CN113755569A - Marker for diagnosing acute lung injury and application thereof - Google Patents
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Abstract
The invention discloses a marker for diagnosing acute lung injury and application thereof, wherein the marker is long-chain non-coding RNA (lncRNA) malat 1. The gene therapy inhibiting the expression of IncRNA MALAT1 has important significance in acute lung injury, and the excessive down-regulation of the expression of MALAT1 can obviously inhibit the reduction of the activity of mouse alveolar epithelial cells induced by LPS, the increase of apoptosis and the excessive secretion of inflammatory factors, thereby having good treatment effect on the acute lung injury.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a long-chain non-coding RNA related to acute lung injury and application thereof in acute lung injury, wherein the long-chain non-coding RNA is LncRNA MALAT 1.
Background
Acute Lung Injury (ALI) is clinically characterized by pulmonary inflammation and increased permeability syndrome, which are caused by various reasons, such as diffuse lung cell injury as the basis, characteristic pathological changes of lung tissue structure, pulmonary edema and pulmonary atony, and rapid influence on gas exchange function. The pathological features are damage of alveolar capillary endothelial cells and alveolar epithelial cells, which are manifested as extensive pulmonary edema and small atelectasis. The pathophysiological changes are mainly increased intrapulmonary bypass and decreased lung compliance. Clinically, hypoxemia, respiratory rate and diffuse infiltration of both lungs in chest X-ray are manifested. The etiology of acute lung injury is complex, the mortality rate is high, and severe infection, shock, trauma, smoking, toxicosis, radiation, high oxygen and the like all can cause acute lung injury, but the pathological mechanism of the acute lung injury is not completely clarified. At present, the treatment of acute lung injury mainly adopts large-dose hormone shock therapy, but lacks compelling experimental evidence, and a large number of clinical studies prove that glucocorticoid can not prevent the occurrence of acute lung injury and has no treatment effect on early acute lung injury. The existing medicine for treating acute lung injury has poor treatment effect, has the problems of treating symptoms and not treating root causes, cannot treat acute lung injury fundamentally, and has long treatment time
Acute lung injury is an acute inflammatory disease characterized by the destruction of lung endothelial and epithelial cells and the release of inflammatory factors and cytotoxic mediators. As a primary defense of the lung, alveolar epithelial cells form a strong natural barrier against ALI caused by infectious and non-infectious stimuli. Therefore, alveolar epithelial cells play a key role in the pathogenesis and treatment of ALI. If the condition of ALI cannot be controlled, it will worsen to Acute Respiratory Distress Syndrome (ARDS). The causes of ALI/ARDS are many, including infection, trauma, hypoxia, etc. Infection is one of the major pathogenic factors of ALI/ARDS, of which Lipopolysaccharide (LPS) is one of the causes inducing severe infectious processes in humans. Despite the current advances in ALI/ARDS, mortality remains high, with mortality rates of 38.5% in ALI patients and 41.1% in ARDS patients. At present, the treatment aiming at acute lung injury comprises mechanical ventilation and pharmacological intervention, the clinical risk factors of a mechanical ventilation strategy are more, including aspiration, septicemia and mechanical ventilation induced lung injury, and clinically used medicines for treating ALI, such as dexamethasone, prednisolone and the like, have adverse reactions of inducing blood coagulation dysfunction and liver function injury and the like. Therefore, there is an urgent need to explore new methods and mechanisms for treating ALI.
Long non-coding RNA (LncRNA) refers to RNA that is greater than 200 nucleotides in length but cannot encode a protein. LncRNA plays a regulatory role in gene expression and cell function, and thus is involved in various biological processes including cell differentiation, senescence, proliferation, apoptosis, etc. In recent years, a plurality of lncrnas have been found to be potential biomarkers for treating ALI, and can reduce inflammation and apoptosis induced by LPS. The lung adenocarcinoma metastasis associated transcript 1 (MALAT 1) is a highly expressed intranuclear lncRNA that is highly conserved in organisms from zebrafish to humans. MALAT1 is one of the members of lncRNA and has been found to be abnormally elevated in ALI rats. In addition, MALAT1 was also found to modulate LPS-induced systemic and pulmonary inflammation. Thus, MALAT1 may be a potential biomarker for predicting ALI.
Disclosure of Invention
The invention provides a marker for diagnosing acute lung injury and application thereof, which solve the problems in the prior art.
In order to solve the technical problems, the invention is realized by the following technical scheme:
a marker for diagnosing acute lung injury, wherein the marker is long-chain non-coding rna (lncrna) malat 1.
The RNA (lncRNA) malat1 was highly expressed in acute lung injury.
The application of the RNA (lncRNA) malat1 in acute lung injury inhibits the LPS-induced reduction of alveolar epithelial cell viability, the increase of apoptosis and the over-secretion of inflammatory factors of mice.
Use as a marker for diagnosing acute lung injury, comprising the following analytical steps:
the method comprises the following steps: selecting mouse alveolar epithelial cells;
step two: culturing mouse alveolar epithelial cells in DMEM medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin;
step three: dividing the culture medium in the second step into five groups, and performing five groups of experiments, including a control group, an LPS + empty carrier control group, an LPS + MALAT1 group and an LPS + simaLAT1 group;
step four: detecting the expression condition of MALAT1 in the ALI model, the gene transfection condition of MALAT1 high expression and low expression vector in the ALI model by RT-qPCR according to five groups of experiments;
step five: the effect of MALAT1 on LPS-induced alveolar epithelial cell viability was detected by MTT according to five sets of experiments; the effect of MALAT1 on LPS-induced apoptosis of alveolar epithelial cells was examined by flow cytometry and the effect of MALAT1 on the inflammatory factors IL-6 and IL-8 in LPS-induced alveolar epithelial cells was examined by ELISA.
The control group is normal cultured MLE12 cells; the LPS group stimulates MLE12 cells for 24h with 25 μ g/mL LPS; the LPS + empty vector control group is MLE12 cells transfected by EVC and stimulated by 25 mu g/mL LPS for 24 h; LPS + MALAT1 group was MLE12 cells transfected with MALAT1 overexpression vector), and then stimulated with 25 μ g/mL LPS for 24 h; the LPS + SiMALAT1 group was MLE12 cells transfected with siMALAT1, then stimulated with 25. mu.g/mL LPS for 24 h.
A medicine for treating acute lung injury, which is prepared from a marker for diagnosing acute lung injury, contains siRNA or shRNA interfering MALAT1 expression.
The interfering MALAT1 expression inhibits MALAT1 expression.
The invention shows that MALAT1 is obviously up-regulated in ALI model, and a new biomarker of ALI is excavated. By down-regulating the expression of MALAT1, the method can obviously inhibit the LPS-induced reduction of the activity of mouse alveolar epithelial cells, the increase of apoptosis and the over-secretion of inflammatory factors, and has good treatment effect on acute lung injury.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art 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 for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a graph of RT-qPCR assay for LPS-induced expression of MALAT1 in alveolar epithelial cells;
FIG. 2 is a diagram of RT-qPCR detection of MALAT1 gene vector expression of MALAT1 in LPS-induced alveolar epithelial cells;
FIG. 3 is a graph of MTT assay for the effect of MALAT1 on LPS-induced viability of alveolar epithelial cells;
FIG. 4 is a flow cytometer measuring the effect of MALAT1 on LPS-induced apoptosis of alveolar epithelial cells;
FIG. 5 is an ELISA test of the effect of MALAT1 on LPS-induced secretion of inflammatory factors from alveolar epithelial cells.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
A marker for diagnosing acute lung injury, wherein the marker is long-chain non-coding rna (lncrna) malat 1.
The RNA (lncRNA) malat1 was highly expressed in acute lung injury.
The application of the RNA (lncRNA) malat1 in acute lung injury inhibits the LPS-induced reduction of alveolar epithelial cell viability, the increase of apoptosis and the over-secretion of inflammatory factors of mice.
Use as a marker for diagnosing acute lung injury, comprising the following analytical steps:
the method comprises the following steps: selecting mouse alveolar epithelial cells; mouse alveolar epithelial cell line MLE12 was purchased from Nanfeng Hui organisms in China.
Step two: culturing mouse alveolar epithelial cells in DMEM medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin;
step three: to test the effect of MALAT1 on LPS-induced MLE12, the medium from step two was divided into five groups and five experiments were performed, including control, LPS + empty vector control, LPS + MALAT1 and LPS + siMALAT 1; simalat1 and MALAT1 overexpression vectors were synthesized by Producer.
Step four: detecting the expression condition of MALAT1 in the ALI model, the gene transfection condition of MALAT1 high expression and low expression vector in the ALI model by RT-qPCR according to five groups of experiments;
step five: the effect of MALAT1 on LPS-induced alveolar epithelial cell viability was detected by MTT according to five sets of experiments; the effect of MALAT1 on LPS-induced apoptosis of alveolar epithelial cells was examined by flow cytometry and the effect of MALAT1 on the inflammatory factors IL-6 and IL-8 in LPS-induced alveolar epithelial cells was examined by ELISA.
The control group is normal cultured MLE12 cells; the LPS group stimulates MLE12 cells for 24h with 25 μ g/mL LPS; the LPS + empty vector control group is MLE12 cells transfected by EVC and stimulated by 25 mu g/mL LPS for 24 h; LPS + MALAT1 group was MLE12 cells transfected with MALAT1 overexpression vector), and then stimulated with 25 μ g/mL LPS for 24 h; the LPS + SiMALAT1 group was MLE12 cells transfected with siMALAT1, then stimulated with 25. mu.g/mL LPS for 24 h.
A medicine for treating acute lung injury, which is prepared from a marker for diagnosing acute lung injury, contains siRNA or shRNA interfering MALAT1 expression. The interfering MALAT1 expression inhibits MALAT1 expression.
Example 1:
the expression condition of MALAT1 in ALI model is detected by RT-qPCR in the experiment. The method comprises the following steps:
cells were seeded on 100mm dishes and then treated differently according to experimental groups. Cells were digested with trypsin, centrifuged, collected and RNA extracted.
1. The reverse transcription reagent comprises nuclease-free pure water (RNase free ddH2O), reverse transcription Reaction Buffer (Reaction Buffer), Random Primer, dNTP Mix, RevertAid RT and RiboLock RI.
2. Reverse transcription reagent each component use table
3. Mu.g of RNA was added and RNase free ddH2O was added to 20. mu.L.
4. Reaction conditions are as follows: 25 ℃ for 5 minutes, 42 ℃ for 60 minutes, 70 ℃ for 5 minutes, 4 ℃ Forever.
Amplification:
1. the amplification reagents include 2X SYBR Green Supermix, forward primer, reverse primer, cDNA, and nuclease free pure water (RNase free ddH 2O).
2. Usage table of components of amplification reagent
RNase free ddH2O was added to 10. mu.L.
4. Reaction conditions are as follows: 95 degrees celsius for 3 minutes. 95 degrees celsius for 10 seconds, 51 degrees celsius for 30 seconds, 72 degrees celsius for 30 seconds, for a total of 40 cycles.
Primer sequences
The results are shown in figure 1, LPS induced alveolar epithelial cells to form an acute lung injury model in which MALAT1 is highly expressed.
Example 2:
the experiment adopts RT-qPCR to detect the gene transfection condition of the MALAT1 high expression and low expression vector in the ALI model. The experimental procedure was as described above and was not repeated.
The results are shown in FIG. 2, where the MALAT1 high expression vector promotes the expression of MALAT1 in alveolar epithelial cells in the ALI model; the MALAT1 low expression vector inhibited alveolar epithelial cell MALAT1 expression in the ALI model.
Example 3:
this experiment examined the effect of MALAT1 on LPS-induced alveolar epithelial cell viability by MTT. The method comprises the following steps: cells were processed according to different groups. To each well, 50. mu.l of serum-free medium and 50. mu.l of MTT reagent were added. After 3 hours of incubation, 150. mu.l of MTT solvent was added per well to stop the reaction. The absorbance at 590nm was measured.
The results are shown in fig. 2, the viability of the cells in the LPS group is significantly lower than that in the control group, the LPS + MALAT1 group further enhances the LPS-induced decrease in the viability of the alveolar epithelial cells, while the LPS + siMALAT1 reverses the LPS-induced decrease in the viability of the alveolar epithelial cells, which indicates that silencing MALAT1 can effectively promote the LPS-induced viability of the alveolar epithelial cells.
Example 4:
in the experiment, the influence of MALAT1 on the apoptosis of alveolar epithelial cells induced by LPS is detected by a flow cytometer. The method comprises the following steps: collecting the cells after the drug treatment in a flow tube; PBS washed cells 1 time; on ice, 1 × Annexin-binding buffer was prepared and used to resuspend the cells. Resuspended cells were added 5. mu.Lannexin V and 10. mu.L PI and incubated at room temperature for 5 min in the dark. Detecting apoptosis on a flow cytometer.
The results are shown in fig. 3, compared with the control group, the apoptosis rate of the cells in the LPS group is obviously increased, the LPS + MALAT1 group further enhances the increase of the apoptosis rate of the alveolar epithelial cells caused by LPS, and the LPS + siMALAT1 reverses the increase of the viability of the alveolar epithelial cells caused by LPS, which indicates that the silencing MALAT1 can effectively inhibit the apoptosis of the alveolar epithelial cells caused by LPS.
Example 5:
this experiment examined the effect of MALAT1 on the inflammatory factors IL-6 and IL-8 in LPS-induced alveolar epithelial cells by ELISA. The method comprises the following steps: the experiments were performed according to the manufacturer's instructions. Briefly, the cell supernatants of each group were diluted to appropriate concentrations and then added to 96-well plates. To each well in turn was added a detection antibody, streptavidin-HRP, substrate solution, and then the reaction was stopped using a stop solution. The absorbance at 540nm was measured.
As a result, as shown in FIG. 4, the level of IL-6 and IL-8 in the cell supernatant of LPS group was significantly increased compared to the control group, while the LPS + MALAT1 group further enhanced the increase of IL-6 and IL-8 in the alveolar epithelial cells caused by LPS, while LPS + SimALT 1 reversed the increase of IL-6 and IL-8 in the alveolar epithelial cells caused by LPS, indicating that silencing MALAT1 was effective in suppressing the secretion of inflammatory factors in the alveolar epithelial cell supernatant caused by LPS.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Claims (7)
1. A marker for diagnosing acute lung injury, characterized by: the marker is long non-coding RNA (lncRNA) malat 1.
2. A marker as a diagnosis of acute lung injury according to claim 1, wherein: the RNA (lncRNA) malat1 was highly expressed in acute lung injury.
3. Use according to claim 1 as a marker for the diagnosis of acute lung injury, characterized in that: the application of the RNA (lncRNA) malat1 in acute lung injury inhibits the reduction of the activity of mouse alveolar epithelial cells induced by LPS, the increase of apoptosis and the over-secretion of inflammatory factors.
4. Use according to claim 3 as a marker for the diagnosis of acute lung injury, characterized in that: the method comprises the following analysis steps:
the method comprises the following steps: selecting mouse alveolar epithelial cells;
step two: culturing mouse alveolar epithelial cells in DMEM medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin;
step three: dividing the culture medium in the second step into five groups, and performing five groups of experiments, including a control group, an LPS + empty carrier control group, an LPS + MALAT1 group and an LPS + simaLAT1 group;
step four: detecting the expression condition of MALAT1 in the ALI model, the gene transfection condition of MALAT1 high expression and low expression vector in the ALI model by RT-qPCR according to five groups of experiments;
step five: the effect of MALAT1 on LPS-induced alveolar epithelial cell viability was detected by MTT according to five sets of experiments; the effect of MALAT1 on LPS-induced apoptosis of alveolar epithelial cells was examined by flow cytometry and the effect of MALAT1 on the inflammatory factors IL-6 and IL-8 in LPS-induced alveolar epithelial cells was examined by ELISA.
5. The use according to claim 4 as a marker for the diagnosis of acute lung injury, wherein: the control group is normal cultured MLE12 cells; the LPS group stimulates MLE12 cells for 24h with 25 μ g/mL LPS; the LPS + empty vector control group is MLE12 cells transfected by EVC and stimulated by 25 mu g/mL LPS for 24 h; LPS + MALAT1 group was MLE12 cells transfected with MALAT1 overexpression vector), and then stimulated with 25 μ g/mL LPS for 24 h; the LPS + SiMALAT1 group was MLE12 cells transfected with siMALAT1, then stimulated with 25. mu.g/mL LPS for 24 h.
6. A medicament for treating acute lung injury prepared according to claim 1 as a marker for diagnosing acute lung injury, wherein: the medicine contains siRNA or shRNA interfering with MALAT1 expression.
7. The drug for treating acute lung injury, which is prepared as a marker for diagnosing acute lung injury according to claim 6, wherein: the interfering MALAT1 expression inhibits MALAT1 expression.
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| CN114634932A (en) * | 2022-03-11 | 2022-06-17 | 安徽医科大学 | Novel circRNA, kit and application |
| CN114634932B (en) * | 2022-03-11 | 2023-09-05 | 安徽医科大学 | Novel circRNA, kit and application |
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