CN115944647A - Application of has-miR-129-5p in preparation of anti-aging drugs - Google Patents
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Abstract
The invention belongs to the technical field of biological medicines, and relates to application of a has-miR-129-5p small molecular reagent in delaying senescence, wherein the nucleotide sequence of the has-miR-129-5p is shown as SEQ ID No.1, the drug inhibits the expression of the drug by combining a 3' UTR region (the combination region is shown as SEQ ID No. 2) of ITPR2, so that the signal transduction of calcium ions in cells from endoplasmic reticulum to mitochondria is regulated, the calcium ion transfer causes the decrease of mitochondrial membrane potential, the accumulation of active oxide (ROS) is reduced, DNA damage is reduced, and finally the senescence of cells and organisms is delayed. The invention provides a new medicine source for preventing, diagnosing, protecting, delaying and researching cell and organism aging, and the medicine for controlling cell and organism aging provided by the invention is safe, strong in pharmacological action, small in side effect and definite in curative effect.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to application of has-miR-129-5p in preparation of a medicine for delaying cell senescence.
Background
Cellular senescence refers to a stable inhibition of cell proliferation accompanied by a cell senescence-associated secretory phenotype (SASP), which occurs mainly due to the consumption of telomerase during cell replication and external senescence-associated stimuli such as oncogene activation, reactive Oxygen Species (ROS) accumulation, DNA damage, and the like. Cell senescence is mainly manifested by cell cycle arrest and morphological changes of the cells such as flattening, enlargement, nuclear condensation; and senescence-associated beta-galactosidase activity (SA-beta-GAL), and secretion of senescence-or inflammatory-related factors, and the like. Senescent cells remain metabolically active and can survive for years or more. The generation of aging cells in a short period is beneficial to the embryonic development, damage repair, tumor inhibition and the like of an organism; the long-term accumulation of senescent cells in the body can lead to tumorigenesis, body aging and aging-related diseases. Therefore, more and more research is being devoted to finding, identifying and attempting to understand the mechanisms of cellular senescence; however, the molecular and cellular mechanisms that regulate aging are still not fully understood at present. In addition, due to the lack of specific markers for senescence and the limitations in studying mechanisms of senescence, screening for new senescence regulators and studying their regulatory networks is imminent.
The function of intracellular calcium signaling pathways in cellular senescence has been more and more emphasized in recent years and is gradually considered as an important factor in the regulation of senescence; our previous studies found that the nuclear receptor RXRA regulates intracellular calcium signaling and regulates cellular senescence via the inositol 1,4, 5-triphosphate receptor type 2 (ITPR 2); in addition, ITPR2 knockout mice demonstrate efficacy in prolonging the lifespan of the mice by reducing exposure to the mitochondrial endoplasmic reticulum. However, the specific role of intracellular calcium signaling in cellular senescence is not fully understood. Therefore, further elucidating the route mechanism of intracellular calcium signal regulation of cell aging is of great significance for further understanding the occurrence and development mechanism of cell and body aging.
The regulation of senescence-associated gene expression by conserved small non-coding RNAs (miRNA) (about 22 bp) has been studied for decades. Studies have shown that miRNA-24 (miR-24) inhibits replicative senescence of cells by binding the 3' UTR of p16 mRNA. Another study showed that miR-124, miR-34a and miR-29a/b/c, as a p53 downstream senescence-regulating factor, regulates cellular senescence by inhibiting cyclin A2 (CCNA 2). However, no studies are currently being made regarding the effect of mirnas on ITPR 2.
The function of has-miR-129-5p in the cell senescence process is unknown, and no prior art reports the inhibiting effect of has-miR-129-5p on ITPR2 in cell senescence and the treatment effect on delaying cell senescence, so that the has-miR-129-5p can be found to have a great promoting effect on the treatment of cell senescence and senescence-related diseases.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the application of has-miR-129-5p in the preparation of the medicines for preventing and/or treating cell aging and aging-related diseases.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the application of has-miR-129-5p in the preparation of the medicine for preventing or treating the diseases related to cell aging is disclosed, wherein the nucleotide sequence of the has-miR-129-5p is shown in SEQ ID No.1, and the medicine prevents or treats the diseases related to cell aging by inhibiting the expression of ITPR 2. The drug inhibits the expression of the drug by combining with a 3' UTR region of ITPR2, so that the signal transduction of calcium ions in cells from endoplasmic reticulum to mitochondria is regulated, the calcium ion transfer causes the decrease of mitochondrial membrane potential, the accumulation of Reactive Oxygen Species (ROS) is reduced, the DNA damage is reduced, and finally the aging of cells and organisms is delayed. The sequence of the 3' UTR region is shown as SEQ ID NO.2, and the sequence of the binding region is GCAAAAAA.
SEQ ID NO.1
3’-CGUUCGGGUCUGGCGUUUUUC-5’。
SEQ ID NO.2
TAAATATTTCTGAGTTGTATTTTTAATGCCTCTTTCTTTTCTGCCTAAATGCTTAGAAATTATAAA(GCAAAAAA)AAAAAAAACAGCAACAAAAAATCGAAGCAGACAAAAAAGGCACTTTTCAGAACATCAAATTCCTAATGAAGAAGAGGAGAAGATAATGGGGAAATTTGACATTTGATATAAATTTATATTTGTTATGTGTATGTTTGTTAATGCAACTGGAATATTTGACTTAGGTGAGTATCAGTTAACATCCTTGTATTTATATAGTGACATC。
Further, the application includes any one of:
delaying the aging of fibroblasts;
improving the aging symptoms in the body of the mouse and delaying natural aging;
delaying the onset or development of diseases associated with cell aging.
Further, the use includes ameliorating bleomycin-induced pulmonary aging and natural aging.
Further, the medicament inhibits aging of senescence-associated cells, including fibroblasts, naive CD4+ T cells, memory CD8+ T cells, and the like.
Further, the drug reduces bleomycin-induced lung aging of mice and lung tissue senescence-associated beta galactosidase staining and naive T cell failure, memory T cell accumulation in naturally aging mice, and expression levels of senescence markers CDKN1A, CDKN 2A.
Further, the drug is introduced into the body tissue by an intraperitoneal injection method.
Further, the medicine comprises an active ingredient has-miR-129-5p and a pharmaceutically acceptable carrier.
Furthermore, the medicine is an injection and is prepared by dissolving has-miR-129-5p in PBS buffer solution.
Further, the concentration of the has-miR-129-5p is 5mg/kg.
Further, the intraperitoneal injection is performed for more than 2 times.
Further, the invention discloses application of has-miR-129-5p in preparation of a medicine for preventing and/or treating diseases related to cell aging and aging
The has-miR-129-5p is conservative small molecular non-coding RNA (about 22 bp).
The medicine is used for controlling cell and organism aging, and the aging-related cells comprise fibroblasts, naive CD4+ T cells, memory CD8+ T cells and other cells.
The medicine is a medicine for inhibiting the expression of ITPR 2.
The invention also discloses a product, the active component of which is has-miR-129-5p, and the application of the product at least comprises one of the following applications:
a) Delaying the aging of fibroblasts;
b) Improving bleomycin-induced lung aging;
c) Delaying natural aging.
The invention also discloses a medicament which can be used for preventing/treating the aging (TIS) induced by chemotherapy medicaments commonly occurring in clinic, wherein the medicament consists of an active ingredient has-miR-129-5p and auxiliary materials which can be added in pharmacy.
The drug can be introduced into body tissues by injection absorption and physical or chemical mediated methods; or mixed or coated with other materials and introduced into body.
Advantageous effects
The invention provides application of has-miR-129-5p in preparation of a medicine for delaying cell aging and body aging. The invention simulates and clinically treats the frequently-occurring medicament-induced aging (TIS) model and the natural aging model: intratracheal instillation (i.i) of bleomycin (1.875 mg/kg) in C57BL/6 mice induced a model of lung aging; model of natural aging in C57BL/6 mice aged 20 months. Experiments prove that the ha-miR-129-5 p is injected into a mouse body in an abdominal cavity, and the occurrence and development of the lung aging and the natural aging can be obviously improved. Meanwhile, the in vitro cell aging model adopted by the invention is as follows: the primary human embryonic lung fibroblast MRC5 is characterized in that a cell senescence model is constructed by adopting bleomycin (1 mu g/ml), and the expression of transfected has-miR-129-5p is inhibited by combining with a 3' UTR region of ITPR2, so that the signal transduction of calcium ions in cells from endoplasmic reticulum to mitochondria is regulated, the calcium ion transfer causes the decrease of mitochondrial membrane potential, the accumulation of Reactive Oxygen Species (ROS) is reduced, the DNA damage reaction is reduced, and finally the cell senescence is delayed. Therefore, the invention provides the research, development and preparation of the drug that has-miR-129-5p can be applied to preventing/treating clinically common drug-induced aging (TIS) for the first time.
The medicine for controlling cell and organism aging provided by the invention is safe, strong in pharmacological action, definite in curative effect and small in side effect. The invention provides a new medicine source for preventing, detecting, protecting, treating and researching cell and organism aging, and the medicine for controlling cell and organism aging provided by the invention is safe, strong in pharmacological action, small in side effect and clear in curative effect; and the method is easy to popularize and apply clinically, and can generate great clinical application prospect and social benefit in a short time.
Drawings
FIG. 1 is a mechanism of has-miR-129-5p in regulating cell senescence via ITPR 2-intracellular calcium signaling-mitochondrial membrane potential-active oxygen-DNA damage axis.
FIG. 2 shows that has-miR-129-5p is used as a direct inhibitor of ITPR 2. Wherein (a) is miRNA micromolecules which are screened from four miRNA databases and are possible to inhibit ITPR 2; (b-d) determining mRNA expression of has-miR-129-5p for inhibiting ITPR2 after miRNA is overexpressed and knocked down in different fibroblasts; (e-f) is a dual-luciferase reporter experiment that has-miR-129-5p can directly target the 3' UTR region of ITPR 2.
FIG. 3 is the axis of injury of has-miR-129-5p in regulating intracellular calcium signaling-mitochondrial membrane potential-reactive oxygen species-DNA. miR-Ctrl/miR-129 (has-miR-129-5 p analog (miR-129, the same below)) or imiR-Ctrl/imiR-129 (has-miR-129-5 p inhibitor (imiR-129, the same below)) +/-siITPR2 or siMCU (small interfering RNA, supplied by Ribo Biotech, guangzhou) transfects MRC5 cells. 4 days after transfection, the cells were incubated with cytoplasmic calcium indicator Fluo4 (a) or mitochondrial calcium indicator Rhod2 (b) or mitochondrial membrane potential drop indicator Rhodamine123 (c) or mitoSOX RED mitochondal (mitoROS) (d) or Dihydroethium (ROS) (e), respectively, for 30min and their frequency deviations were analyzed by flow cytometry. The left panel shows the partial graphs, the middle panel represents the mean of three independent experiments ± SEM, and the right panel represents the mean of six biological replicates ± SEM. Cells were fixed 4 days after transfection and immunofluorescent staining was performed with anti-53 BP1 antibody. Representative pictures and relative percentages of nuclei greater than or equal to 5 and above points 53BP1 (3 independent replicates of overexpression experiment, 4 replicates of inhibitor experiment, ± SEM) show (f, g).
FIG. 4 shows that calcium signaling and antioxidants are involved in the reactive oxygen species and DNA damage regulated by has-miR-129-5 p. BAPTA (5. Mu.M) or NAC (5. Mu.M) was treated every 2 days with IP3 (5. Mu.M) or imiR-Ctrl or imiR-129 following miR-Ctrl or miR-129 transfection of MRC5 cells. 4 days after transfection, cells were incubated with mitoSOX RED mitochondal (mitoROS) (a, c) or Dihydroxyethylium (ROS) (b, d) for 30min, respectively. Representative frequency deviation photographs and mean values of 3 biological replicates were plotted using ± SEM. Cells were fixed 4d after transfection and immunofluorescent stained with an antibody to 53BP1. (e, f) is a representative picture of nuclei at greater than or equal to 5 BP1 points or more (± SEM 3 independent repeats) and relative percentage of nuclear quantification.
FIG. 5 shows that has-miR-129-5p regulates cell senescence by ITPR2 and MCU. miR-Ctrl or miR-129 transfects MRC5 cells. 6 days after transfection, cells were stained with either crystal violet (a) or SA- β -GAL solution, the percentage of SA- β -GAL positive cells for each condition (left) and representative pictures (right) (mean of 3 independent repeats of. + -. SEM) (b). (c-f) 3 days after transfection, the mRNA levels of ITPR2 (c), ki67 (d), CDKN1A (e) and CDKN2A (f) were examined by RT-qPCR (mean of 5 biological replicates,. + -. SEM). MRC5 cells were transfected with imiR-Ctrl or imiR-129 and siITPR2 or siMCU. 6 days after transfection, cells were stained with either crystal violet (g) or SA- β -GAL solution, representative picture of the percentage of SA- β -GAL positive cells for each condition (left side) (mean of + -SEM independent replicates 3 times) (h). (i-m) 3 days after transfection, the mRNA levels of ITPR2 (i), MCU (j), ki67 (k), CDKN1A (l) and CDKN2A (m) were detected by RT-qPCR (mean of 5 independent replicates,. + -. SEM).
FIG. 6 shows that has-miR-129-5p retards bleomycin-induced cell senescence. MRC5 cells were transfected with miR-Ctrl or miR-129 and treated with bleomycin (1. Mu.g/ml). 4 days after transfection, cells were collected, incubated for 30min with cytoplasmic calcium indicator Fluo4 (a) or mitochondrial calcium indicator Rhod2 (b) or mitochondrial membrane potential drop indicator Rhodamine123 (c) or mitoSOX RED mitochondal (mitoROS) (d) or Dihydroethidium (ROS) (e), and subjected to flow cytometry; the left panel is representative image and the right panel is mean of ± SEM triplicates independently. (f) Cells were fixed 4 days after transfection and immunofluorescent staining was performed with 53BP1 antibody. Representative pictures of nuclei at greater than or equal to 5 points of 53BP1 (± SEM,3 independent replicates) and relative percentage of nuclear quantification. 6 days after transfection, cells were stained with either crystal violet (g) or SA- β -GAL solution, representing the picture (left) and the percentage of SA- β -GAL positive cells in each condition (right panel is the average of 3 independent replicates of + -SEM) (h). (i-l) 3 days after transfection, RT-qPCR was used to measure the mRNA levels of ITPR2 (i), ki67 (j), CDKN1A (k) and CDKN2A (l) (mean of 5 independent replicates,. + -. SEM).
FIG. 7 shows that has-miR-129-5p retards bleomycin-induced mouse aging and natural aging. (a) Linear regression analysis between miR-129 and ITPR2 mRNA levels in lung tissue of 8 week old female mice (N = 8). (b) mouse experimental timeline: mice were instilled with bleomycin (1.875 mg/kg) intraductally on day 1, and were intraperitoneally injected with miR-NC or miR-129agomir twice (250 nM/kg) on days 2 and 5, respectively; sacrificed on day 8. (c) Representative pictures of the level of SA- β -GAL staining in lung tissue and the percentage of SA- β -GAL positive cells under each condition (N =6, mean ± SEM). (d-e) lung tissue naive or memory CD4+ and CD8+ T cell immunophenotypes (N =6, mean ± SEM). (f) RT-qPCR detected CDKN1A mRNA levels (N =6, mean ± SEM). (g) mouse experimental timeline: mice aged 8 weeks or 20 months were intraperitoneally injected 3 times a week with miR-NC or miR-129agomir at a dose of 250nM/kg and sacrificed on day 21. (h) Lung tissue SA- β -GAL staining levels and representative pictures (left) and percentage of SA- β -GAL positive cells (right) under each condition (N =5, mean ± SEM). (i-j) naive or memory CD4+ and CD8+ T cell lung tissue immunophenotypes (N =4, mean ± SEM). RT-qPCR detected CDKN2A mRNA levels (N =4, mean ± SEM) of lung (k), liver (l), and kidney (m).
Detailed Description
In order to make those skilled in the art better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the accompanying drawings:
the experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The reagent materials used in the examples are all available in routine purchase, and the quantitative experiments referred to in the examples are all set up in at least three replicates and the results are averaged.
The C57BL/6 mice used were purchased from the Yangzhou university center for comparative medicine.
The bleomycin was purchased from MCE under the designation HY-17565.
The miR-129 used was purchased from Ribo Biotech, guangzhou.
The human primer sequences are shown in Table 1, and the murine primer sequences are shown in Table 2.
The sequences of siRNAs and miRNAs used are shown in Table 3.
The information on the antibodies and probes used is shown in Table 4.
TABLE 1
TABLE 2
TABLE 3
TABLE 4
Example 1: miR-129 direct targeting inhibition of ITPR2 expression
As shown in fig. 2, to screen for endogenous miRNAs that modulate ITPR2 expression, we performed a preliminary screening for ITPR2 regulation using Venny analysis based on four miRNA databases (Targetscan, miRDB, mirDIP, and miRanda). This method initially screened 8 miRNAs that inhibited ITPR2 transcription. Further we retested the regulation of ITPR2 by these miRNAs in two ways: (1) The 8 mimnas mimics were transfected into primary human lung fibroblasts MRC5, 4 of which significantly inhibited ITPR2 expression; (2) We again transfected inhibitors of these 4 miRNAs (imiRNAs) into MRC5, ultimately determined imiR-129 to be the most significant one inducing upregulation of ITPR2 mRNA levels. To further validate our observations, we transfected miR-129 and imiR-129 into two additional primary human lung fibroblasts (IMR 90 and WI 38), also with similar results to MRC 5. Since miRNAs generally control target gene expression by targeting the 3'-UTR region, we queried the miRbase database and found that there are three potential binding sites for miR-129 in the 3' -UTR region of ITPR 2. Then, we constructed 3 recombinant plasmids: wild type (wt) contains all 3 binding peaks, two mutations, one of which mutated at all 3 binding sites (mut 1) and the other mutated at only the longest one (mut 2). Luciferase detection assays showed that miR-129 preferentially binds to region 1 of ITPR 2. Taken together, these data support that miR-129 binds directly to ITPR2 and inhibits its expression.
Example 2: miR-129 regulates calcium signal-membrane potential-active oxygen-DNA damage signal path
ITPR2 is known to be a calcium release channel located in the endoplasmic reticulum. Therefore, as shown in FIG. 3, we investigated whether miR-129 can control ITPR 2-mediated intracellular calcium signaling. By using a cytosolic calcium indicator Fluo4, we observed a decrease in intracellular calcium levels after transfection of a miR-129 analog by flow cytometry; while intracellular calcium levels increased following transfection with imiR-129, such increased intracellular calcium levels could be reversed by knock-down of ITPR 2. Similarly, we have also found the same trend using Rhod2 indicators to measure mitochondrial calcium levels.
Mitochondrial calcium accumulation triggers a decrease in mitochondrial membrane potential, leading to overproduction of mitochondrial ROS (mitoROS) and total intracellular ROS. Therefore, we applied Rhodamine123 as a reporter factor for mitochondrial membrane potential decline and used mitoSOX RED mitochondrial and Dihydroethidium indicators to measure mitoROS and total intracellular ROS levels. Compared with a miRNA control group (miR-Ctrl), the miR-129 analogue increases mitochondrial membrane potential and reduces mitoROS and total ROS levels; while imiR-129 is the opposite. Furthermore, the effect of miR-129 inhibitors can be partially reversed when ITPR2 or MCU is simultaneously knocked down.
The accumulation of ROS can trigger DNA damage. Thus, we introduced the DNA damage marker 53BP1.miR-129 analogs resulted in a significant reduction in DNA damage, but their inhibitors resulted in a significant increase in 53BP1. Furthermore, the effect of imiR-129 can be reduced by knocking down ITPR2 or MCU. Taken together, the results of these studies indicate that miR-129 regulates intracellular calcium signaling-mitochondrial membrane potential-active oxygen-DNA damage signaling pathways through ITPR2 and MCU.
Example 3: miR-129 regulated active oxygen-DNA damage is regulated by calcium signaling and antioxidants
As shown in fig. 4, to verify whether miR-129-regulated active oxygen accumulation and DNA damage can be mediated by calcium signaling and anti-oxidation. We used inositol 1,4, 5-trisphosphate (IP 3) (ITPR ligand) to activate ITPRs. The miR-129 analogs induced reductions in mitoROS, ROS, and DNA damage levels were all partially upregulated after IP3 treatment. Furthermore, we used the calcium chelator BAPTA and the antioxidant N-acetyl-L-cysteine (NAC) in imiR-129 transfected cells. As a result, both the calcium chelator and the antioxidant were found to prevent increased mitoROS, ROS and DNA damage caused by imiR-129. These observations support that miR-129 controls reactive oxygen species and corresponding DNA damage through an upstream calcium signaling pathway.
Example 4 has-miR-129-5p inhibits cell senescence in vitro.
As shown in fig. 5-6.
miR-129 delays cell senescence by inhibiting ITPR2
Human embryonic lung fibroblast MRC5 cells were cultured and inoculated in 12-well plates (Crystal Violet or SA-. Beta. -GAL staining: 2.5X 10) 5 Individual cells/well; IF staining: 4 x 10 5 Individual cells/well) in DMEM (containing 10% FBS) medium, at 37 ℃ and 5% 2 Cultured in a cell culture box. The following grouping processing is performed:
transfection of imiR-Ctrl or imiR-129+/-siITPR2 or siMCU (Small interfering RNA, supplied by Ribo Biotech, guangzhou, same.) per well (80 nM per well) for 4 days; 3 days after transfection, the mRNA levels of ITPR2, ki67, CDKN1A and CDKN2A were detected by RT-qPCR (mean ± SEM of five independent replicates); after 4 days of transfection, each set of cells was collected and examined by flow cytometry for either the cytoplasmic calcium indicator Fluo4 (a) or the mitochondrial calcium reporter Rhod2 (b) or the mitochondrial membrane potential drop indicator Rhodamine123 (c) or mitoSOX RED mitochondal (mitoROS) (d) or Dihydroethidium (ROS) (e). In addition, cells were fixed in 12-well plates and immunofluorescent staining was performed using an anti-53 BP1 antibody; 6 days after transfection, cells were stained with crystal violet (g) or SA- β -GAL solution. As a result, the imiR-129 combined with siITPR2 or siMCU can obviously delay the cellular senescence induced by the imiR-129.
Bleomycin-induced cell senescence model construction
Human embryonic lung fibroblast MRC5 cells were cultured and seeded in 12-well plates (Crystal Violet or SA-beta-GAL staining: 2.5X 10) 5 Individual cells/well; IF staining: 4X 10 5 Individual cells/well) in DMEM (containing 10% FBS) medium, at 37 ℃ and 5% CO 2 Cultured in a cell culture box.
Treating cells with bleomycin (1 μ g/ml) for three days to construct a cell aging model; after bleomycin treatment, a significant increase in intracellular calcium accumulation was observed, mitochondrial calcium levels increased, mitochondrial membrane potential decreased, and a large accumulation of reactive oxygen species in the mitochondria and cytoplasm. DNA damage, cell proliferation inhibition, SA-beta-GAL activity increase, ki67 proliferation factor inhibition, and obvious up-regulation of aging markers CDKN1A and CDKN 2A. The results show that bleomycin can induce human embryonic lung fibroblast MRC5 senescence.
(III) miR-129 delays bleomycin-induced cell senescence
Model group: the transfected miR-Ctrl in each well is cultured for 4 days, added with bleomycin (1 mu g/ml) and cultured for 3 days, and the same detection as that of the control group is carried out.
Control + miR-129 group: miR-129 was transfected into each well and cultured for 4 days, and the same assay as that of the control group was performed.
Model group + miR-129 group, miR-129 is transfected into each well and cultured for 4 days, bleomycin (1 μ g/ml) is added and cultured for 3 days, and detection is carried out in the same way as in the control group.
As a result, as shown in FIG. 6, a large increase in intracellular calcium levels and subsequent increase in mitochondrial calcium levels, decrease in mitochondrial membrane potential, accumulation of mitochondrial and intracellular reactive oxygen species was observed after bleomycin treatment. These phenomena were significantly reduced when the miR-129 analogs were added to bleomycin-treated cells (fig. 6a-6 e). In addition, bleomycin-induced DNA damage, proliferation arrest, increased levels of SA- β -GAL staining, and Ki67 inhibition, ITPR2, CDKN1A, and CDKN2A upregulation were all improved by miR-129 (fig. 6f-6 l). The results show that bleomycin can induce MRC5 cell aging, and miR-129 slows down bleomycin-induced cell aging through a calcium signal-delta psi (m) -ROS-DDR axis.
Example 5: application of miR-129 in bleomycin-induced lung aging
Bleomycin-induced lung aging model
Experimental animals: 8 weeks female C57BL/6J mice (weight average 20 g).
The experimental animals are divided into a normal control group and a model group, the model group mice adopt an intratracheal instillation (i.i.) bleomycin (10 mg/kg) method to construct a lung aging model, the control group mice are given strict PBS control instillation, and the mice are perfused in the heart of the body on the 8 th day to remove blood in circulation. Extracting lung cells for immunosenescence index detection, simultaneously extracting lung tissues for freezing, and preparing a frozen section for immunohistochemical staining. Referring to fig. 7, in bleomycin-induced lung aging, the levels of SA- β -GAL staining and aging index CDKN1A in the lungs of model mice were significantly higher than those of the control group, demonstrating that bleomycin can indeed induce lung aging. The model group presented both naive T cell failure and memory T cell accumulation, which are typical manifestations of immunosenescence, which is considered an important feature of senescence.
(II) has-miR-129-5p intraperitoneal injection
has-miR-129-5p (miR-129) is dissolved in PBS buffer. The mice in the model group are randomly divided into a model group, a miR-NC group and a miR-129 group (8 mice in each group), the miR-NC or miR-129 (5 mg/kg) (containing PBS solvent) is injected into the abdominal cavity on the 2 nd day and the 5 th day after the bleomycin molding, and only the solvent buffer containing PBS is injected into the abdominal cavity of the model group. On day 8, mice were sacrificed and perfused into the heart to remove circulating blood, lung cells were extracted for immunosenescence index detection, and lung tissues were simultaneously extracted and frozen to prepare frozen sections for immunohistochemical staining. The result is shown in FIG. 7, and it can be seen that miR-129 significantly reduces the pulmonary SA-beta-GAL staining ratio of the mice in the model group, while the miR-NC group has no obvious change. miR-129 can reverse bleomycin-induced naive T cell failure and memory T cell accumulation, especially memory CD8+ T cells are reduced to a level close to that of a normal control group. And (4) prompting: miR-129 can improve the pulmonary aging condition induced by bleomycin.
Example 6 application of has-miR-129-5p in natural aging
The experimental animals were divided into young (8 weeks) and old (20 months) groups according to age, and miR-NC or miR-129 (250 nM/kg) (containing PBS solvent) was intraperitoneally injected into each of the two groups 1 time per week for 3 times, each group containing 5 animals. In total, 4 groups of mice were perfused in vivo at day 21 to remove circulating blood and lung cells were extracted for immunosenescence indicator detection; meanwhile, extracting lung tissues and freezing to prepare frozen sections for immunohistochemical staining.
The results are shown in FIG. 7, the lung SA-beta-GAL staining ratio of the aged mice is obviously higher than that of the young mice, which indicates that the aged mice have obvious lung aging; after miR-129 is injected into the abdominal cavity, SA-beta-GAL staining of the lung of an old mouse is obviously reduced, and the SA-beta-GAL staining of the lung of a young mouse is basically unchanged, so that the miR-129 can relieve the natural lung aging condition; the change in naive T cell failure is substantially consistent with the change; the miR-129 is proved to be capable of relieving the natural immunity aging condition; the mRNA expression level of cyclin-dependent kinase 2A (CDKN 2A) in lung, liver and kidney is also changed consistently, which indicates that miR-129 can partially reverse natural aging.
These in vivo data support that miR-129 can improve the senescence phenotype in a model of natural senescence.
The above contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention should not be limited thereby, and any modification made on the basis of the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.
Claims (10)
- The application of has-miR-129-5p in the preparation of the anti-aging drug is characterized in that the nucleotide sequence of the has-miR-129-5p is shown in SEQ ID No.1, the drug inhibits the expression of the drug by combining with a 3' UTR region of ITPR2, so that the signal transduction of intracellular calcium ions from endoplasmic reticulum to mitochondria is regulated, the calcium ion transfer causes the decrease of mitochondrial membrane potential, the decrease of active oxide accumulation and the decrease of DNA damage reaction, and finally the aging of cells and organisms is delayed; the sequence of the 3' UTR region is shown as SEQ ID NO. 2.
- 2. The use of claim 1, wherein the sequence of the binding region of the drug to the 3' UTR region is GCAAAAAA.
- 3. The application according to claim 1, wherein the application comprises any one of:delaying the aging of fibroblasts;improving aging symptoms;delaying the onset or development of diseases associated with cell aging.
- 4. The use according to claim 1, wherein the medicament ameliorates bleomycin-induced lung aging and natural aging.
- 5. The use of claim 1, wherein the medicament inhibits the aging of senescence-associated cells, which are fibroblasts, naive CD4+ T cells, or memory CD8+ T cells.
- 6. The use according to claim 1, wherein the medicament reduces bleomycin-induced lung aging in mice and lung tissue senescence-associated β -galactosidase staining and naive T cell failure, memory T cell accumulation in naturally aging mice, and expression levels of senescence markers CDKN1A, CDKN 2A.
- 7. The use of claim 1, wherein the medicament is introduced into the body tissue by intraperitoneal injection.
- 8. The use according to claim 1, wherein the medicament comprises the active ingredient has-miR-129-5p and a pharmaceutically acceptable carrier.
- 9. The use of claim 1, wherein the medicament is an injection prepared by dissolving has-miR-129-5p in PBS buffer solution.
- 10. The use according to claim 1, wherein the concentration of has-miR-129-5p is 5mg/kg.
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