CN115747214A - Application of MLPH gene in preparing medicine for treating pneumoconiosis - Google Patents
Application of MLPH gene in preparing medicine for treating pneumoconiosis Download PDFInfo
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Abstract
The invention discloses an application of MLPH gene in preparing a medicine for treating pneumoconiosis, belonging to the field of biological medicine. The invention utilizes the small interfering RNA to target and silence the MLPH gene in the macrophage and utilizes the dust particles to stimulate the macrophage, and the result shows that the MLPH is used as the upstream gene of TGF-beta, and the dust particles can induce the macrophage to generate TGF-beta by regulating the MLPH, thereby participating in the EMT process of the pneumoconiosis fibrosis. The invention discovers an important molecular mechanism in the process of the fibrosis of the pneumoconiosis and provides a new immune target for preventing and treating the pneumoconiosis.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to application of MLPH gene in preparing a medicine for treating pneumoconiosis.
Background
The pneumoconiosis is a pulmonary fibrosis disease caused by dust, is an occupational lung disease with the most serious harm and the highest incidence rate at present, but the fibrosis mechanism is not clear and an effective treatment means is lacked. The research on the molecular mechanism causing the fibrosis of the pneumoconiosis has important significance for early intervention, prevention and treatment targets of diseases. Miners exposed to high levels of silica and coal dust for a long period of time cause long-term inflammation of the lungs, which eventually progresses to irreversible pneumoconiosis under the long-term effects of various inflammatory factors and cells.
Macrophages play a very important role in the progression of pneumoconiosis, fulfilling the role of "scavenger" in lung tissue by phagocytosing various particles, however, various inflammatory factors produced during particle phagocytosis cause epithelial-to-mesenchymal transition (EMT) of epithelial and fibroblasts in the lung to accelerate the progression of pulmonary fibrosis.
Transforming growth factor-beta (TGF-beta) belongs to a group of TGF-beta super families for regulating cell growth and differentiation, and plays an important role in early embryonic development, tissue organ formation, immune supervision, tissue repair and adult homeostasis balance. Abnormal TGF- β signaling may lead to diseases such as tissue fibrosis. Melanophilins (MLPH) play a crucial role in regulating skin pigmentation through the process of melanosome transport. Melanosome transport involves intracellular distribution of melanosomes and their mobile Mlph complex from around the nucleus to the dendritic apical end of melanocytes, and is closely associated with melanosome transport through actin filaments. There are no reports of melanoavidin in relation to pneumoconiosis.
Disclosure of Invention
The invention aims to provide application of MLPH gene in preparation of drugs for treating pneumoconiosis, which aims to solve the problems in the prior art, and verifies that MLPH is used as an upstream gene of TGF-beta by targeting MLPH gene of silenced macrophages, and dust particles can induce macrophages to generate TGF-beta by regulating MLPH, so that the MLPH gene participates in the EMT process of pneumoconiosis fibrosis, and a new immune target is provided for prevention and treatment of pneumoconiosis.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides an application of MLPH gene in preparing a medicine for treating pneumoconiosis; the nucleotide sequence of the MLPH gene is shown as SEQ ID NO:1, and the following components:
atggggaaaaggttggaccttt ccacgctcac agacgaggag gctgagcacg tgtgggcagt ggttcagcgg gactttgacc tcaggaggcg agaggaagaa agactccagg ggctgaaggg caaaatacaa aaggagagct ccaagaggga gctgctgtcg gacacagccc atctgaatga gactcactgt gcccgctgcc tgcagcccta ccggctgctc ctgaacagca gacgacagtg cctagagtgc agcctcttcg tctgcaaaag ctgcagccac gcccacccag aagagcaggg ctggctctgc gacccctgcc acctggccag ggtcgtgaag atcggttctc tggagtggta ctaccagcac gtgagggctc gcttcaagcg tttcgggagt gccaaagtga tccggtctct ctgtgggcgg ctgcagggtg gaggtggatc tgagccaagc ttggaagaag gaaatggaga cagtgagcag actgatgagg atggagacct ggacacagag gccagagacc agcccctcaa cagcaaaaag aaaaagcgcc tgctctcctt ccgagatgtg gactttgagg aagactcaga ccacttggtg cagccttgca gccaaacctt gggcctgtcc tcagtccctg agtctgcaca cagcctgcag tccctctcag gtgagcccta ctctgaggac accacctctc tggagcccga gggcctagag gagactggtg caagggcttt gggatgtcat cccagtcctg aagtgcagcc atgtagccct ttaccctctg gggaggatgc tcacgctgaa ctggactcgc ctgcagcatc ctgcaagagt gcctttggga ccacagctat gcctggaaca gacgatgtca ggggcaaaca tctgccctca cagtacctgg ctgatgtaga cacctctgat gaagacagta tccagggtcc cagggcagcc tcccagcata gcaagaggag ggcccggact gtgcctgaga ctcagatctt ggagttgaac aagcgaatgt cagctgtgga gcacctgcta gtccacctgg agaatacggt tctgccaccc tcagcccagg aaccaactgt ggagacacac cccagtgctg acacagagga ggagacactc aggaggaggc tggaggagct gaccagcaac atcagtggtt ccagcacctc atcagaagat gagaccaagc cagatggcac cttccttgga gggtccccaa aggtgtgcac agacacaggg cacatggaga cacaggaaag gaaccctcgg agccctggga accctgctcg gcctacaaaa agcacagatg aggagctctc tgagatggag gatagagtgg ccatgacagc ctctgaagtt cagcaggctg agagtgagat ctcagacatc cagtccagga tcgcagctct gagagccgca ggactcacag tgaagccctc gggaaaacct cggagaaagt caggcatccc gatctttctt ccccgcgtta ctgagaaact tgacaggatc ccaaagactc cacctgcaga ccctgatgac caagccaaga tgcccaaggc aacaacagct gtgccctctc tcctgaggag gaagtattct cccagcagcc aaggcgtaga cagtggttct tttgatcgga aatcagtgta ccgtggctcg ctgacacaaa ggaaccctaa cgggaggaga gggacagcca gacacatctt cgcgaaaccc gtgatggccc agcagcccta a。
preferably, inhibiting the expression of said MLPH gene effects treatment of said pneumoconiosis.
The invention also provides a small interfering RNA of the target MLPH gene, and the nucleotide sequence of the small interfering RNA is shown as SEQ ID NO:3 and the sense strand shown in SEQ ID NO: 4.
The amino acid sequence of SEQ ID NO:3, the following steps:
5’-GATCCGTTCAGCGGGACTTTGACCTCCTTCCTGTCAGA GAGGTCAAAG TCCCGCTGAAC TTTTTG-3’；
SEQ ID NO:4, the following steps:
5’-GATCCGGGCAAAATACAAAAGGAGCTTCCTGTCAGA CTCCTTTTGTATT TTGCCC TTTTTG-3’。
the invention also provides a short hairpin RNA of the targeted MLPH gene, and the sequence of the short hairpin RNA is shown as SEQ ID NO:2-4 of any one of the sequences:
shRNA-1(SEQ ID NO：2)：
5’-GATCCGCTGTGATTCTTTAACCCGATCTTCCTGTCAGA ATCGGGTTAAA GAATCACAGCTTTTTG-3’；
shRNA-2(SEQ ID NO：3)：
5’-GATCCGTTCAGCGGGACTTTGACCTCCTTCCTGTCAGA GAGGTCAAAG TCCCGCTGAAC TTTTTG-3’；
shRNA-3(SEQ ID NO：4)：
5’-GATCCGGGCAAAATACAAAAGGAGCTTCCTGTCAGA CTCCTTTTGTATT TTGCCC TTTTTG-3’。
the invention also provides a recombinant vector containing the small interfering RNA.
Preferably, the recombinant vector further comprises a lentiviral or adenoviral vector.
The invention also provides the small interfering RNA, or the short hairpin RNA, or the recombinant vector in silencing MLPH gene application.
The invention also provides the small interfering RNA, or the short hairpin RNA, or the recombinant vector in the preparation of drugs for treating pneumoconiosis, and the treatment of pneumoconiosis is realized by silencing MLPH through the small interfering RNA or the recombinant vector.
Preferably, the small interfering RNA target interferes with the MLPH gene to slow down the EMT progression of alveolar epithelial cells, so as to prevent and treat pulmonary fibrosis.
The invention also provides a medicine for treating pneumoconiosis, which comprises the small interfering RNA and a pharmaceutically acceptable carrier or auxiliary agent.
The invention discloses the following technical effects:
1) The invention provides a small interfering RNA of a targeted MLPH gene, and provides a new strategy for developing targeted pneumoconiosis fibrosis by directionally knocking out the MLPH gene of macrophages.
2) The invention reduces the expression of the TGF-beta of the fibrosis promoting factor after the macrophage is stimulated by coal dust and silicon dioxide by infecting the macrophage with the small interfering RNA lentiviral vector of the MLPH gene.
3) The invention also discloses an important molecular mechanism in the process of the fibrosis of the pneumoconiosis based on RNAi targeted interference of macrophages and reduction of EMT (acute respiratory syndrome) progression of alveolar epithelial cells, and provides a new therapeutic target for research and development of targeted pneumoconiosis fibrosis.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows that lentivirus infects macrophages and drug-sifts to obtain stable transformants;
FIG. 2 is the expression of MLPH of lentivirus-infected macrophages; a: qPCR verification results; b: WB verification result;
FIG. 3 is a graph of the effect of different stimuli to stimulate MLPH targeted knockout macrophages on secretion of TGF- β; a: analyzing the level of TGF-beta secretion stimulated by coal dust at the transcription level; b: analyzing the level of silica-stimulated secretion of TGF-beta at the transcriptional level; c: analyzing the level of TGF-beta secretion stimulated by coal dust at the protein level; d: assaying silica-stimulated secretion of TGF- β levels at the protein level;
fig. 4 is a graph of WB demonstrating the effect of coal dust and silica stimulation of macrophages targeting knockout MLPH on alveolar epithelial EMT.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1 construction of macrophages stably expressing targeted silenced MLPH genes
1. Construction of recombinant plasmid pGreenpuro-MLPHshRNA-GFP
Based on the sequence of the mouse MLPH gene (SEQ ID NO: 1), with reference to the siRNA target sequence design rules, an MLPH-specific shRNA sequence (SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO: 4) was designed: blank vector (pGreenpuro) was digested with EcoRI, bamHI TM vector), then inserting the siRNA target sequence into the H1 promoter to control the expression of the siRNA target sequence, wherein the lentiviral vector contains GFP gene expression controlled by a CMV promoter. Wherein the expression of Green Fluorescent Protein (GFP) is controlled by CMV promotor respectively, as the sign of transduction efficiency, shanghai Ji Ma Biotechnology Limited company is entrusted to synthesize complete shRNA sequence. The general steps of shRNA template annealing, linearized expression vector preparation, interference fragment ligation into expression vector, competent cell preparation for ligation product transformation, validation and sequencing are not detailed here.
2. Viral packaging, purification and titer determination
1) Recovering 293T cells, carrying out passage for 2-3 times, and preparing to prepare viruses when the cell state is stable and the cells grow well;
2) Plasmid and PEI transfection reagents were added to DMEM and left for 5 minutes, respectively, in a ratio of (pdR8.1: pcmv-vsvg: target plasma) = (4.5;
3) Adding the plasmids into PEI, quickly blowing and uniformly mixing by using a gun, standing the uniformly mixed transfection reagent at room temperature for 15 minutes, and uniformly dripping the uniformly mixed transfection reagent into cells to be transfected;
4) Continuously culturing for two days, collecting a cell culture medium, and filtering the DEME culture medium containing the virus by using a 0.45um filter membrane;
5) The virus was collected after solubilization by centrifugation at 1000rpm for 10 minutes.
3. Infecting cells with slow virus and screening to obtain stable transformant
1) When Raw264.7 cells to be infected with the virus grow to 80-90% of density, passage is carried out, and then a proper amount of purified lentivirus is added into the cells after passage and Polybrene with the final concentration of 1 mu g/ml is added to increase the infection efficiency;
2) After about 24 hours, the culture medium containing the lentivirus is discarded and replaced by fresh DEME culture medium, and Puromycin with the final concentration of 2 mu g/ml is added 48 hours after infection to start screening;
3) Changing to a new culture medium every two days and supplementing puromycin, continuously screening until no obvious cell death exists, continuously using the screening culture medium for screening twice, and determining that stable transformants are obtained when all blank cells are dead (the result is shown in figure 1);
4) After screening, carrying out qPCR (quantitative polymerase chain reaction) on the stable transgenic cells and carrying out MLPH verification on WB (white cell line);
the primer sequences used for qPCR were as follows:
TABLE 1 primers
| Gene | Sequence (5 '-3') |
| GAPDH-F | AGGTCGGTGTGAACGGATTTG |
| GAPDH-R | TGTAGACCATGTAGTTGAGGTCA |
| MLPH-F | GTTCAGCGGGACTTTGACCTC |
| MLPH-R | GGCACAGTGAGTCTCATTCAGA |
The specific steps of qPCR detection are as follows:
(a) Denaturation of RNA template
The following mixture was prepared in RNase free PCR tube:
TABLE 2 denaturation mixture of RNA templates
| Reagent | Volume of |
| DEPC-H 2 O | 13μl |
| Oligo dT(50uM) | 1μl |
| Random hexamers(50ng/ul) | 1μl |
| Total RNA | 1μg |
Heating at 65 deg.C for 5min, rapidly placing on ice, and standing on ice for 2min.
(b) Preparing first chain cDNA synthetic reaction liquid
TABLE 3 first Strand cDNA Synthesis reaction solution
The reaction solutions were added as in Table 3, and gently pipetted and mixed.
(c) First Strand cDNA Synthesis reaction was carried out under the following conditions
TABLE 4 first Strand cDNA Synthesis reaction conditions
| Temperature of | Time |
| 25℃ | 5min |
| 50℃ | 45min |
| 85℃ | 5min |
The reverse transcription product can be used immediately for QRT-PCR reaction, or stored at-20 ℃.
(d) The following real-time fluorescent quantitative PCR reaction system is prepared in a qPCR tube
TABLE 5 fluorescent quantitative PCR reaction System
| 2×AceQ Universal SYBR qPCR Master Mix | 10.0μl |
| Upstream primer F (10 μm) | 0.4μl |
| Downstream primer R (10 μm) | 0.4μl |
| cDNA template | 1.2μl |
| ddH 2 O | Make up to 20.0. Mu.l |
(e) The qPCR reaction was carried out under the following conditions
TABLE 6 qPCR reaction procedure
The WB detection method specifically comprises the following steps:
cells were lysed using cell lysate RIP, the supernatant collected and centrifuged to determine protein concentration using BCA method. Then, 30. Mu.g of the protein sample was subjected to SDS-PAGE gel electrophoresis. Subsequently PVDF membrane was transferred and antibody blocked followed by an anti-incubation overnight with MLPH antibody (1. The next day, PBST was washed 5 times, 5min after each time, with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1 10000, sa00001-2, proteintech), room temperature 1h, enhanced chemiluminescence was shown. GAPDH was used as a protein loading control and band gray values were determined to assess protein levels.
The results are shown in FIG. 2: MLPH can be stably expressed in the selected stable transformants.
Example 2 targeting of macrophage silencing MLPH-shRNA Gene on coal dust and SiO 2 Expression and effect on EMT of alveolar epithelial cells of the stimulating profibrotic factor TGF-beta
1) Mixing 200 mu g/mL coal dust and SiO 2 Respectively stimulate macrophages stably transcribing MLPH-shRNA genes,the intracellular TGF-beta RNA transcript level and the TGF-beta content of the cell supernatant were measured after 24 hours.
Synthesis of TGF-. Beta.cDNA and qPCR method the same experimental procedure as in example 1 was used to verify MLPH gene expression in stably transfected strains.
The results are shown in FIG. 3: the macrophage is infected by the small interfering RNA lentiviral vector of the MLPH gene, so that the expression of the TGF-beta factor is reduced after the macrophage is stimulated by coal dust and silicon dioxide.
2) Mixing 200 mu g/mL coal dust and SiO 2 Separately stimulating macrophages capable of stably transcribing MLPH-shRNA genes, planting the macrophages with MLE-shRNA genes in the upper chamber and the lower layer by using a Transwell chamber and alveolar epithelial cells MLE-12, and researching cell coal dust and SiO 2 Stimulated macrophage secretion of MLPH-shRNA gene on MLE-12 cells E-Cadherin and N-Cadherin expression.
3) The expression of E-Cadherin and N-Cadherin of MLE-12 cells was examined using WB, and the results are shown in FIG. 4, by transwell experiment, coal dust and SiO 2 The stimulated macrophage RAW264.7 cell enables E-Cadherin of MLE-12 cells to be down-regulated and N-Cadherin of the MLE-12 cells to be up-regulated, and EMT transformation of alveolar epithelial cells is promoted; while macrophage with MLPH knocked out is on coal dust and SiO 2 Stimulated MLPH-shRNA RAW264.7 cells preserved the original expression of E-Cadherin and N-Cadherin of MLE-12 cells without promoting EMT transformation of alveolar epithelial cells.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
- Application of MLPH gene in preparing medicine for treating pneumoconiosis is disclosed.
- 2. The use of claim 1, wherein inhibiting the expression of said MLPH gene effects treatment of said pneumoconiosis.
- 3. A small interfering RNA targeting an MLPH gene is characterized in that the nucleotide sequence of the small interfering RNA is shown as SEQ ID NO:3 and the sense strand shown in SEQ ID NO: 4.
- 4. A MLPH gene-targeted short hairpin RNA, characterized in that the sequence of the short hairpin RNA is shown in SEQ ID NO:2-4 of any one of the sequences set forth herein.
- 5. A recombinant vector comprising the small interfering RNA of claim 3 or the short hairpin RNA of claim 4.
- 6. The recombinant vector of claim 5, wherein the recombinant vector further comprises a lentiviral or adenoviral vector.
- 7. The small interfering RNA of claim 3, or the short hairpin RNA of claim 4, or the recombinant vector of claim 5 or 6 for use in silencing the MLPH gene.
- 8. Use of a small interfering RNA according to claim 3, or a short hairpin RNA according to claim 4, or a recombinant vector according to claim 5 or 6 for the preparation of a medicament for the treatment of pneumoconiosis, wherein the treatment of pneumoconiosis is effected by silencing MLPH in said small interfering RNA or in said recombinant vector.
- 9. The use of claim 8, wherein the small interfering RNA targets and interferes with the MLPH gene to slow EMT progression in alveolar epithelial cells, thereby preventing pulmonary fibrosis.
- 10. A medicament for treating pneumoconiosis, comprising the small interfering RNA of claim 3 and a pharmaceutically acceptable carrier or adjuvant.
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| PCT/CN2023/105890 WO2024012325A1 (en) | 2022-07-12 | 2023-07-05 | Use of mlph gene in preparation of drug for treating pneumoconiosis |
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| US20220363720A1 (en) * | 2019-07-18 | 2022-11-17 | Olipass Corporation | Melanophilin antisense oligonucleotides |
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