLAS, #HPA006161), anti-FLOT2 (CST, #3436), anti-CD81 (Proteintech, #sc-166029), anti-GAPDH (Proteintech, #HRP-60004).

[0060] 7. qRT-PCR detection

[0061] Total RNA was extracted from 293T and NRVMs cells using an RNA extraction solution (Servicebio) according to the manufacturer's instructions.

[0062] RNA was quantified using NanoDrop (Thermo Fisher Scientific). Cells were washed twice with PBS buffer for 3 minutes each time, then Trizol was added. The cells were scraped off the six-well plate using a cell scraper; left standing at room temperature for 5 minutes, pre-chilled chloroform at 4°C was added, shaken vigorously for 30 seconds, left standing at room temperature for 3 minutes, then centrifuged at 12,000 rpm at 4°C for 15 minutes; the liquid stratified, with the upper aqueous layer and the lower organic layer. The upper aqueous layer was carefully aspirated, an equal volume of isopropanol was added, gently mixed, left standing at room temperature for 10 minutes, then centrifuged at 12,000 rpm at 4°C for 60 minutes; RNA formed a precipitate and sank to the bottom of the tube. 75% alcohol prepared with DEPC water was added for one wash, then centrifuged at 12,000 rpm at 4°C for 10 minutes; the 75% alcohol was aspirated off, left standing at room temperature to dry, an appropriate amount of DEPC water was added to dissolve the RNA, and the concentration was measured using a Nanodrop instrument. RNA was reverse transcribed into cDNA using a reverse transcription kit (RevertAid First Strand cDNA Synthesis kit). The reaction system was as follows: 5×Reaction Buffer 2 μL, 10 mM dNTP Mix 1 μL, Random Primer 1 μL, RiboLock RNase Inhibitor 0.5 μL, RevertAid M-MuLV RT 0.5 μL, RNA 1 μg, nuclease-free Water was made up to 10 μL; PCR reaction: 25°C for 5 minutes; 42°C for 60 minutes; 70°C for 5 minutes. Real-time fluorescence quantitative PCR was performed using specific primers and ChamQ SYBR qPCR Master Mix (Vazyme) on a QuantStudio 7 Pro detection instrument (Thermo Fisher Scientific). The reaction system for qRT-PCR was as follows:

[0063] 2×SYBR Green Master MIX 5 μL Water 3 μL Forward Primer 0.5 μL Reserve Primer 0.5 μL cDNA 1 μL

[0064] First, the reaction system except for cDNA was added to a 384-well plate, then 1 μL of cDNA was added to the corresponding wells, and the 384-well plate was sealed with a transparent film. After centrifuging the 384-well plate at 1000 rpm at room temperature for 1 min, it was placed in a real-time quantitative PCR instrument for detection. The reaction program was as follows:

[0065]

[0066]

[0067] After the reaction, the specificity of the primers was judged by the melting curve, and the content of the target gene was calculated from the Ct value. The sequences of the PCR primers used in this application are shown in the sequence information table. Using hGapdh and rActb as endogenous controls, the relative expression levels of the genes were measured.

[0068] 8. Statistical analysis

[0069] Statistical analysis was performed using GraphPad Prism 8 software. All experimental data were expressed as the mean ± SEM of at least three independent experiments. One-way ANOVA or two-way ANOVA was used for the statistical significance of multiple comparisons, followed by Tukey's test. Bonferroni adjustment was used for post hoc analysis. The t-test was used for comparisons between two groups. P < 0.05 was considered statistically significant.

[0070] II. Experimental results

[0071] 1. Detection of the mRNA level of overexpressed Pxdc1 in 293T cells by qRT-PCR ( Figure 1 a)

[0072] In this example, qRT-PCR technology was used to detect the mRNA level of overexpressed Pxdc1 in 293T cells. Among them, oe-Vector represents the control group transfected with the empty vector, and oe-Pxdc1 represents the experimental group transfected with the Pxdc1 plasmid. The ordinate represents the expression level of Pxdc1 relative to Gapdh, and the data show that the Pxdc1 mRNA level in the experimental group was significantly higher than that in the control group (*** indicates P < 0.001).

[0073] 2. Detection of the protein level of overexpressed Pxdc1 in 293T cells by Western blot ( Figure 1 b)

[0074] The Western blot method was used to detect the protein level after overexpression of Pxdc1 in 293T cells, with Gapdh as the internal reference. Figure 1 The right graph (bar graph) in b shows the quantitative analysis of the relative protein expression levels. The results show that the expression level of Pxdc1 protein in the experimental group was significantly higher than that in the control group (*** indicates P < 0.001).

[0075] 3. Detection of the effect of overexpressed Pxdc1 on exosome secretion in 293T cells by Western blot ( Figure 1 c)

[0076] Western blot was used to detect the levels of related proteins in whole cell lysates (WCL) and exosomes of 293T cells overexpressing Pxdc1. The proteins detected included ALIX, TSG101, FLOT2, and CD81 (these four protein molecules are involved in the formation, secretion, and functional regulation of exosomes through different mechanisms; their expression levels can reflect the activity of exosome secretion and the purity of exosomes, and are also marker proteins of exosome biogenesis); Gapdh was used as an internal reference. The detection results showed that overexpression of Pxdc1 increased the expression of exosome-related markers, indicating that overexpression of Pxdc1 promoted exosome secretion.

[0077] 4. Detection of the mRNA level of adenovirus overexpressing Pxdc1 in NRVMs by qRT-PCR Figure 2 a)

[0078] In the detection results of the mRNA level of adenovirus overexpressing Pxdc1 in primary rat ventricular myocytes (NRVMs), Ad-Vector represents the control group transfected with the adenovirus empty vector, and Ad-Pxdc1 represents the experimental group transfected with the adenovirus overexpressing Pxdc1. The ordinate is the relative expression of Pxdc1 to Actb. The detection results showed that the Pxdc1 mRNA level in the experimental group was significantly higher than that in the control group (*** indicates P < 0.001).

[0079] 5. Detection of the protein level of adenovirus overexpressing Pxdc1 in NRVMs by Western blot Figure 2 b)

[0080] The protein level of adenovirus overexpressing Pxdc1 in NRVMs was detected by Western blot. Gapdh was used as an internal reference; the right bar graph is the quantitative analysis of the relative protein expression. The results showed that the expression level of Pxdc1 protein in the experimental group was higher than that in the control group (*** indicates P < 0.001).

[0081] 6. Detection of the effect of adenovirus overexpressing Pxdc1 in NRVMs on exosome secretion Figure 2 c)

[0082] The effect of overexpressing Pxdc1 by adenovirus on exosome secretion in NRVMs was detected by Western blot. The figure shows the expression of related proteins (ALIX, TSG101, FLOT2, CD81) in the whole cell lysate (WCL) and exosomes of NRVMs. Gapdh was used as an internal reference. There were significant differences between the Ad-Pxdc1 group and the Ad-Vector group, indicating that overexpressing Pxdc1 adenovirus affected the expression of exosome-related proteins. Thus, it can be known that overexpressing Pxdc1 by adenovirus in primary rat ventricular myocytes (NRVMs) promoted exosome secretion.

[0083] 7. Detection of the mRNA level of Pxdc1 knocked down by siRNA in NRVMs cells by qRT-PCR ( Figure 3 a)

[0084] The mRNA level of Pxdc1 knocked down by siRNA in NRVMs was detected by qRT-PCR. siNeg represents the control group transfected with negative control siRNA, and siPxdc1 represents the experimental group transfected with siRNA that knocks down Pxdc1. The ordinate is the relative expression level of Pxdc1 to Actb. The results show that the mRNA level of the experimental group was significantly lower than that of the control group (** indicates P < 0.01).

[0085] 8. Detection of the protein level of Pxdc1 knocked down by siRNA in NRVMs cells by Western blot ( Figure 3 b)

[0086] The protein level of Pxdc1 knocked down by siRNA in NRVMs cells was detected by Western blot, with Gapdh used as an internal reference; Figure 3 The bar graph on the right side of b is the quantitative analysis of the relative protein expression. The results show that the protein expression level of Pxdc1 in the experimental group was significantly lower than that of the control group (** indicates P < 0.01).

[0087] 9. Detection of the effect of knocking down Pxdc1 by siRNA on exosome secretion in NRVMs cells by Western blot ( Figure 3 c)

[0088] The Western blot detection results show that there were significant differences between the siPxdc1 group and the siNeg group, indicating that knocking down Pxdc1 by siRNA affected the expression of exosome-related proteins, and knocking down Pxdc1 by siRNA inhibited exosome secretion.

[0089] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of the Pxdc1 gene in regulating exosome secretion, characterized in that, The application is for non-disease treatment purposes.

2. The application according to claim 1, characterized in that, The Pxdc1 gene is of human or rat origin; the full-length cDNA sequence of human Pxdc1 has the ID of NM_183373.4 in the NCBI database; the full-length cDNA sequence of rat-derived Pxdc1 has the ID of NM_001025719.1 in the NCBI database.

3. The application according to claim 1 or 2, characterized in that, Overexpression of the Pxdc1 gene promotes exosome secretion.

4. The application according to claim 3, characterized in that, Overexpression of the Pxdc1 gene is carried out by adenovirus infection of primary rat cardiomyocytes.

5. The application according to claim 3, wherein Overexpression of the Pxdc1 gene is carried out by plasmid transfection of 293T cells.

6. The application according to claim 1 or 2, characterized in that, Knockdown of the expression of the Pxdc1 gene inhibits exosome secretion.

7. The application according to claim 6, characterized in that, Knockdown of the expression of the Pxdc1 gene is achieved by an RNA interference molecule targeting the Pxdc1 gene, and the RNA interference molecule includes at least one of siRNA, shRNA, and miRNA mimics.

8. Application of the Pxdc1 gene as a target in the preparation of a reagent for regulating exosome secretion.

9. The application according to claim 8, characterized in that, The Pxdc1 gene is of human or rat origin; the full-length cDNA sequence of human Pxdc1 has the ID of NM_183373.4 in the NCBI database; the full-length cDNA sequence of rat-derived Pxdc1 has the ID of NM_001025719.1 in the NCBI database; the reagent regulates exosome secretion through the following approach (1) or (2): (1) promoting exosome secretion by upregulating the expression of the Pxdc1 gene; (2) inhibiting exosome secretion by knocking down the expression of the Pxdc1 gene.

10. A method for in vitro regulating exosome secretion, characterized in that, In cells cultured in vitro, exosome secretion is regulated through the following approach (1) or (2): (1) promoting exosome secretion by upregulating the expression of the Pxdc1 gene; (2) inhibiting exosome secretion by knocking down the expression of the Pxdc1 gene.

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