siRNA for down-regulating PRMT2 gene expression and its application

Abstract

The present invention relates to the field of biomedicine technology, and in particular to siRNA and its application for down-regulating PRMT2 gene expression. The present invention finds through research and analysis that PRMT2 gene expression is positively correlated with IFN-β production, specifically, down-regulation of PRMT2 gene expression causes TLR4 / IRF3 signaling pathway to be negatively regulated, and it is found that when siRNA fragments are used to down-regulate PRMT2 gene expression, the production of IFN-β is affected, and the development of disseminated intravascular coagulation caused by endotoxemia is finally inhibited, and the harm caused by endotoxemia is reduced. In addition, the present invention also particularly finds that PRMT2siRNA1 is the most effective fragment among the above-mentioned siRNA fragments; siRNA can be used to prepare corresponding drugs to inhibit disseminated intravascular coagulation caused by endotoxemia. Therefore, the present invention provides a new treatment strategy for controlling complications of endotoxemia.

Description

[Technical field]

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to siRNA for down-regulating PRMT2 gene expression and application thereof. [Background technology]

[0002] Endotoxemia is caused by bacteria in the blood or in the lesions releasing a large amount of endotoxin into the blood, or by the transfusion of a large amount of endotoxin-contaminated fluids. It is divided into two types: endogenous and exogenous. Surgical diseases such as acute respiratory distress syndrome, hepatobiliary surgical diseases, severe burns, inflammation, abscesses, necrosis of other organs, etc. can all lead to endotoxemia, and its further development may cause septic shock, systemic inflammatory response syndrome or multifunctional organ failure and lead to death. In the United States alone, 30,000 to 100,000 patients die each year due to lack of effective treatment for endotoxemia. Therefore, the study of drugs related to the treatment of endotoxemia and its complications is a technical problem that needs to be solved urgently in our current research. Bacterial infection is the main cause of endotoxemia, of which Gram-negative bacteria infection accounts for 45% to 60%. Endotoxin in bacterial infection (endotoxinemia) can bind to TLR4 and activate two downstream MyD88-dependent and -independent signaling pathways, activating transcription factors NF-κB and IRF3, leading to the production of a large number of pro-inflammatory cytokines such as IL-6, IL-12, TNFα and type I interferons such as IFN-α / β, which can lead to complications such as shock, disseminated intravascular coagulation, cell damage and potential multiple organ failure, and can induce epilepsy, coma, and even death.

[0003] Therefore, in-depth exploration of the mechanism of action during the pathological changes of endotoxemia will help to develop new prevention, diagnosis and treatment strategies. [Summary of the invention]

[0004] In view of the above, the present application provides siRNA for down-regulating PRMT2 gene expression, regulating IFN-β produced by endotoxemia, thereby providing a new treatment strategy for controlling complications of endotoxemia.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] siRNA for down-regulating PRMT2 gene expression, wherein the nucleotide sequence of the siRNA is any one of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3

[0007] Furthermore, the nucleotide sequence of the siRNA is SEQ ID NO.1.

[0008] Furthermore, the PRMT2 gene expression is positively correlated with IFN-β production.

[0009] The present invention also includes the use of siRNA for down-regulating PRMT2 gene expression in inhibiting the production of IFN-β via the natural immune TLR4 / IRF3 signaling pathway.

[0010] The present invention also includes a method for inhibiting the production of IFN-β through the natural immune TLR4 / IRF3 signaling pathway by down-regulating PRMT2 gene expression with siRNA, the method comprising: transfecting the siRNA in Raw264.7 cells, down-regulating PRMT2 gene expression, achieving a reduction in the arginine methylation level of the natural immune TLR4 receptor, and ultimately inhibiting the production of IFN-β

[0011] The IFN-β promotes the complication of endotoxemia, namely disseminated intravascular coagulation, which in turn endangers the patient's life. Therefore, by down-regulating the expression of the PPRMT2 gene, the complication of endotoxemia, namely disseminated intravascular coagulation, can be ultimately inhibited, and the harm caused by endotoxemia can be reduced.

[0012] The present invention also includes the use of siRNA for down-regulating PRMT2 gene expression in the preparation of a drug for inhibiting the natural immune TLR4 / IRF3 signaling pathway.

[0013] The present invention has the following beneficial effects:

[0014] The present invention has found through research and analysis that PRMT2 is an arginine methyltransferase of the natural immune TLR4 receptor and the transcription factor IRF3, and that arginine methylation produces an IFN-β model through the TLR4 / IRF3 signaling pathway, that is, PRMT2 gene expression is positively correlated with IFN-β production, specifically, down-regulation of PRMT2 gene expression leads to negative regulation of the TLR4 / IRF3 signaling pathway, and it is also found that when siRNA fragments are used to down-regulate PRMT2 gene expression, the production of IFN-β is affected, and ultimately inhibits the development of disseminated intravascular coagulation, a complication caused by endotoxemia, and reduces the harm caused by endotoxemia, thereby saving the patient's life. In addition, the present invention has also particularly found that PRMT2 siRNA1 is the most effective fragment of the above siRNA fragments; siRNA can be used to prepare corresponding drugs to inhibit disseminated intravascular coagulation caused by endotoxemia. Therefore, the present invention provides a new treatment strategy for controlling disseminated intravascular coagulation, a complication of endotoxemia.

Brief Description of the Drawings

[0015] Figure 1 is the expression graph of siRNA immunoblot analysis of down-regulated PRMT2 gene;

[0016] Figure 2This is a graph showing the expression of siRNA down-regulating the PRMT2 gene using RT-qPCR technology analysis;

[0017] Figure 3 This is the expression diagram of the immunoblot analysis of the arginine methylation modification of the innate immune signaling pathway receptor TLR4 after downregulation of PRMT2;

[0018] Figure 4 yes Figure 3 Quantitative analysis diagram of

[0019] Figure 5 This is the expression diagram of the immunoblot analysis of the arginine methylation modification of the innate immune signaling pathway transcription factor IRF3 after downregulation of PRMT2;

[0020] Figure 6 yes Figure 5 Quantitative analysis diagram of

[0021] Figure 7 This is an analysis diagram of the effect of PRMT2 on the activity of the transcription factor IRF3 response element;

[0022] Figure 8 This is a graph showing the detection of IFN-β expression after down-regulating PRMT2. [Specific implementation method]

[0023] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0024] Embodiment 1:

[0025] Human PRMT2 siRNA fragment sequence

[0026] The siRNA for down-regulating PRMT2 gene expression, wherein the nucleotide sequence of the siRNA is any one of PRMT2 siRNA1: 5'GGACUGGGAUCAUCAGUCUDTDT 3'; (SEQ ID NO.1), PRMT2 siRNA2: 5'GUGUCAUCCUGCAGAAUAADTDT 3'; (SEQ ID NO.2), and PRMT2 siRNA3: 5'GGUACAUUCCGGCAAACCADTDT 3'; (SEQ ID NO.3).

[0027] Embodiment 2:

[0028] Screening of effective siRNA fragments for down-regulating human PRMT2 gene expression

[0029] In addition to the fragments described in Example 1, another negative control group was set up: Control siRNA, the nucleotide sequence of which was: 5'UUCUCCGAACGUGUCACGUTT 3'.

[0030] The above-mentioned Control siRNA, PRMT2 siRNA1, PRMT2 siRNA2, and PRMT2 siRNA3 fragments were transfected into mouse macrophage Raw264.7 cells. After 72 hours, the cells were collected and analyzed by immunoblotting and RT-qPCR. The results are as follows: Figure 1-Figure 2 shown.

[0031] according to Figure 1-2 The results showed that the down-regulation effect of PRMT2 siRNA1 fragment was the most significant, therefore, PRMT2 siRNA1 was selected for subsequent experiments.

[0032] Examples 3-6 will further study the regulatory relationship between PRMT2 and the innate immune TLR4 / IRF3 signaling pathway.

[0033] Example 3

[0034] Regulatory relationship between PRMT2 and arginine methylation modification of innate immune signaling pathway receptor TLR4

[0035] Control siRNA was used as the control group and PRMT2 siRNA was used as the experimental group. Raw264.7 cells were transfected with TLR4 receptor activator lipopolysaccharide (LPS) for 48 hours and then treated or not treated for 20 hours. The cells were then collected and lysed, and immunoblot analysis was performed after immunoprecipitation. The results are shown in Figure 2. Figure 3-4 shown.

[0036] according to Figure 3-4 It can be seen that compared with the cells in the control group, the expression of PRMT2 gene in the experimental group cells was significantly downregulated after siRNA interference; in the control group, compared with the cells not treated with LPS, the TLR4 arginine methylation level was significantly increased after LPS treatment; and in the PRMT2 gene expression downregulated group, the TLR4 arginine methylation level was significantly decreased before and after LPS treatment.

[0037] The results showed that PRMT2 mediated the arginine methylation modification of TLR4, that is, PRMT2 was the arginine methyltransferase of TLR4. Figure 3 shown. Figure 3 For quantitative analysis, see Figure 4 shown.

[0038] Example 4

[0039] Regulatory relationship between PRMT2 and arginine methylation modification of the transcription factor IRF3 in the innate immune signaling pathway

[0040] Control siRNA was used as the control group and PRMT2 siRNA was used as the experimental group. Raw264.7 cells were transfected with LPS for 48 hours and then treated with LPS for 20 hours or without treatment. The cells were then collected and lysed. Immunoprecipitation was performed and immunoblot analysis was performed. The results are shown in Figure 5-6 shown.

[0041] according to Figure 5-6 It can be seen that compared with the cells in the control group, the expression of PRMT2 gene in the experimental group cells was significantly downregulated after siRNA interference, and the arginine methylation level of IRF3 in this group of cells was significantly reduced whether treated with LPS or not.

[0042] The results showed that PRMT2 mediated the arginine methylation modification of IRF3, that is, PRMT2 is the arginine methyltransferase of IRF3. Figure 5 As shown, Figure 5 For quantitative analysis, see Figure 6 shown.

[0043] Example 5

[0044] Regulatory relationship between PRMT2 and the activity of the transcription factor IRF3 in the innate immune signaling pathway

[0045] Empty vector and TLR4 were co-transfected with PRMT2 wild type (PRMT2-WT) and two mutant (PRMT2-H112Q, PRMT2-M115I) plasmids into human embryonic kidney 293T cells, and then treated with LPS or not for 6 hours, respectively. After that, the cells were collected and lysed, and the activity of IRF3 response element was analyzed using dual reporter gene detection kit. The results are shown in Figure 7 shown.

[0046] according to Figure 7 It can be seen that compared with cells transfected with empty vector plasmid, the activity of IRF3 response element was significantly increased after transfection with TLR4 plasmid and treatment with LPS or without treatment of cells, while the activity of IRF3 response element of cells co-transfected with TLR4 and wild-type PRMT2 plasmid was higher than that of cells transfected with TLR4 plasmid alone, and the activity of IRF3 response element of cells co-transfected with TLR4 and PRMT2 two mutant plasmids was significantly lower than that of cells co-transfected with TLR4 and wild-type PRMT2 plasmid.

[0047] The results showed that PRMT2 enhanced the activity of the transcription factor IRF3 response element. Figure 7shown.

[0048] Example 6

[0049] Effect of downregulating PRMT2 on IFN-β production

[0050] IFN-β is one of the main cytokines produced by endotoxemia and one of the end products of the innate immune TLR4 / IRF3 signaling pathway. Therefore, this example further studies the effect of PRMT2 on IFN-β production;

[0051] Similarly, the Control siRNA was used as the control group and the PRMT2 siRNA was used as the experimental group. Raw264.7 cells were transfected with LPS 48 hours later and not treated with LPS for 20 hours. The cells were then harvested and lysed. After immunoprecipitation, immunoblot analysis was performed. The results are shown in Figure 8 shown.

[0052] according to Figure 8 The results showed that compared with the cells in the control group, the expression of IFN-β in the experimental group was significantly downregulated after siRNA interference. In the control group, compared with the cells not treated with LPS, the expression of IFN-β was significantly increased after LPS treatment.

[0053] The results showed that downregulating PRMT2 gene expression inhibited the production of IFN-β.

[0054] Based on Examples 3-6, it can be seen that PRMT2 is involved in regulating the TLR4 / IRF3 signaling pathway, indicating that arginine methylation modification mediated by PRMT2 has a positive regulatory effect on the TLR4 / IRF3 signaling pathway.

[0055] Example 7

[0056] Application Examples

[0057] According to the above embodiments, the siRNA interference fragment of the present application can inhibit the production of IFN-β produced by endotoxemia. Therefore, it can be prepared into a medicine for preventing and treating the complication of endotoxemia, namely disseminated intravascular coagulation, and thus saving the patient's life.

[0058] In summary, the present application can ultimately inhibit the complication of endotoxemia, namely, mass intravascular coagulation, and reduce the harm caused by endotoxemia by down-regulating the expression of PPRMT2 gene.

[0059] The above-mentioned embodiments merely express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it should not be understood as limiting the scope of the present invention. Sequence Listing <110> Youjiang Medical College for Nationalities <120> siRNA for down-regulating PRMT2 gene expression and its application <141> 2021-09-07 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty three <212> DNA / RNA <213> Artificial Sequence <400> 1 ggacugggau caucagucud tdt 23 <210> 2 <211> twenty three <212> DNA / RNA <213> Artificial Sequence <400> 2 gugucauccu gcagaauaad tdt 23 <210> 3 <211> twenty three <212> DNA / RNA <213> Artificial Sequence <400> 3 gguacauucc ggcaaaccad tdt 23

Claims

1. Downward adjustment PRMT2 The siRNA for gene expression is characterized in that The nucleotide sequence of the siRNA is SEQ ID NO.1.

Images